The Quest Diagnostics Manual
Endocrinology Test Selection and Interpretation
Fourth Edition
The Quest Diagnostics Manual
Endocrinology Test Selection and Interpretation
Fourth Edition
Edited by: Delbert A. Fisher, MD Senior Science Officer Quest Diagnostics Nichols Institute Professor Emeritus, Pediatrics and Medicine UCLA School of Medicine Consulting Editors: Wael Salameh, MD, FACP Medical Director, Endocrinology/Metabolism Quest Diagnostics Nichols Institute San Juan Capistrano, CA Associate Clinical Professor of Medicine, David Geffen School of Medicine at UCLA Richard W. Furlanetto, MD, PhD Medical Director, Endocrinology/Metabolism Quest Diagnostics Nichols Institute Chantilly, VA ©2007 Quest Diagnostics Incorporated. All rights reserved. Fourth Edition Printed in the United States of America
Quest, Quest Diagnostics, the associated logo, Nichols Institute, and all associated Quest Diagnostics marks are the trademarks of Quest Diagnostics. All third party marks − ®' and ™' − are the property of their respective owners.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, and information storage and retrieval system, without permission in writing from the publisher. Address inquiries to the Medical Information Department, Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92690-6130.
Previous editions copyrighted in 1996, 1998, and 2004. Re-order # IG1984 Forward
Quest Diagnostics Nichols Institute has been committed to providing state of the art endocrine testing for nearly 4 decades. During this time, we have introduced assays for most of the endocrine systems hormones, important metabolites and carrier proteins, free hormones, many hormone receptors, endocrine system autoantibodies, relevant chromosomal abnormalities, and selected genetic mutations. Our commitment to state of the art diagnostics and our dedication to quality and service have always included both adult and pediatric patients, and we have an ongoing program providing adult and pediatric reference range and clinical correlations data. Additionally, we have special procedures for identification and handling of pediatric samples, including reduced sample volume, whenever possible.
In this manual, we have consolidated detailed technical information for more than 150 of the endocrine and endocrine-related tests performed at Nichols Institute. The manual includes an overview of the physiology of each endocrine system, the diagnostic applications of each test, interpretation of test results, and a catalog of the endocrine-related dynamic or perturbation tests that facilitate diagnosis and patient management. For your convenience, we have included a guide for interconversion of conventional and SI units for our endocrine tests. In addition, we have indexed our endocrine tests in several formats to provide easier access to the information.
National Quest Diagnostics test codes are listed for each of the tests described in the Alphabetical Test section. Methodologies may differ for the non-Nichols Institute regional laboratory tests; information and reference ranges for these tests are available from your sales representative or regional laboratory personnel.
We trust that the manual will assist you in the diagnosis and management of your adult and pediatric patients and reiterate our commitment at Quest Diagnostics to provide you with the highest quality test results and ready availability of consultative services. Our laboratory staff and our Medical and Scientific Directors are available for consultation to help assure the most efficacious resolution of complex or confusing endocrine problems. We welcome your feedback and suggestions for improving our service to you.
Delbert A. Fisher, MD Senior Science Officer Quest Diagnostics Nichols Institute
v Acknowledgments
Normative data were developed by the Clinical Correlations department under direction of Dr. Richard Reitz, Esther Carlton, and Collette Scheele. Text development, overall composition, and project direction were accomplished by Patricia Vendely. Mary Jane Michel assisted with typing; Lorraine Saunders was responsible for formatting; and Lynn Ricard helped with proofing. For their dedicated contributions, the editor is indeed grateful.
Delbert A. Fisher, MD
vi Contents
Page
Forward ...... v
Acknowledgments ...... vi
Endocrine Test Categories and Associated Tests Adrenocortical Function ...... 3 Cardiovascular/Lipids ...... 4 Catecholamines...... 5 Congenital Adrenal Hyperplasia (CAH)...... 5 Endocrine Autoimmunity...... 5 Fluid, Electrolyte, and Renal...... 6 Genetic (Biochemical and Cytogenetic) ...... 7 Gonadal Function ...... 8 Growth and Growth Hormone...... 9 Hypothalamic and Pituitary Function ...... 9 Metabolic (Including Diabetes Mellitus) and Gastrointestinal Disorders...... 10 Multiple Endocrine Neoplasia ...... 11 Parathyroid and Mineral Metabolism...... 12 Thyroid Function ...... 12 Additional Diagnostic Profiles...... 13 Perturbation Tests...... 14 Alphabetical Test Section See Test Index for list of tests included in this section...... 15 Test Application and Interpretation Preface...... 189 Disorders of Adrenal Function Background Physiology ...... 190 Adrenal Insufficiency ...... 193 Adrenal Biosynthetic Enzyme Deficiencies ...... 193 Classical Congenital Adrenal Hyperplasia (CAH) ...... 193 Nonclassical Disease (Partial Deficiency in Steroid Biosynthesis) ...... 205 Cushing's Syndrome ...... 206 Renin-Angiotensin – Aldosterone System (RAAS) ...... 207 Background Physiology...... 207 Assessment of Disorders of the RAAS ...... 208 Primary Aldosteronism ...... 210 Renovascular Hypertension...... 210 Dexamethasone-suppressible Hyperaldosteronism ...... 211 Pseudohypoaldosteronism...... 211 Adrenal Diagnosis via Urine GC/MS Metabolite Analysis ...... 211
vii Contents
Page References ...... 215
Disorders of Anterior Pituitary Function Background Physiology ...... 217 Hypopituitarism ...... 218 Pituitary Adenomas...... 220 General Features ...... 220 Prolactinoma ...... 221 Acromegaly...... 222 Cushing's Disease ...... 223 Thyrotropinoma...... 223 Gonadotropinoma ...... 223 References ...... 224
Disorders of Calcium and Bone Metabolism Background Physiology ...... 225 Laboratory Assessment of Disorders of Calcium Metabolism ...... 226 Evaluation of Hypercalcemia ...... 228 Evaluation of Hypocalcemia...... 229 Vitamin D Deficiency...... 230 Vitamin D Metabolism in Renal Failure...... 230 Metabolic Bone Disease ...... 232 Clinical Application of Bone Markers...... 233 Diagnosis of Bone Disorders ...... 233 Diagnosis of Osteoporosis ...... 236 Efficacy of Therapy ...... 236 Follow-up to Therapy...... 236 References ...... 236
Disorders of Carbohydrate Metabolism Background Physiology ...... 239 Hyperglycemic Disorders ...... 239 Type 1 Diabetes ...... 240 Maturity Onset Diabetes of the Young (MODY) ...... 243 Type 2 Diabetes ...... 243 Diagnosis of Diabetes Mellitus...... 244 Preventing and Managing Complications ...... 244 Hypoglycemic Disorders...... 248 Diagnostic Approach ...... 249 References ...... 251
Disorders of Gonadal Function Background Physiology ...... 255
viii Contents
Page Development of the Hypothalamic–Pituitary Axis and Isolated GnRH Deficiency ...... 255 Regulation of Pubertal Onset...... 256 Regulation of the Mature Hypothalamic–Pituitary–Gonadal Axis...... 256 Gonadal Determination and Differentiation ...... 259 Measurement of Pituitary–Gonadal Axis Hormones...... 261 Luteinizing Hormone ...... 261 Follicle Stimulating Hormone...... 261 Estrogens...... 261 Testosterone...... 262 Sex Hormone Binding Globulin ...... 263 Inhibin ...... 263 Abnormalities of Sexual Determination and Differentiation ...... 263 Disorders of Sexual Maturation ...... 264 Normal Puberty Staging...... 264 Timing of Physical Changes of Puberty ...... 264 Normal Variants of Puberty ...... 266 Precocious Puberty...... 267 Delayed or Absent Puberty ...... 267 Disorders of Testicular Function...... 269 Developmental Disorders ...... 269 Disorders of the Germinal Cells...... 269 Testicular Aging...... 271 Disorders of Ovarian Function...... 272 Premature Ovarian Failure...... 273 Polycystic Ovarian Syndrome ...... 274 Assessment of Functional Gonadal Tumors ...... 275 References ...... 276
Disorders of the Growth Hormone–Insulin-like Growth Factor Axis Background Physiology ...... 279 Growth Hormone Deficiency in Children...... 282 Growth Hormone Deficiency in Adults...... 283 Growth Hormone Excess ...... 284 Laboratory Assessment of the Growth Hormone – IGF Axis ...... 284 Insulin-like Growth Factors and Binding Proteins ...... 285 Growth Hormone Dynamic Testing...... 289 References ...... 290
Disorders of Thyroid Function Background Physiology ...... 293 Differential Diagnosis of Thyroid Disorders ...... 295 Primary Hypothyroidism...... 298
ix Contents
Page Hyperthyroidism ...... 298 Hypothalamic-Pituitary Hypothyroidism...... 299 Inappropriate TSH Secretion...... 299 Nonthyroidal Illness...... 299 Autoimmune Thyroid Disease ...... 300 Thyroid Dysfunction in Infancy and Early Childhood ...... 304 Thyroid Neoplasia...... 304 Thyroid Protein-Binding Abnormalities...... 308 References ...... 308
Fluid & Electrolyte Disorders Background Physiology ...... 311 Primary Disorders ...... 312 Secondary Disorders...... 314 Disorders of Potassium ...... 316 References ...... 317
Pheochromocytoma, Medullary Thyroid Carcinoma, and Multiple Endocrine Neoplasia Pheochromoctyoma...... 319 Diagnosis of Pheochromocytoma...... 320 Genetic Testing for Pheochromocytoma...... 321 Medullary Thyroid Carcinoma (MTC)...... 322 Hereditary MTC Diagnosis and Management...... 323 Sporadic MTC Diagnosis and Management...... 324 MEN Type 1...... 325 References ...... 327
Dynamic Tests Adrenal ...... 329 ACTH Stimulation Test, Standard...... 329 ACTH Stimulation Test, Prolonged ...... 329 ACTH Stimulation Test, Low Dose ...... 330 Aldosterone Suppression Test ...... 330 CRH Stimulation Test ...... 330 CRH Stimulation Test, Adrenal Venous Sampling...... 331 CRH Stimulation Test, Petrosal Venous Sampling...... 331 Dexamethasone Suppression Tests ...... 332 Carbohydrate Metabolic...... 332 Glucose Tolerance Test...... 332 Pancreatic Hormone Response Test...... 333 Prolonged Fasting Test...... 334
x Contents
Page Gonadal ...... 335 GnRH Stimulation Test...... 335 Growth Hormone ...... 336 Glucose Suppression Test ...... 336 Growth Hormone Stimulation Test ...... 336 Multiple Endocrine Neoplasia ...... 337 Secretin Stimulation Test...... 337 Posterior Pituitary ...... 337 Combined Anterior Pituitary (CAP) Test...... 337 Water Deprivation Test ...... 338 Thyroid ...... 338 Calcitonin-Calcium Stimulation Test ...... 338 Thyrotropin Releasing Hormone (TRH) Stimulation Test...... 339 References ...... 339
SI Units...... 345 Test Index ...... 353 Subject Index ...... 359
xi Contents
Page
xii Endocrine Test Categories
and Associated Tests
Endocrine Endocrine Tests Endocrine Test Categories Category by and Associated Tests
Page Adrenocortical Function ACTH, Plasma ...... 17 Adrenal Antibody Screen with Reflex to Titer ...... 18 Aldosterone, 24-Hour Urine...... 19 Aldosterone, LC/MS/MS, Serum ...... 20 Aldosterone/Cortisol Ratio, Adrenal Vein Serum or Plasma...... ‡ Aldosterone/Plasma Renin Activity Ratio*...... 21 3α-Androstanediol Glucuronide (3α-diol G) ...... 24 Angiotensin Converting Enzyme (ACE)...... ‡ Androstenedione, LC/MS/MS...... 25 CAH (21-Hydroxylase Deficiency) Common Mutations*...... 29 CAH (21-Hydroxylase Deficiency) Rare Mutations*...... 30 Corticosterone, LC/MS/MS...... 50 Corticotropin Releasing Hormone (CRH)† ...... 51 Cortisol Binding Globulin (Transcortin) ...... 52 Cortisol, Free, LC/MS/MS, 24-Hour Urine ...... 54 Cortisol, Free, Saliva...... 55 Cortisol, Free, Serum ...... 56 Cortisol, Free and Cortisone, 24-Hour Urine...... 53 Cortisol, Total, LC/MS/MS...... 57 Cortisone, 24-Hour Urine...... 58 Cortisone, Serum*...... 59 Deoxycorticosterone (DOC) ...... 63 11-Deoxycortisol, LC/MS/MS ...... 64 Dexamethasone ...... 66 DHEA (Dehydroepiandrosterone) ...... 67 DHEA Sulfate...... 68 Dihydrotestosterone (DHT) ...... 69 Dihydrotestosterone, Free ...... 70 17-Hydroxycorticosteroids with Creatinine, 24-Hour Urine ...... 95 18-Hydroxycorticosterone...... 96 6β-Hydroxycortisol, 24-Hour Urine...... 97 18-Hydroxycortisol, Free, 24-Hour Urine ...... 98 21-Hydroxylase Antibody†...... 99
3 Endocrine Test Categories and Associated Tests
Page
17-Hydroxypregnenolone...... 100 17-Hydroxyprogesterone, LC/MS/MS ...... 101 17-Hydroxyprogesterone, Neonatal/Infant...... 101 17-Ketosteroids with Creatinine, 24-Hour Urine ...... 119 17-Ketosteroids, Fractionated, Urine ...... 120 Plasma Renin Activity (PRA)* ...... 137 Pregnanetriol, Urine...... 138 Pregnenolone ...... 139 Progesterone, LC/MS/MS ...... 140 Sex Hormone Binding Globulin ...... 149 Testosterone, Free and Total...... 168 Testosterone, Free, Bioavailable and Total, LC/MS/MS ...... 166 Testosterone, Total (Women, Children, Hypogonadal Males)...... 170 Testosterone, Total* ...... 169 Testosterone, Urine ...... 171 Tetrahydroaldosterone, 24-Hour Urine ...... 172 Cardiovascular/Lipids Angiotensin Converting Enzyme (ACE)...... ‡ Angiotensin Converting Enzyme (ACE) Polymorphism (Insertion/Deletion)* ..‡ Angiotnsin II Type 1 Receptor (AGTR1) Gene 1166A→C Polymorphism* ...... ‡ Apolipoprotein A1 ...... ‡ Apolipoprotein B...... ‡ B-Type Natriuretic Peptide (BNP) ...... ‡ Cardio CRP™ (high-sensitivity C-reactive protein)...... ‡ Cholesterol, HDL ...... ‡ Cholesterol, HDL Subclasses ...... ‡ Cholesterol, LDL Subparticles ...... ‡ Cholesterol, LDL, Direct...... ‡ Cholesterol, Total...... ‡ Homocysteine (Cardiovascular), Serum...... ‡ Homocysteine, Total, Urine ...... ‡ Lipid Panel Includes: cholesterol, HDL, cholesterol/HDL ratio, LDL (calculated), and triglycerides...... ‡
4 Endocrine Endocrine Tests Endocrine Test Categories Category by and Associated Tests
Page
Lipoprotein (a)...... ‡ Lipoprotein Fractionation, Ion Mobility with Reflex to Direct LDL ...... ‡ Lipoprotein Fractionation, Ultracentrifugation...... ‡ Lp-PLA2 (PLAC™′)...... ‡ Phospholipids†...... ‡ proBNP, N-terminal ...... ‡ Triglycerides ...... ‡ Catecholamines Catecholamines, Fractionated, 24-Hour Urine ...... 43 Catecholamines, Fractionated, Plasma ...... 44 Catecholamines, Fractionated, Random Urine...... 45 Chromogranin A ...... 47 Homovanillic Acid (HVA), Urine ...... 94 Metanephrines, Fractionated, LC/MS/MS, Plasma...... 127 Metanephrines, Fractionated, LC/MS/MS, Urine ...... 128 Pheochromocytoma Gene Mutations (SDHB, SDHD, VHL)* ...... 136 VMA (Vanillylmandelic Acid), Urine...... 186 Congenital Adrenal Hyperplasia (CAH) CAH (21-Hydroxylase Deficiency) Common Mutations*...... 29 CAH (21-Hydroxylase Deficiency) Rare Mutations*...... 30 CAH Panel 1 (21-hydroxylase and 11-hydroxylase deficiencies)...... 31 CAH Panel 3 (aldosterone synthase deficiency) ...... 32 CAH Panel 4 (17α-hydroxylase deficiency in females)...... 33 CAH Panel 6 (StAR deficiency)...... 34 CAH Panel 6B (comprehensive screen) ...... 35 CAH Panel 7 (21-OH deficiency therapeutic monitoring) ...... 36 CAH Panel 8 (17-hydroxylase deficiency in males)...... 33 CAH Panel 9 (3β-hydroxysteroid dehydrogenase deficiency)...... 37 CAH Panel 11 (neonatal random urine)...... 38 Endocrine Autoimmunity Adrenal Antibody Screen with Reflex to Titer ...... 18 Gastric Parietal Cell Antibody...... 80 Glutamic Acid Decarboxylase-65 Autoantibodies (GAD-65)* ...... 84
5 Endocrine Test Categories and Associated Tests
Page
Growth Hormone Antibody*...... 87 21-Hydroxylase Antibody†...... 99 IA-2 Antibody*...... 106 Insulin Antibody*...... 114 Islet Cell Antibody Screen with Reflex to Titer* ...... 118 PTH Antibody...... 143 T3 (Triiodothyronine) Antibody ...... 151 T4 (Thyroxine) Antibody ...... 157 TBII (Thyrotropin-binding Inhibitory Immunoglobulin) ...... 165 Thyroglobulin Antibody ...... 173 Thyroid Peroxidase Antibody (Anti-TPO) ...... 177 TSH Antibody...... 179 TSI (Thyroid Stimulating Immunoglobulin)* ...... 182 Fluid, Electrolyte, and Renal Aldosterone, 24-Hour Urine...... 19 Aldosterone, LC/MS/MS, Serum ...... 20 Aldosterone/Plasma Renin Activity Ratio* ...... 21 Angiotensin II...... 26 Angiotensin Converting Enzyme (ACE)...... ‡ Arginine Vasopressin (AVP, ADH), Plasma*...... 27 Arginine Vasopressin (AVP, ADH) and Osmolality, Urine ...... 28 Central Diabetes Insipidus (CDI) Mutations*...... 46 Corticosterone, LC/MS/MS...... 50 Cortisol Binding Globulin (Transcortin) ...... 52 Cortisol, Free, 24-Hour Urine ...... 54 Cortisol, Free, Saliva...... 55 Cortisol, Free, Serum ...... 56 Cortisol, Free and Cortisone, 24-Hour Urine...... 53 Cortisol, Total, LC/MS/MS...... 57 Cortisone, 24-Hour Urine...... 58 Cortisone, Serum*...... 59 Cystatin C ...... 62 Deoxycorticosterone (DOC) ...... 63 18-Hydroxycorticosterone...... 96
6 Endocrine Endocrine Tests Endocrine Test Categories Category by and Associated Tests
Page
Nephrogenic Diabetes Insipidus (Autosomal) Mutations* ...... 130 Nephrogenic Diabetes Insipidus (X-linked) Mutations*...... 131 Osmolality, Random Urine...... 132 Osmolality, Serum ...... 133 Plasma Renin Activity (PRA)* ...... 137 Genetic (Biochemical and Cytogenetic) Acylcarnitine, Plasma ...... ‡ Amino Acid Analysis for MSUD, LC/MS, Plasma ...... ‡ Amino Acid Analysis for Nutritional Status, LC/MS, Plasma ...... ‡ Amino Acid Analysis, LC/MS, CSF...... ‡ Amino Acid Analysis, LC/MS, Plasma...... ‡ Amino Acid Analysis, LC/MS, Urine ...... ‡ Amino Acid Analysis, Limited, LC/MS, Plasma ...... ‡ Carnitine, LC/MS/MS...... ‡ Chromosome Analysis, Blood...... ‡ Chromosome Analysis, High Resolution ...... ‡ Chromosome Analysis, Tissue ...... ‡ Cystine, Qualitative, Urine...... ‡ Cystine, Quantitative, 24-Hour Urine ...... ‡ Cystine, Quantitative, Random Urine ...... ‡ FISH, Angelman* ...... ‡ FISH, Chromosome-Specific Probe* Choose one of the following: Chromosome-Specific [1-22, X and Y] Centromere or Chromosome-Specific [1-22, X and Y] Painting...... ‡ FISH, DiGeorge, Velocardiofacial (VCFS)*...... ‡ FISH, Kallmann* ...... ‡ FISH, Microdeletion Syndromes Panel*...... ‡ FISH, Neonatal Screen...... ‡ FISH, Prader Willi* ...... ‡ FISH, SRY/X Centromere* ...... ‡ FISH, Subtelomere Screen*...... ‡ FISH, Williams* ...... ‡ FISH, X-Linked Ichthyosis Steroid Sulfatase Deficiency*...... ‡ Glycogen Storage Disease Type Ia Mutation Analysis (Ashkenazi Jewish)* ...... ‡
7 Endocrine Test Categories and Associated Tests
Page
Hydroxyproline, LC/MS, Plasma ...... ‡ Methylmalonic Acid ...... ‡ Organic Acids, Qualitative, Urine ...... ‡ Organic Acids, Quantitative, Full Panel, Urine...... ‡ Phenylalanine ...... ‡ Phenylalanine and Tyrosine ...... ‡ Prader-Willi/Angelman Syndrome* ...... ‡ Tryptophan, LC/MS ...... ‡ Tyrosine ...... ‡ Y Chromosome Microdeletion, DNA Analysis† ...... ‡ Gonadal Function Alpha Subunit*...... 23 3α-Androstanediol Glucuronide (3α-diol G) ...... 24 Androstenedione, LC/MS/MS ...... 25 DHEA (Dehydroepiandrosterone) ...... 67 DHEA Sulfate ...... 68 Dihydrotestosterone (DHT)...... 69 Dihydrotestosterone, Free ...... 70 Estradiol, Free, LC/MS/MS ...... 71 Estradiol, Ultra Sensitive, LC/MS/MS...... 72 Estrogen, Total, Serum ...... 73 Estrogens, Fractionated, LC/MS/MS ...... 74 Estrone, LC/MS/MS...... 75 Estrone Sulfate ...... 76 FSH (Follicle Stimulating Hormone) ...... 78 FSH (Follicle Stimulating Hormone), Pediatrics...... 79 hCG, Total with HAMA Treatment...... 90 hCG, Total, Quantitative...... 91 17-Hydroxypregnenolone...... 100 17-Hydroxyprogesterone, LC/MS/MS ...... 101 17-Hydroxyprogesterone, Neonatal/Infant...... 101 Inhibin A...... 112 Inhibin B† ...... 113 Invasive Trophoblast Antigen (ITA)(Pregnancy) ...... 117
8 Endocrine Endocrine Tests Endocrine Test Categories Category by and Associated Tests
Page
17-Ketosteroids with Creatinine, 24-Hour Urine...... 119 17-Ketosteroids, Fractionated, Urine ...... 120 LH (Luteinizing Hormone) ...... 123 LH (Luteinizing Hormone), Pediatrics ...... 124 Macroprolactin ...... 125 Pregnenolone ...... 139 Progesterone, LC/MS/MS...... 140 Prolactin...... 142 Sex Hormone Binding Globulin ...... 149 Testosterone, Free and Total...... 168 Testosterone, Free, Bioavailable and Total, LC/MS/MS ...... 166 Testosterone, Total (Women, Children, Hypogonadal Males)...... 170 Testosterone, Total*...... 169 Testosterone, Urine...... 171 Growth and Growth Hormone Growth Hormone (GH)...... 86 Growth Hormone Antibody*...... 87 Growth Hormone Binding Protein ...... 88 Growth Hormone Releasing Hormone* ...... 89 IGF Binding Protein-1 (IGFBP-1)*...... 107 IGF Binding Protein-2 (IGFBP-2)*...... 108 IGF Binding Protein-3 (IGFBP-3)...... 109 IGF-I ...... 110 IGF-II (Insulin-Like Growth Factor II)...... 111 Hypothalamic and Pituitary Function ACTH, Plasma...... 17 Alpha Subunit*...... 23 Arginine Vasopressin (AVP, ADH), Plasma*...... 27 Arginine Vasopressin (AVP, ADH) and Osmolality, Urine ...... 28 Central Diabetes Insipidus (CDI) Mutations*...... 46 Corticotropin Releasing Hormone (CRH)† ...... 51 FSH (Follicle Stimulating Hormone) ...... 78 FSH (Follicle Stimulating Hormone), Pediatrics ...... 79 Growth Hormone (GH)...... 86
9 Endocrine Test Categories and Associated Tests
Page
Growth Hormone Antibody*...... 87 Growth Hormone Binding Protein...... 88 Growth Hormone Releasing Hormone (GHRH)* ...... 89 LH (Luteinizing Hormone) ...... 123 LH (Luteinizing Hormone), Pediatrics...... 124 Macroprolactin ...... 125 Prolactin...... 142 Somatostatin* ...... 150 Thyrotropin-releasing Hormone (TRH)* ...... 178 TSH Antibody...... 179 TSH with HAMA Treatment...... 181 TSH, Ultrasensitive...... 180 Metabolic (Including Diabetes Mellitus) and Gastrointestinal Disorders C-peptide, Serum or Urine ...... 60 Fructosamine ...... 77 Gastrin...... 81 Glucagon*...... 82 Glucose...... 83 Glutamic Acid Decarboxylase-65 Autoantibodies (GAD-65)*...... 84 Glycated Albumin...... 85 Glycogen Storage Disease Type Ia Mutation Analysis (Ashkenazi Jewish) ...... ‡
Hemoglobin A1c ...... 92 5-HIAA (5-Hydroxyindoleacetic Acid), Urine...... 93 IA-2 Antibody*...... 106 IGF Binding Protein-1 (IGFBP-1)*...... 107 IGF Binding Protein-2 (IGFBP-2)*...... 108 IGF Binding Protein-3 (IGFBP-3) ...... 109 IGF-I ...... 110 IGF-II (Insulin Like Growth Factor II) ...... 111 Insulin Antibody*...... 114 Insulin, Free (Bioactive)* ...... 115 Insulin, Total (Free and Antibody Bound)* ...... 116 Islet Cell Antibody Screen with Reflex to Titer* ...... 118
10 Endocrine Endocrine Tests Endocrine Test Categories Category by and Associated Tests
Page
Leptin† ...... 122 Microalbumin, Intact with Creatinine, HPLC, Urine ...... 129 Osmolality, Random Urine...... 132 Osmolality, Serum ...... 133 Pancreatic Polypeptide*...... 135 Proinsulin† ...... 141 Somatostatin* ...... 150 Vasoactive Intestinal Polypeptide (VIP)*...... 183 Multiple Endocrine Neoplasia ACTH, Plasma...... 17 Calcitonin...... 39 Calcium, 24-Hour Urine ...... 42 Calcium, Pediatric Urine ...... 42 Calcium, Ionized ...... 40 Calcium, Total, Serum ...... 41 Catecholamines, Fractionated, 24-Hour Urine ...... 43 Catecholamines, Fractionated, Plasma ...... 44 Catecholamines, Fractionated, Random Urine...... 45 Chromogranin A ...... 47 C-Peptide, Serum or Urine ...... 60 Gastrin...... 81 Glucagon*...... 82 Growth Hormone (GH)...... 86 5-HIAA (5-Hydroxyindoleacetic Acid), Urine ...... 93 Homovanillic Acid (HVA), Urine ...... 94 Insulin, Free (Bioactive)* ...... 115 Macroprolactin ...... 125 MEN 2 and FMTC Mutations, Exons 10, 11, 13-16* ...... 126 Metanephrines, Fractionated, LC/MS/MS, Plasma...... 127 Metanephrines, Fractionated, LC/MS/MS, Urine ...... 128 Pancreatic Polypeptide*...... 135 Proinsulin† ...... 141 Prolactin...... 142 PTH, Intact and Calcium ...... 144
11 Endocrine Test Categories and Associated Tests
Page
PTH, Intact and Ionized Calcium ...... 144 PTH-related Protein (PTH-RP)† ...... 145 Serotonin, Blood ...... 148 Serotonin, Serum ...... 148 Vasoactive Intestinal Polypeptide (VIP)*...... 183 VMA (Vanillylmandelic Acid), Urine...... 186 Parathyroid and Mineral Metabolism Alkaline Phosphatase Isoenzymes (ALP Isoenzymes)...... ‡ Alkaline Phosphatase, Bone Specific ...... 22 Calcitonin ...... 39 Collagen Cross-Linked N-Telopeptide (NTx), Urine...... 48 Collagen Type I C-Telopeptide (CTx)...... 49 Cyclic Adenosine Monophosphate (Cyclic AMP) ...... 61 Cyclic Adenosine Monophosphate (Cyclic AMP), Nephrogenous ...... 61 Deoxypyridinoline (DPD, DPYD), Free ...... 65 Hydroxyproline, Free, Urine ...... 103, 104 Hydroxyproline, Free, Plasma ...... 102 Hydroxyproline, Total, Urine...... 103, 104 Hydroxyproline, Total & Free, 24-Hour Urine ...... 103 Osteocalcin (Bone Gla Protein, BGP)†...... 134 PTH Antibody...... 143 PTH, Intact and Calcium...... 144 PTH, Intact and Ionized Calcium ...... 144 PTH-related Protein (PTH-RP)† ...... 145 Pyridinium Collagen Cross-Links (PYD and DPYD), Urine ...... 146 Tartrate Resistant Acid Phosphatase...... 163 Vitamin D, 1,25-Dihydroxy ...... 184 Vitamin D, 25-Hydroxy, LC/MS/MS ...... 185 Thyroid Function Calcitonin ...... 39 Resistance to Thyroid Hormone (RTH) Mutation Analysis*...... 147 Somatostatin* ...... 150 T3 (Triiodothyronine) Antibody ...... 151 T3 Uptake ...... 152
12 Endocrine Endocrine Tests Endocrine Test Categories Category by and Associated Tests
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T3, Free, Non-dialysis ...... 153 T3, Free, Tracer Dialysis...... 154 T3, Reverse† ...... 155 T3, Total (Triiodothyronine) ...... 156 T4 (Thyroxine) Antibody ...... 157 T4 Binding Proteins ...... 158 T4, Free, Direct Dialysis ...... 160 T4, Free, Non-dialysis ...... 161 T4, Total (Thyroxine)...... 162 TBG (Thyroxine Binding Globulin) ...... 164 TBII (Thyrotropin-binding Inhibitory Immunoglobulin)...... 165 Thyroglobulin Antibody...... 173 Thyroglobulin Panel Includes thyroglobulin and thyroglobulin antibody...... 174 Thyroglobulin Panel with HAMA Treatment Includes thyroglobulin and thyroglobulin antibody...... 175 Thyroid Peroxidase and Thyroglobulin Antibodies...... 176 Thyroid Peroxidase Antibody (Anti-TPO)...... 177 Thyrotropin-releasing Hormone...... 178 TSH Antibody ...... 179 TSH with HAMA Treatment...... 181 TSH, Ultrasensitive...... 180 TSI (Thyroid Stimulating Immunoglobulin)* ...... 182 Additional Diagnostic Profiles Hypertension, Endocrine Includes 31 steroid analytes for detection of treatable causes of low-renin hypertension in children and adults...... 105 Metabolic Syndrome Panel Includes glucose, HDL cholesterol, triglycerides, blood pressure, and waist circumference...... ‡ Pheochromocytoma Evaluation Includes creatinine, total catecholamines, catecholamine/ creatinine ratio, total metanephrines, and metanephrines/ creatinine ratio...... ‡
13 Endocrine Test Categories and Associated Tests
Page Dynamic/Perturbation Tests Aldosterone, Response to ACTH Stimulation, LC/MS/MS...... ‡ Androstenedione Response to ACTH Stimulation, LC/MS/MS...... ‡ Catecholamines, Total, Suppression by Clonidine, Plasma...... ‡ Cortisol Stimulation by Adrenocorticotropic Hormone (ACTH) ...... ‡ C-Peptide Response to Glucose...... ‡ C-Peptide Stimulation by Glucagon ...... ‡ 11-Deoxycortisol Response to ACTH Stimulation ...... ‡ DHEA (Dehydroepiandrosterone) Response ...... ‡ Growth Hormone Response (Supression or Stimulation) ...... ‡ 17-Hydroxypregnenolone Response ...... ‡ 17-Hydroxyprogesterone Response to ACTH Stimulation...... ‡ Insulin Response to Glucose...... ‡ Proinsulin Response to Glucose† ...... ‡
*This test was developed and its performance characteristics determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and Drug Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test. †This test is performed using a kit that has not been approved or cleared by the FDA. The analytical performance characteristics of this test have been determined by Quest Diagnostics Nichols Institute. This test should not be used for diagnosis without confirmation by other medically established means. ‡Refer to the Quest Diagnostics Nichols Institute Directory of Services for ordering information. Reflex tests are performed at an additional charge and are associated with an additional CPT code.
14 Alphabetical Test Section
Test components are disclosed in the individual test write-ups in this section.
Alphabetical Test Section 211X Immunochemiluminometric assay Immunochemiluminometric (ICMA) intact ACTH Detects 5 pg/mL sensitivity: Analytical Clinical Background hormone Adrenocorticotropic polypeptide acid 39-amino is a (ACTH) by the anterior mainly secreted pituitaryThe hypothalamus gland. by secretion pituitarycontrols ACTH releasing of corticotropin means acid a 41-amino (CRH), hormone pain, to in response released peptide is a There also and stress. anxiety, Cortisol exerts variation. diurnal the control on feedback negative at the pituitary of ACTH secretion levels. and hypothalamic gland are measurements ACTH Plasma the of disorders in diagnosing useful system. hypothalamic-pituitary-adrenal Method • • • Specimen Requirements plasma frozen EDTA 1.5 mL minimum 0.3 mL 10 AM. between 7 and samples Collect 17 1997; pg/mL Euro J Endocrin. Euro Adrenal carcinoma Adrenal Adrenal adenoma Adrenal Stress Addison’s disease Addison’s Pituitary Cushing’s disease Pituitary Cushing’s ACTH-secreting tumor ACTH-secreting Secondary insuffiency adrenal • • • • • • • PrepubertalPubertal 7-28 2-49 Differentiate Cushing's syndrome syndrome Cushing's Differentiate are levels ACTH normal when from low Diagnose disorders of the of the disorders Diagnose hypothalamic-pituitary-adrenal system 137:635-647. MenWomenChildren* *Pediatric data from 5-27 7-50 Interpretive Information Interpretive Reference Range Reference Clinical Use ACTH, Plasma • • Adrenal Antibody 4645X
Clinical Use Clinical Background • Confirm autoimmune nature of Autoimmune Addison's disease is Addison's disease characterized by the presence of serum • Identify adrenal gland involvement autoantibodies to adrenal antigens. in polyglandular autoimmune Recent studies have identified the syndrome adrenal biosynthetic enzymes P450c21 and P450c17 as 2 of the synthetic Reference Range antigens. Immunofluorescence is Negative useful for adrenal autoantibody Titer: <10 detection. Addison's disease is seen as part of 2 types of polyglandular auto- Interpretive Information immune syndromes (PAS): type I • Polyglandular autoimmune includes candidiasis and hypo- disease, type I parathyroidism and type II includes autoimmune thyroiditis and type 1 • Polyglandular autoimmune disease, type II diabetes. Type II is more frequent. Measurement of other glandular • Isolated autoimmune Addison’s autoantibodies may provide useful disease diagnostic and management information.
Method • Immunofluorescence assay (IFA) • Titer provided if antibody is present (at additional charge)
Specimen Requirements 2 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable
18 Alphabetical Test Section 19552X Acid hydrolysis, extraction, hydrolysis, Acid (RIA) radioimmunoassay 1 μg/L sensitivity: Analytical also concentration Creatinine reported. Clinical Background cortex adrenal an Aldosterone, of major regulator is the hormone, excretion. and potassium sodium by the largely is modulated Production response system in renin-angiotensin and balance in sodium to changes aldosterone Urine blood flow. renal in increased is commonly excretion of heart secondary renin) states (high In renal disease. or liver or failure due to primary hyperaldosteronism urine secretion, adrenal autonomous plasma and is increased aldosterone Aldosterone is suppressed. renin in Addison's is reduced excretion and in hyporeninemic disease by renal caused hypoaldosteronism urinary The major disorders. is aldosterone- metabolite aldosterone of Measurement 18 glucuronide. for corrected in urine, aldosterone of an estimate provides creatinine, period. a 24-hour rate over secretion Method • • • Specimen Requirements a 24-h aliquot of refrigerated 5 mL 0.8 mL minimum urine; acid. 10 g of boric urine in Collect collection. during Refrigerate and on vial 24-h volume Record form. request 19 5 < 5.7 10.2 15.6 g/24-h μg/24-h suppression suppression ®´ Very high sodium diet high sodium Very Addison's disease Addison's Hyporeninemic hypoaldostero- Hyporeninemic Adrenal hypoplasia Adrenal Congenital deficiency of deficiency Congenital Congenital adrenal hyperplasia adrenal Congenital Bartter syndrome Bartter Pregnancy Very low sodium diet low sodium Very Secondary hyperaldosteronism Primary hyperaldosteronism nism aldosterone synthetase aldosterone • • • • • • • • • • • 12-16 y12-16 Adults < 2.3-21.0 Diagnose primaryDiagnose hyperaldostero- or adenomas due to adrenal nism hyperplasia adrenal bilateral Assess adrenal aldosterone aldosterone adrenal Assess production 2-7 y < 8-11 y8-11 < Post-Florinef Results affected by recent sodium by recent affected Results intake or IV saline infusion or IV Aldosterone diet sodium Random 3-8 y y9-12 y13-17 Adults 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 Creatinine Interpretive Information Interpretive Reference Range Reference Clinical Use Aldosterone, 24-Hour Urine 24-Hour Aldosterone, • • Aldosterone, LC/MS/MS, Serum 17181X
Clinical Use Clinical Background • Assess adrenal aldosterone Aldosterone is the major sodium- production retaining hormone produced by the • Diagnose primary hyperaldostero- adrenal gland. Secretion is normally nism regulated by renin-angiotensin system • Differential diagnosis of fluid and monitoring of extracellular fluid vol- electrolyte disorders ume. A low- salt diet will decrease vol- Reference Range ume, increase renin-angiotensin, and increase aldosterone. Potassium is a Adults ng/dL secondary regulator. Diseases of the Upright (8-10 AM) <28 heart, liver, and kidneys commonly Upright (4-6 PM) <21 increase renin, leading to secondary Supine (8-10 AM) 3-16 aldosteronism. Primary hyper- Children aldosteronism due to autonomous Premature infants <144 secretion from an adrenal adenoma or (31-35 wks)* secondary to bilateral hyperplasia Term infants* <217 produces sodium retention, hyper- 1-12 mo 2-70 tension, and renal potassium wasting. An elevated serum aldosterone with 1-4 y 2-37 low renin (PRA or direct renin) in the ≤ 5-9 y 9 face of hypokalemia is diagnostic of 10-13 y ≤21 primary hyperaldosteronism. 14-17 y ≤35 Aldosterone deficiency occurs in Tanner II-III Addison's disease (associated with a Males 1-13 high renin) and in patients with Females 2-20 isolated hypoaldosteronism, which Tanner IV-V Males 3-14 may be hyporeninemic (eg, diabetes) Females 4-32 or hyperreninemic (eg, AIDS). In *Data from J Clin Endocrinol Metab. 1992;75: children, adrenal biosynthetic enzyme 1491-1496 and J Clin Endocrinol Metab. 1989; deficiencies may produce aldosterone 69:1133-1136. deficiency.
Interpretive Information Method • Primary hyperaldosteronism • Liquid chromatography tandem • Secondary hyperaldosteronism mass spectrometry (LC/MS/MS) • Very low sodium diet • Analytical sensitivity: 1.0 ng/dL • Pregnancy • Bartter syndrome Specimen Requirements 1.0 mL refrigerated serum (no additive • Congenital adrenal hyperplasia red top tube); 0.25 mL minimum • Aldosterone synthetase SST red top tube unacceptable. deficiency Draw upright samples ½ hour after • Very high sodium diet patient sits up. • Addison's disease • Hyporeninemic hypoaldostero- nism Levels are affected by recent sodium intake and posture.
20 Alphabetical Test Section 11183Z Liquid chromatography tandem tandem chromatography Liquid spectrometry mass (LC/MS/MS) 1.0 ng/dL sensitivity: Analytical I generation, Angiotensin (RIA) radioimmunoassay 0.37 ng/mL/h sensitivity: Analytical Clinical Background to due Primary hyperaldosteronism an adrenal from secretion autonomous or secondary adenoma to bilateral sodium produces hyperplasia renal and hypertension, retention, serum An elevated wasting. potassium or (PRA low renin with aldosterone hypo- the face of renin) in direct of primary is diagnostic kalemia the of Diseases hyperaldosteronism. commonly kidneys and liver, heart, to secondary renin, leading increase normal aldosterone/ A aldosteronism. presence of ratio in the PRA consistent with is hyperkalemia hypoaldosteronism. hyporeninemic Method Aldosterone • • PRA • • Specimen Requirements plasma frozen EDTA 3.0 mL minimum 1.0 mL refrigerate. Do not ambulatory for 30 preparation: Patient diet; sodium prior; moderate minutes prior. for 3 weeks medications avoid 21 for for Addison’s disease Addison’s Aldosterone synthetase synthetase Aldosterone Congenital adrenal hyperplasia adrenal Congenital Bartter syndrome Bartter Pregnancy Hyporeninemic hypoaldostero- Hyporeninemic Secondary hyperaldosteronism Primary hyperaldosteronism deficiency nism • • • • • • • Ratio • Plasma Renin Activity (PRA) Plasma Renin Aldosterone, LC/MS/MS, Serum LC/MS/MS, Aldosterone, Diagnose primaryDiagnose hyperaldostero- nism aldosterone reference ranges. aldosterone ranges. reference PRA Levels are affected by recent sodium by recent are affected Levels and posture. intake Normal or decreased ratio or decreased Normal See See Information Interpretive Reference Range Reference Ratio Aldosterone/PRA / ng/mL/h ng/dL 1.5-18.2 See Clinical Use Aldosterone/PRA Ratio Aldosterone/PRA • Alkaline Phosphatase, 29498X Bone Specific
Clinical Use Clinical Background • Therapeutic monitoring of Bone-specific alkaline phosphatase postmenopausal osteoporosis and (BSAP) is one of a group of alkaline Paget's disease phosphatase enzymes produced in various tissues including bone, liver, Reference Range placenta, intestine, spleen, and kidney. Males Females The bone-specific isoenzyme is Adults μg/L μg/L synthesized and released by osteo- blastic cells during the process of bone 18-29 y 8.4-29.3 4.7-17.8 formation. The processes of bone 30-39 y 7.7-21.3 5.3-19.5 formation and bone dissolution are 40-49 y 7.0-18.3 5.0-18.8 linked. Thus, BSAP levels reflect 50-68 y 7.6-14.9 osteoblast cellular activity due to either 50-76 y 5.6-29 increased bone formation or osteo- Children blastic stimulation associated with 2-24 mo 25.4-124.0 25.4-124.0 excessive bone destruction. 6-9 y 41.0-134.6 41.0-134.6 Method 10-13 y 43.8-177.4 24.2-154.2 14-17 y 13.7-128.0 10.5-75.2 • Immunochemiluminometric assay (ICMA) Pediatric data from Int J Biol Markers. 1996;11:159-164. • Analytical sensitivity: 0.1 μg/L
Interpretive Information Specimen Requirements • Hyperthyroidism 1 mL frozen serum • Osteomalacia 0.3 mL minimum • Osteoporosis No additive red top preferred • Other metabolic bone diseases SST red top acceptable • Paget's disease (osteitis deformans) • Primary hyperparathyroidism
• Response to therapy
22 Alphabetical Test Section 8658X 1. Radioimmunoassay (RIA) Radioimmunoassay at 0.1 ng/mL sensitivity: Analytical B/Bo 90% cross-reactivity specificity: Analytical with <0.01% hCG, with intact is 1.4% LH, 4.5% with subunit hCG, beta TSH and 2.0% with with FSH, 3.4% Clinical Background pituitaryThe hormones– glycoprotein (TSH), hormone stimulating thyroid and follicle hormone (LH), luteinizing (FSH)–are hormone stimulating subunits alpha of identical comprised confer subunits that unique beta and pituitary Some specificity. biological a clinical with unassociated adenomas secretion over of hormonal syndrome which subunit, the alpha produce servesmarker. tumor useful as a can subunit alpha of the Measurement TSH- in differentiating be useful pituitary in which secreting adenomas, serum TSH of alpha-to-intact the ratio >1, from thyroid is concentrations in syndromes, resistance hormone is < the ratio which Gonadotropin-secreting pituitary Gonadotropin-secreting alpha excessive producing adenomas Non- reported. have been subunit pituitary eg, choriocarcinoma tumors, the secreting carcinoid tumors, and also been subunit have alpha described. Method • • • Specimen Requirements serum refrigerated 2 mL minimum 0.3 mL top preferred red No additive red top acceptable SST 23 0.6 1.5 3.7 < 0.9-3.3 ng/mL 1.8-360* Hypopituitarism Pituitary (selected) adenomas Hydatidiform mole Hydatidiform Choriocarcinoma Pregnancy • • • • • Diagnose and manage pituitary, pituitary, and manage Diagnose and pancreatic tumors placental, Premenopausal Postmenopausal Hypothyroid < Women or hCG- Pregnant tumors producing Men < *Varies with concentration of hCG of concentration with *Varies Information Interpretive Reference Range Reference Clinical Use Alpha Subunit Alpha • 3α-Androstanediol Glucuronide 5276X (3α-diol G)
Clinical Use Clinical Background • Marker for disorders of peripheral 3α-Androstanediol glucuronide (3α- androgen formation and action, such diol G) is a metabolite of dihydro- as in hirsutism and acne testosterone (DHT) produced in androgen responsive tissues, such as Reference Range hair follicles. 3α-diol G is a marker of a ng/dL peripheral androgen formation and Men 260-1500 action. Women 60-300 The most important application of this assay is in hirsute states. Patients with Children hirsutism of either the idiopathic type Prepubertal 10-60 or secondary to polycystic ovarian Tanner II-III diseases (PCO) may have markedly Males 19-164 elevated levels. 3α-diol G has also been Females 33-244 shown to be a marker of therapeutic Tanner stage data from J Clin Endocrinol response in congenital adrenal Metab. 1991;72:46-50. hyperplasia (CAH).
Interpretive Information Method • Idiopathic hirsutism (females) • Extraction, enzyme digestion, • PCO-hirsutism (females) chromatography, radioimmunoassay (RIA) • Acne (females) • Analytical sensitivity: 5 ng/dL • 5α-reductase deficiency (males) Specimen Requirements 2 mL refrigerated serum 0.3 mL minimum No additive red top preferred SST red top acceptable
24 Alphabetical Test Section 17182X -hydroxylase deficiencies. In In deficiencies. -hydroxylase β Liquid chromatography tandem tandem chromatography Liquid spectrometrymass (LC/MS/MS) 5 ng/dL sensitivity: Analytical Clinical Background is an androgenic Androstenedione the by both produced hormone steroid In the gonads. cortex and adrenal is activity the compound's males, with compared unimportant, relatively androgen testicular the predominant females, andros- In testosterone. and the testosterone tenedione–and is it to which dihydrotestosterone to contribute metabolized–normally dependent of sexually the growth the In women, axillary hair. and pubic androstenedione of concentration with cycle, with the menstrual varies adrenals contributing and the ovaries phase. At follicular during the equally andros- ovarian increased mid-cycle, for 70% accounts secretion tenedione overall production. of its production androstenedione Excessive to and contribute cause hirsutism may in the can occur This virilization. polycystic ovary syndrome, idiopathic and adrenal ovarian hirsutism, neoplasms, and congenital adrenal or to 21-hydroxylase due hyperplasia 17 with an elevated patients hirsute serial concentration, androstenedione monitor useful to are measurements improvement since clinical therapy, the biochemical lags behind typically by months. response Method • • Specimen Requirements serum refrigerated 1 mL 0.25 mL minimum top tube red No additive red top unacceptable SST 25 6-78 5-51 20-75 6-115 50-220 40-190 50-220 35-250 60-285 30-235 12-221 22-225 ng/dL J ClinJ Endocrinol Metab. J Clin Endocrinol Metab. Addison's disease Addison's Idiopathic hirsutism Idiopathic Polycystic ovarian disease ovarian Polycystic Congenital adrenal hyperplasia adrenal Congenital Cushing’s disease Cushing’s Adrenal tumors Adrenal • • • • • • MalesFemales 57-150 7-68 MalesFemales 43-180 17-82 Diagnose hirsutism, polycystic polycystic hirsutism, Diagnose and virilization, disease, ovarian hyperplasia adrenal congenital 18-30 y 18-30 y 31-50 y 51-60 Follicular Midcycle Luteal Postmenopausal Tanner IV-V Tanner Tanner II-III Tanner 1-12 m 1-12 1-4 y 5-9 y y 10-13 y 14-17 Pediatric data from 1991;73:674-686 and 1989;69:1133-1136. Men Women Children Interpretive Information Interpretive Reference Range Reference Clinical Use Androstenedione, LC/MS/MS Androstenedione, • Angiotensin II 36718X
Clinical Use Clinical Background • Differential diagnosis of renal Angiotensin II is the primary regulator hypertension of renal aldosterone secretion and a potent vasoconstrictor. It is generated Reference Range through the renin angiotensin system Adults 10-50 ng/L (RAS): circulating angiotensinogen is cleaved by renin to form angiotensin I, Interpretive Information which is then converted to angiotensin • High renin hypertension II via angiotensin converting enzyme (ACE). The rate-limiting step in the • Renin-secreting juxta- RAS is renin secretion by the renal glomerular renal tumor juxtaglomerular cells, modulated by • Volume depletion renal blood flow. Low renal blood flow • Congestive heart failure and low perfusion pressure increases • Cirrhosis renin secretion, stimulating angio- tensin II and aldosterone production • Angiotensin II receptor blocking with a resulting increase in blood drug therapy pressure and renal sodium retention. These changes then produce • Anephric patients inhibition of renin secretion and • Primary aldosteronism complete the feedback control loop. • Cushing's syndrome Method • ACE inhibitor therapy • Radioimmunoassay (RIA)
Specimen Requirements 1 mL frozen EDTA plasma 0.3 mL minimum
26 Alphabetical Test Section 252X Extraction, radioimmunoassay (RIA) radioimmunoassay Extraction, 1.0 pg/mL sensitivity: Analytical Clinical Background or (AVP), vasopressin Arginine is a (ADH), hormone antidiuretic hypo- the by produced nonapeptide the from and released thalamus pituitary to posterior in response and hyperosmolarity fluid extracellular concen- promotes AVP hypovolemia. water increasing by urine the of tration tubules. the kidney in reabsorption causes action AVP Inadequate (DI), a syndrome insipidus diabetes nonglycosuric by characterized and dehydration. polydipsia, polyuria, AVP to insufficient DI refers Central hypo- of the due to diseases release pituitary pituitarythalamus, stalk, and result of DI is the Nephrogenic gland. to AVP renal responsiveness impaired to renal or due may be congenital and hypercalcemia, hypokalemia, disease, (eg, multiple disorders systemic or drugs and amyloidosis), myeloma and lithium or demeclocycline (eg, ethanol). presence on the is based DI diagnosis in- serum with of hyperosmolar urine. Central and dilute appropriately can be differentiated DI nephrogenic level and AVP plasma the by measuring simul- light of the it in interpreting osmolality. plasma taneous inappropriate syndrome of The hormone of antidiuretic secretion by hyponatremia is manifest (SIADH) and inappropriately concentrated by is confirmed The diagnosis urine. levels in- AVP or urine plasma serum osmolality. for appropriate Method • • Specimen Requirements plasma frozen EDTA 4 mL minimum 1.1 mL tube; blood in prechilled Draw in refrigerated immediately centrifuge centrifuge. 27 Central DI Central Phenothiazine, carbamazepine Phenothiazine, Nephrogenic DI Nephrogenic Ectopic ADH syndrome Ectopic SIADH • •• • • • Diagnose syndrome of inappropriate inappropriate of syndrome Diagnose (SIADH) secretion ADH Differential diagnosis of central DI diagnosis vs Differential DI nephrogenic Diagnose central diabetes insipidus diabetes central Diagnose (DI) 1.0-13.3 pg/mL 1.0-13.3 1 μU 2.5 pg = Note: Interpretive Information Interpretive Reference Range Reference Clinical Use Arginine Vasopressin, Plasma Vasopressin, Arginine • • • Arginine Vasopressin (AVP) 36572X and Osmolality, Random Urine
Clinical Use Clinical Background • Diagnose central diabetes insipidus Arginine vasopressin (AVP), also (DI) known as antidiuretic hormone • Differential diagnosis of central DI vs (ADH), is a 9-amino acid polypeptide nephrogenic DI synthesized by the hypothalamus and • Confirm syndrome of inappropriate stored and secreted by the posterior ADH secretion (SIADH) diagnosis pituitary. Secretion is regulated by osmoreceptors and volume receptors Reference Range and is increased by dehydration or increased blood osmolarity. Conversely, AVP 1-112 pg/mL secretion is decreased by increased Osmolality 50-1200 mOsm/kg blood volume or decreased blood osmolarity. AVP acts primarily on the Interpretive Information kidney, where it exerts an antidiuretic • Low AVP and urine osmolality = effect. central DI AVP plasma concentrations correlate • High AVP and low urine osmolality = with osmolarity. Significant amounts of nephrogenic DI AVP are filtered via the glomerulus and • Normal AVP despite low serum appear in urine. Concentrations are osmolality = SIADH; in SIADH, the higher in urine than in plasma. Urine kidney responds normally to AVP AVP provides an integrated view of recent blood levels. Urine AVP determinations are useful in the differential diagnosis of hyponatremic states.
Method AVP • Radioimmunoassay (RIA) • Analytical sensitivity: 1.0 pg/mL Osmolality • Freezing point depression
Specimen Requirements 3 mL refrigerated random urine 1.2 mL minimum Do not use a preservative.
28 Alphabetical Test Section
14755X CYP21A2 CYP21A2 Polymerase chain reaction (PCR) reaction (PCR) chain Polymerase mini-sequencing and DNA P30L; sought: mutations Common I172N; 2 "g"; G110del8nt; Intron & I236N, V237E 6 cluster of exon F306+1nt; Q318X; V281L; M239K; R356W; and P453S and of deletions Deduction between recombinations and its pseudogene Clinical Background the most deficiency, 21-Hydroxylase adrenal congenital cause of common (CAH),hyperplasia an autosomal is caused by mutations recessive disorder in the or rearrangements 6. The on chromosome gene by is characterized deficiency and increased cortisol decreased levels. Severe blood androgen activity in 21-hydroxylase reduction classic CAH,causes simple the and/or of cases), form (25% virilizing is further form, which the salt-wasting decreased by characterized Onset occurs levels. aldosterone it prenatally, if detected and, prenatally in reduce virilization treated to can be CAH, Non-classic females. affected on presents postnatally hand, the other signs of hyperandrogenism. with common for the more Testing both relevant can detect mutations in and 1 mutation in 81% mutations individuals. 18% of affected about prenatal testing for Preparation parents analysis of mutation requires offspring. an affected and/or Method • • • Specimen Requirements blood whole room temperature 5 mL minimum 3 mL a lavender-top blood in Collect solution (ACD or yellow-top (EDTA) B) tube. amniotic submit testing, For prenatal cells or cultured CVS sample, fluid, either source. from 29 21-hydroxylase deficiency 21-hydroxylase or affected) (carrier Prenatal diagnosis of 21-OHD diagnosis Prenatal Screen for 21-OHD carrier status in status carrier for 21-OHD Screen individuals at-risk Confirm diagnosis of 21-hydroxylase of 21-hydroxylase diagnosis Confirm (21-OHD) deficiency Interpretive Information Interpretive detected) (mutations Positive Reference Range Reference detected) (no mutations Negative Clinical Use CAH (21-Hydroxylase Deficiency) (21-Hydroxylase CAH Mutations Common • • • • CAH (21-Hydroxylase Deficiency) 16072X Rare Mutations
Clinical Use Clinical Background • Confirm diagnosis of 21-hydroxylase 21-Hydroxylase deficiency, the most deficiency (21-OHD) common cause of congenital adrenal • Determine 21-OHD carrier status in hyperplasia (CAH), is an autosomal at-risk individuals recessive disorder caused by mutations or rearrangements in the CYP21A2 • Prenatal diagnosis of 21-OHD gene on chromosome 6. The deficiency is characterized by decreased cortisol Reference Range and increased androgen blood levels. Negative (no mutations detected) Severe reduction in 21-hydroxylase activity causes classic CAH, the simple Interpretive Information virilizing form (25% of cases), and/or Positive (mutations detected) the salt-wasting form, which is further characterized by decreased aldosterone • 21-hydroxylase deficiency (carrier or affected) levels. Onset occurs prenatally and, if detected prenatally, it can be treated to reduce virilization in affected females. Non-classic CAH, on the other hand, presents postnatally with signs of hyper- androgenism. This test is appropriate for affected individuals in whom only 1 or no muta- tions were detected in the CAH (21-Hydroxylase Deficiency) Common Mutations test. It is also appropriate for carrier testing in family members of such individuals, once a rare mutation has been identified. About 1% of affected individuals have rare muta- tions on both chromosomes, and about 18% have a rare mutation on only 1 chromosome. Method • Polymerase chain reaction (PCR) and DNA sequencing • Entire CYP21A2 gene sequenced Specimen Requirements 5 mL room temperature whole blood 3 mL minimum Collect blood in a lavender-top (EDTA) or yellow-top (ACD solution B) tube. For prenatal testing, submit amniotic fluid, CVS sample, or cultured cells from either source. Call 1-866-GENEINFO (1-866-436- 3436) before collecting or submitting samples.
30 Alphabetical Test Section , 15269X CYP11B1 ed blood pressure - from 21-hydroxylase β ) accounts for 90% to 95% c21 HSD) accounts for 5%8% toof -Hydroxlase deficiency ( β β Liquid chromatography, tandem mass tandem chromatography, Liquid spectrometry (LC/MS/MS) • deficiency. A late-onset form A late-onset typically that deficiency. signs of androgen with excess presents is analogous to nonclassic 21-OHD. CAHdoesnot occur; cases. Salt-wasting of female fetuses virilization however, Elevat can be severe. manifests early in life in approximately approximately in in life early manifests with along and, patients of two-thirds and deoxycorticosterone elevated clinically levels, 11-deoxycortisol distinguishes 11 11 of CAHof cases. is Classic 21-OHD or diminished by markedly characterized Affected activity. absent 21-hydroxylase at individuals typically birth present a with either period neonatal the or in Female form. salt-wasting or virilizing have 21-OHD virilizing infants with varying of masculinization degrees clitoral to from enlargement ranging external of male complete development have normal male infants Male genitalia. Symptoms of hyponatremia, genitalia. and depletion, volume hyperkalemia, blood generally pressure decreased of life in 2 weeks the first appear within salt-wastingpatients with form. the by characterized 21-OHD is Nonclassic decreased 21-hydroxylase marginally present usually females Affected activity. childhoodpostin puberty or with (eg, of androgen excess evidence growth and/orincreased pubic hair In women, non- enlargement). clitoral classic21-OHD is frequently confused ovary polycystic with syndrome. Diagnosis stimulation. ACTH require may 11 Method Specimen Requirements serum refrigerated mL 0.6 minimum mL 0.3 top tube No additive red preferred. is specimen morning An early and sex on test request form. age Specify 21-Hydroxylase deficiency (21-OHD, (21-OHD, deficiency 21-Hydroxylase P450 Clinical Background 31 * * * * * ↑ ↑ OH: - β 11 21-OH: >6 21-OH: prematures) >100 (except Hydroxylase Deficiency) Deficiency) Hydroxylase - -hydroxylase deficiency β β Diagnose 21-hydroxylase or or 21-hydroxylase Diagnose 11 11-deoxycortisol ratio* 11-deoxycortisol cortisol ratio* cortisol *See Test Application and Interpretation, *See Test 6-8. Disorders of Adrenal Function, Tables 17-OH progesterone/ 17-OH *See individualassays for age-related reference ranges. 11-Deoxycortisol 17-Hydroxyprogesterone Testosterone 11-Deoxycortisol/ Androstenedione Cortisol Androstenedione Testosterone Interpretive Information Interpretive Reference Range Range Reference Clinical Use CAH Panel 1 (21-Hydroxylase vs 1 (21-Hydroxylase Panel CAH 11 • CAH Panel 3 (Aldosterone 15273X Synthase Deficiency)
Clinical Use Clinical Background • Diagnose aldosterone synthase Aldosterone synthase deficiency deficiency (CYP11B2, P450c11ase) is a rare form of CAH in which only aldosterone Reference Range synthesis is affected. Two forms have Androstenedione * been identified; type I is characterized 11-Deoxycortisol * by decreased, and type II by increased, 18-Hydroxycorticosterone * 18-hydroxycorticosterone. Infants 17-Hydroxyprogesterone * usually present in the neonatal period with failure to thrive, recurrent de- *See individual assays for age-related reference ranges. hydration secondary to salt-wasting, decreased blood pressure, and Interpretive Information acidosis. 18-OH corticosterone/ >40 Method aldosterone ratio* • Extraction, chromatography, *See Test Application and Interpretation, radioimmunoassay (RIA) Disorders of Adrenal Function, Tables 6-8. • Liquid chromatography, tandem mass spectrometry (LC/MS/MS)
Specimen Requirements 1.8 mL refrigerated serum 0.8 mL minimum No additive red top tube An early morning specimen is preferred. Specify age and sex on test request form.
32 Alphabetical Test Section , 15274X 15279X CYP17 nin, low aldoster- s, individuals are -hydroxylation and -hydroxylation α ) accounts for approximately for) accounts approximately gene encodes an enzyme that c17 -Hydroxylase deficiency -Hydroxylase ( α Liquid chromatography, tandem mass mass tandem Liquid chromatography, spectrometry (LC/MS/MS) mass tandem Liquid chromatography, spectrometry (LC/MS/MS) usually found to be hypertensive and (low re hypokalemic 17,20-lyase reactions. Isolated deficiency deficiency Isolated reactions. 17,20-lyase reported; been has activity of either a combined deficiency in which however, there is failure of catalysis of both is the commonmost reactions form. individualsAffected have decreased levels of cortisol, androgens, and presentestrogens and typically during primary with adolescence amenorrhea and/or lack of secondary sexual characteristics. Males (XY) may be at birthdetected with pseudo- hermaphroditism (ie, female external fallopian and of uterus genitalia,absence tubes, and intra-abdominal testes). At time of the diagnosi such individuals should be All one). tested for17-hydroxylase deficiency. Subjects with combined 21- and 17-hydroxylase/17,20-lyase deficiencies syndrome with or without Antley-Bixler (type been shown to have the 2) have mutation. P450 gene oxidoreductase Specimen Requirements CAH Panel 4 (15274X) refrigerated1.2 mL serum minimum 0.6 mL CAH Panel 8 (15279X) refrigerated0.8 mL serum minimum 0.4 mL red top tube No additive An early morning specimen is preferred. Specify age, sex, and time of day sample was collected on test requestform. Method 4 (15274X) CAH Panel • 8 (15279X) CAH Panel • 17 P450 Clinical Background 1% of all CAH 1% of all with an estimated cases, incidence of 1:50,000 newborns. The CYP17 catalyzes both 17 33 ↓ ↑ ↓ ↓ ↓ ↑ ↓ ↓ >6 >6 progesterone ratio* progesterone ratio* progesterone Diagnose 17-hydroxylase deficiency deficiency 17-hydroxylase Diagnose (15279X) in males Diagnose 17-hydroxylase deficiency deficiency 17-hydroxylase Diagnose (15274X) in females *See Test Application and Interpretation, *See Test 6-8 Disorders of Adrenal Function, Tables CAH Panel 4 (15274X) CAH Progesterone/17-OH *See individualassays for age-related reference ranges. Aldosterone Corticosterone Cortisol Estradiol Panel 8 (15279X) CAH Progesterone/17-OH Aldosterone Corticosterone Cortisol Testosterone CAH Panel 4 (15274X) CAH AldosteroneCorticosteroneCortisol17-HydroxyprogesteroneProgesteroneEstradiol Panel 8 (15279X) CAH Aldosterone * Corticosterone * * Cortisol17-HydroxyprogesteroneProgesteroneTestosterone * * * * * * * * * Interpretive Information Interpretive Reference Range Range Reference Clinical Use CAH Panels 4 and 8 and 4 Panels CAH Deficiency) (17-Hydroxylase • • CAH Panel 6 (StAR Deficiency) 15276X
Clinical Use Clinical Background • Diagnose StAR deficiency Steroid acute regulatory protein (StAR) deficiency is responsible for Reference Range congenital lipoid adrenal hyperplasia, Androstenedione * a defect of cholesterol transport Cortisol * resulting in deficiency of all adrenal DHEA * steroids. It is the rarest of the congenital adrenal steroid defects Pregnenolone * and has been fatal in two-thirds of the *See individual assays for age-related reported cases. The defect was thought reference ranges. to reside in CYP11A, the gene that codes for the cholesterol side-chain Interpretive Information cleavage enzyme. However, recent Androstenedione ↓ molecular studies indicate the defect Cortisol ↓ resides on chromosome 8 in the StAR DHEA ↓ gene, which encodes a phosphoprotein Pregnenolone ↓ that enhances cholesterol transport from the outer to inner mitochondrial membrane. Affected individuals present in the neonatal period with severe adrenal insufficiency manifested by failure to thrive, vomiting, diarrhea, hyponatremia, and hypokalemia. Males typically have normal female external genitalia.
Method • Extraction, radioimmunoassay (RIA) • Extraction, chromatography, RIA • Liquid chromatography, tandem mass spectrometry (LC/MS/MS)
Specimen Requirements 1.6 mL refrigerated serum 1.1 mL minimum No additive red top tube An early morning specimen is preferred. Specify age and sex on test request form.
34 Alphabetical Test Section ) 10299X CYP11B1 -hydroxylation of -hydroxylation α ) deficiencies ) deficiencies ) deficiency; ) deficiency; ), and P-450 oxido- ), CYPOR ), steroid acute regulatory), steroid ), aldosterone synthase synthase ), aldosterone StAR CYP21A2 -Hydroxysteroid dehydrogenase dehydrogenase -Hydroxysteroid β -hydroxylase (P-450 -hydroxylase β Extraction, radioimmunoassay (RIA) radioimmunoassay Extraction, RIA chromatography, Extraction, tandem chromatography, Liquid spectrometrymass (LC/MS/MS) HSD3B2 CYP11B2 Clinical Background hyperplasia adrenal congenital Classic (CAH) neonatal the in manifests synthesis cortisol with impaired period result that mutations genetic by caused adrenal of 1 of the activity in deficient The most enzymes. biosynthetic to for 90% accounting form, common of CAH 95% is 21-hydroxylase patients, (P-450 11 deficiency accounts for another 5% to for another accounts deficiency 8%. 3 protein ( protein ( ( ( reductase constitute the remainder of cases in of the remainder constitute deficiency 17-hydroxylase infancy. of puberty at the time presents usually and hypokalemia hypertension with 17 to decreased due pregnenolone and progesterone in the in and progesterone pregnenolone of production and increased adrenal steroid Gonadal mineralocorticoids. and LH is blocked, also production Females increased. FSH levels are and absent and primaryhave amenorrhea and males characteristics, sexual pseudoherma- with complete present genitalia, female (external phroditism tubes). no fallopian no uterus, Method • • • Specimen Requirements serum refrigerated 2.4 mL minimum 1.5 mL top tube red No additive is specimen morning An early preferred. request sex on test age and Specify form. 35 deficiency). deficiency). -OH, 17,20-lyase, -OH, 17,20-lyase, α CYPOR -OH, 17 -OH, β -HSD, StAR, or -HSD, StAR, Diagnosis and differential diagnosis differential and Diagnosis hyperplasia adrenal of congenital (CAH) β See Test Application and Interpreta- and Interpreta- Application Test See Function, of Adrenal Disorders tion, for more information. 6-8 Tables Results vary the defect Results on depending 11 (21-OH, *See individualassays for age-related reference ranges. 3 AndrostenedioneCortisolDHEADeoxycorticosterone11-Deoxycortisol pregnenolone17-OH progesterone17-OH Progesterone * Testosterone * * * * * * * * Interpretive Information Interpretive Reference Range Range Reference Clinical Use CAH Panel 6B (Comprehensive Screen) 6B (Comprehensive Panel CAH • CAH Panel 7 (21-Hydroxylase 15277X Deficiency Therapeutic Monitoring)
Clinical Use Clinical Background • Monitor therapy in patients with 21-Hydroxylase deficiency is the most 21-hydroxylase deficiency common cause of congenital adrenal hyperplasia (CAH). Selected steroids Reference Range and steroid precursors, along with Androstenedione * blood pressure, growth rate, skeletal 17-OH progesterone * age, and renin levels, are helpful when Testosterone * monitoring patients with this disorder. *See individual assays for age-related Due to diurnal rhythm of 17-hydroxy- reference ranges. progesterone secretion, serial measure- ments should be standardized to the Interpretive Information time of day and same conditions (eg, fasting 8 AM specimen prior to taking Androstenedione N or ↑ medications). Androstenedione 17-OH progesterone N or ↑ measurements become less useful for Testosterone N or ↑ monitoring purposes after adrenarche.
Method • Liquid chromatography, tandem mass spectrometry (LC/MS/MS)
Specimen Requirements 0.4 mL refrigerated serum 0.2 mL minimum No additive red top tube An early morning specimen is preferred. Specify age and sex on test request form.
36 Alphabetical Test Section 15280X ) is a rare form of ) is a rare HSD3B2 -Hydroxysteroid dehydrogenase dehydrogenase -Hydroxysteroid Extraction, radioimmunoassay (RIA) radioimmunoassay Extraction, RIA chromatography, Extraction, tandem chromatography, Liquid spectrometrymass (LC/MS/MS) β Clinical Background 3 deficiency ( deficiency CAHlevels increased by characterized 17-hydroxy- of pregnenolone, and DHEA and pregnenolone, adrenal of all other levels decreased usually individuals Affected steroids. of with signs in infancy present infants Female insufficiency. adrenal mild virilization. have will typically infants in male variation Phenotypic to hypospadias range from may male pseudohermaphro- complete ditism. Method • • •• Specimen Requirements serum refrigerated 1.0 mL minimum 0.5 mL top tube red No additive is specimen morning An early preferred. request sex on test age and Specify form. 37 ↓ ↓ >40 -Hydroxysteroid β -hydroxysteroid -hydroxysteroid β dehydrogenase deficiency dehydrogenase Diagnose 3 Diagnose 17-OH progesterone 17-OH DHEA/androstenedioneAndrostenedione Cortisol >30 17-OH pregnenolone/ 17-OH *See individualassays for age-related reference ranges. AndrostenedioneCortisolDHEA pregnenolone17-OH progesterone17-OH * * * * * Interpretive Information Interpretive Reference Range Range Reference Clinical Use CAH Panel 9 (3 Panel CAH Deficiency) Dehydrogenase • CAH Panel 11, 10046X Neonatal Random Urine
Clinical Use Clinical Background • Diagnose congenital adrenal Congenital adrenal hyperplasia (CAH) hyperplasia (mild as well as severe is an autosomal recessive disorder forms) in the newborn characterized by deficiency of any 1 of 4 enzymes required for cortisol Reference Range synthesis in the adrenal gland. Clinical symptoms vary according to the Steroid pediatric reference ranges: particular enzyme deficiency and see Table 9* include developmental abnormalities Precursor-product ratio pediatric of the external genitalia and salt reference ranges: see Table 10* wasting (sodium and potassium imbalance). Differential diagnosis and Interpretive Information treatment in the neonatal period is necessary to minimize morbidity and See Table 10* for deficiency-specific mortality. pattern of precursor-product ratios for diagnosis of: CAH diagnosis is based on clinical findings, biochemical and hormonal • 21-hydroxylase deficiency test results, karyotyping, and some- • 17-hydroxylase deficiency times mutation analysis. Although • 11β-hydroxylase deficiency single hormone levels may be useful, ACTH stimulation tests are frequently • 3β-hydroxysteroid dehydrogenase required. In some cases, hormonal deficiency precursor-product ratios are needed to establish the definitive diagnosis. The precursor (ie, substrate of the deficient enzyme) is often elevated, and the product is decreased, leading to an elevated ratio. The differential diagnosis is made based on the pattern of precursor-product ratios. Method • Gas chromatography/mass spectrometry (GC/MS) • Report includes 34 steroid measurements, 11 ratios, and interpretation • Analytical sensitivity: see Table 9* Specimen Requirements 7.0 mL frozen random urine 2.1 mL minimum Use a premature infant diaper to collect a random urine sample: 1) remove the wet diaper from the baby; 2) remove and discard the top liner of the diaper; 3) insert the wetted diaper pad into a 20 cc syringe barrel; 4) replace the syringe plunger and press down to squeeze out urine; *See Test Application and Interpretation, 5) mix well and freeze without pre- Disorders of Adrenal Function, section. servatives. 38 Alphabetical Test Section 30742X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 2 pg/mL sensitivity: Analytical Clinical Background acid is a 32-amino Calcitonin produced by parafollicular polypeptide of Secretion thyroid. in the cells or C by calcium. is stimulated calcitonin bone osteoclastic decreases Calcitonin role physiological but the resorption, is uncertain. in man indicated is measurement Calcitonin of and follow-up diagnosis for the with medullary patients thyroid of the majority (MTC), carcinoma Settings the hormone. produce whom include in which the test is appropriate with in patients nodule 1) thyroid to attributable potentially symptoms of 2) screening hypercalcitoninemia, known or with in families individuals or the multiple MTC suspected types syndrome neoplasia endocrine of patients follow-up IIb, and 3) IIa or family Screening known MTC. with calcitonin basal with normal members additional should include levels after measurements calcitonin calcium and/or pentagastrin Dynamic Tests). (see stimulation Method • • Specimen Requirements frozen serum 1.0 mL minimum 0.5 mL top red No additive is preferred. fasting Overnight 39 5 6 10 41 14 ≤ ≤ ≤ ≤ ≤ pg/mL have normal basal normal basal have 2004;50:1828-1829. 1/3 Clin Chem. Medullary carcinoma of the Medullary of the carcinoma Thyroid agenesis Thyroid Newborn infants Newborn Pregnancy (term) Pregnancy Pernicious anemia Pernicious Zollinger-Ellison syndrome Zollinger-Ellison Renal failure Renal Thyroiditis Pancreatitis Lung, breast, pancreatic cancer cancer breast, pancreatic Lung, thyroid ( thyroid (some patients) (some levels and require provocative provocative and require levels levels) to reveal abnormal test • • • • • • • • • • Diagnose and monitor medullary and monitor Diagnose carcinoma thyroid <6 mo* y* 6 mo-3 y 3-17 *From Men Women Children Reference Range Reference Clinical Use Calcitonin • Calcium, Ionized 306X
Clinical Use Clinical Background • Diagnose hypo- and hypercalcemia Ionized calcium represents the concentration of free and biologically Reference Range active calcium in blood. Calcium mg/dL circulates in approximately equal free Adults 4.8-5.6 and bound fractions. Most (70%) of the bound calcium is bound to Children albumin. A portion of the non-protein- 8 mo-10 y 4.9-5.4 bound calcium is complexed to anions 11-17 y 4.8-5.3 (bicarbonate, phosphate, and citrate). Pediatric data from J Pediatr. 1989;114: In the presence of an abnormal 952-956 and Eur J Pediatr. 1991;150: circulating albumin concentration, 464-467. measurement of the plasma ionized Interpretive Information calcium provides a more precise assessment of the biologically relevant • Primary hyperparathyroidism calcium level. It permits more accurate • Vitamin D intoxication diagnosis in hypocalcemic and • Hypercalcemia of malignancy hypercalcemic states.
• Hypoparathyroidism Method • Secondary hyperparathyroidism • Ion specific electrode • Analytical sensitivity: 0.4 mg/dL
Specimen Requirements 2 mL refrigerated serum 0.6 mL minimum Sample must be collected anaerobic- ally in a gel-barrier tube. Allow to clot, then centrifuge with the cap on.
40 Alphabetical Test Section 303X Spectrophotometry 0.2 mg/dL sensitivity: Analytical Clinical Background precisely are levels calcium Blood D- vitamin by principally maintained, of absorption intestinal regulated dietaryand parathyroid calcium D- and vitamin (PTH) hormone calcium skeletal of exchange mediated is most Hypercalcemia stores. and malignancy due to commonly and less often the hyperparathyroidism D intoxication, of vitamin result several disorders, and granulomatous Clinical rare conditions. other can hypercalcemia of manifestations muscular, neuropsychological, include is Hypocalcemia cardiac effects. and caused by renal commonly most hyperphosphatemia, with insufficiency D and vitamin hypoparathyroidism, of manifestations Clinical deficiency. include paresthesia, can hypocalcemia and seizures. The tetany, muscle spasm, the ionized but not calcium level, total the serum is low when fraction, is decreased. concentration albumin of the measurement Simultaneous concentrations, PTH and calcium total level assess- vitamin D with occasionally of diagnosis permits differential ment, circu- an abnormal patients with most calcium concentration. lating Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST is preferred. fasting Overnight 41 mg/dL 8.4-10.6 8.7-10.5 8.5-10.6 8.9-10.4 Secondary hyperparathyroidism Secondary hyperparathyroidism Vitamin D deficiency Vitamin Hypoparathyroidism Vitamin D intoxication Vitamin Hypercalcemia of malignancy Hypercalcemia Primary hyperparathyroidism (renal disease) (renal • • •• • • • Manage parathyroid disorders parathyroid Manage Diagnose hypocalcemia or hyper- or hypocalcemia Diagnose calcemia <1 mo mo 1-11 1-3 y y 4-19 AdultsChildren 8.6-10.2 Interpretive Information Interpretive Reference Range Reference Clinical Use Calcium, Total Calcium, • • Calcium, Urine 1635X 11216X
Clinical Use Clinical Background • Assess metabolic disorders of calcium Urinary calcium excretion is the major metabolism route of calcium elimination. Renal tubular calcium reabsorption is closely Reference Range regulated to maintain serum calcium Calcium (1635X) mg/24-h concentrations within a narrow Men 50-300 physiologic range. The daily filtered Women 50-250 load of calcium at the glomerulus mg/g creat approximates 9,000 mg/day in adults; Men 30-210 only 1% to 3% of filtered calcium is Women 30-275 excreted. Urinary calcium is bound in part by citrate, sulfate, and oxalate Creatinine (1635X) g/24-h anions, and binding varies with pH, 3-8 y 0.11-0.68 urine flow, and composition. It is 9-12 y 0.17-1.41 difficult to ascertain whether changes 13-17 y 0.29-1.87 in urine calcium relate to changes in Adults 0.63-2.50 filtered load, tubular reabsorption, Calcium (11216X)* mg/mg creat or both. The usual urine calcium 1-11 mo 0.03-0.81 (mg/dL) to creatinine (mg/dL) ratio 1 y 0.03-0.56 is 0.14 or less; values above 0.20 suggest 2 y 0.02-0.50 hypercalciuria. 3-4 y 0.02-0.41 5-6 y 0.01-0.30 Method 7-9 y 0.01-0.25 • Spectrophotometry 10-17 y 0.01-0.24 • Analytical sensitivity: 0.2 mg/dL Creatinine (11216X) mg/dL • Creatinine concentration also 0-6 mo 2-32 reported 7-11 mo 2-36 1-2 y 2-128 Specimen Requirements 3-8 y 2-149 Urine, 24-h (1635X) 9-12 y 2-183 10 mL refrigerated aliquot of a 24-h >12 y urine; 1 mL minimum Males 20-370 Collect urine in 25 mL of 6N HCl Females 20-320 (preferred), 10 g boric acid, or 10 mL *Matos V, et al. J Pediatr. 1997;131:252-257. concentrated glacial acetic acid. Interpretive Information Refrigerate during collection. Record 24-h volume on vial and request form. • Hyperparathyroidism • Vitamin D intoxication Urine, 2nd void (11216X) • Paget's disease 10 mL refrigerated aliquot of a 2nd • Myeloma void urine; 1 mL minimum • Hypoparathyroidism Adjust pH to <3.0 with 6N HCl prior to • Rickets aliquoting. • Osteomalacia • Hypocalcemia • Hypocalciuric hypocalcemia
42 Alphabetical Test Section 39627X g/L for μ g/L for dopamine g/L μ Creatinine concentration also also concentration Creatinine reported epinephrine and norepinephrine; norepinephrine; and epinephrine 10 High-performance liquid chromato- electrochemical (HPLC), graphy detection 2 sensitivity: Analytical • Specimen Requirements of a aliquot room temperature 10 mL minimum urine; 4.5 mL 24-h 6N HCl. 25 mL of urine with Collect and on vial total volume Record form. request be off to the patient for is preferable It for 3 medications d prior to collection; common antihypertensives however, no interference. minimal or cause coffee, alcohol, should avoid Patient exercise strenuous and tea, tobacco, to collection. prior Clinical Background in are synthesized Catecholamines of the sympathetic cells chromaffin nervous by the epinephrine system: and norepinephrine medulla, adrenal medulla the adrenal dopamine by and and postganglionic sympathetic excreted are Catecholamines neurons. metabolites and as intact urine both in and vanillylmandelic (metanephrines acid). urinary Elevated catecholamine in the detected are concentrations with pheochromo- of patients majority with paroxysmal In patients cytoma. the or hypertension, symptoms increased can be of testing sensitivity imme- collection urine by beginning The an episode. following diately secretion of catecholamine pattern types: among tumor differs generally pheochromocytomas and both norepinephrine produce secrete paragangliomas epinephrine; and neuroblastomas norepinephrine; dopamine. excessive produce Method • • 43 8 11 g/24-h μg/24-h
Neuroblastoma Stress Pheochromocytoma Paragangliomas • • • • Diagnose catecholamine-secreting catecholamine-secreting Diagnose tumors 3-8 y y9-12 1-7 < 13-17 y13-17 < AdultsNorepinephrine 3-8 y y9-12 y13-17 Adults N+E Total 3-8 y y9-12 2-24 y13-17 AdultsDopamine 5-41 5-50 12-88 3-8 y 15-100 y9-12 y13-17 Adults 9-51 Creatinine 9-71 13-90 3-8 y 26-121 y9-12 y13-17 Adults 80-378 51-474 51-645 52-480 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 Interpretive Information Interpretive Reference Range Reference Clinical Use Epinephrine Catecholamines, Fractionated, Fractionated, Catecholamines, Urine 24-Hour • Catecholamines, Fractionated, 314X Plasma
Clinical Use Clinical Background • Diagnose pheochromocytoma, Catecholamines are synthesized in paraganglioma, neuroblastoma, or chromaffin cells of the sympathetic postural hypotension nervous system: epinephrine by the adrenal medulla, and norepinephrine Reference Range and dopamine by the adrenal medulla and postganglionic sympathetic pg/mL pg/mL neurons. Catecholamines circulate in Adults Supine Upright plasma in both free and bound forms Epinephrine <50 <95 complexed to albumin, globulins, and Norepinephrine 112-658 217-1109 lipoproteins. Assays measure the free Dopamine <10 <20 and protein-bound, but not conjugated Total (N+E) 123-671 242-1125 fractions. Plasma catecholamines exhibit Children (3-15 y) Supine considerable physiological variability, Epinephrine <464 increasing with pain and psychological Norephinephrine <1251 stress. Consequently, samples should Dopamine <60 be obtained from patients in the supine position and basal state, ideally Due to stress, plasma catecholamine by using an indwelling venous catheter levels are generally unreliable in at least 30 minutes after venipuncture. infants and small children. Urinary Specificity of elevated plasma cate- catecholamine assays are more cholamine measurements may be reliable. increased by suppression and/or Pediatric data from J Chromatogr. 1993; stimulation testing (see Dynamic Test 617:304-307. section). Certain medications may increase the catecholamine measure- Interpretive Information ment either by assay interference (eg, methyldopa, isoproterenol) or by • Pheochromocytoma increasing endogenous catecholamine • Ganglioneuroma release (eg, nitrates, minoxidil, • Neuroblastoma hydralazine); cigarette smoking and caffeine ingestion may do so as well. • Severe stress Method • Hypoglycemia • HPLC, electrochemical detection • Postural hypotension • Analytical sensitivity: 20 pg/mL for epinephrine and norepinephrine; • Shy-Drager syndrome 10 pg/mL for dopamine • Familial dysautonomia Specimen Requirements 4 mL frozen sodium heparin plasma 2.5 mL minimum Collect in pre-chilled vacutainer. Centrifuge in refrigerated centrifuge within 30 minutes of collection. Separate plasma and freeze immediately. Patient should avoid alcohol, coffee, tea, tobacco, and strenuous exercise prior to collection. Overnight fasting is required.
44 Alphabetical Test Section 5244X HPLC, electrochemical detection electrochemical HPLC, for 2 μg/L sensitivity: Analytical norepinephrine; and epinephrine for dopamine 10 μg/L also concentration Creatinine reported Clinical Background in are synthesized Catecholamines of the sympathetic cells chromaffin nervous by the epinephrine system: and norepinephrine medulla, adrenal medulla the adrenal dopamine by and and postganglionic sympathetic excreted are Catecholamines neurons. metabolites and as intact urine both in and vanillylmandelic (metanephrines acid). urinary Elevated catecholamine in the detected are concentrations with pheochromo- of patients majority with paroxysmal In patients cytoma. the or hypertension, symptoms increased can be of testing sensitivity collection urine by beginning an episode. following immediately pattern of catecholamine The types: among tumor differs secretion generally pheochromocytomas and both norepinephrine produce secrete paragangliomas epinephrine; and neuroblastomas norepinephrine; dopamine. excessive produce Method • • • Specimen Requirements urine room temperature 10 mL minimum 4 mL a HCl to maintain 25 mL of 6N Add 3. pH below be off to the patient for is preferable It for 3 medications d prior to collection; common antihypertensives however, no interference. minimal or cause coffee, alcohol, should avoid Patient exercise strenuous and tea, tobacco, to collection. prior 45 D N mg/dL μg/g creat μg/g creat E Total N+E Total μg/g creat μg/g creat Stress Paragangliomas Neuroblastoma Pheochromocytoma • • • • MalesFemales 20-370 20-320 Diagnose catecholamine-secreting catecholamine-secreting Diagnose tumors Birth to 6 moBirth mo7-11 24-3221-2 y 107-2180 3-8 y y9-12 10-295 y13-17 96-2441 Adults 30-263Creatinine 30-130 86-1861 0-6 mo 25-90 295-1123 20-70 mo7-11 164-744 1-2 y 156-551 9-743-8 y y9-12 40-390 y >12 2-32 2-36 2-128 2-149 2-183 Birth to 6 moBirth mo7-11 1-2 y 2-453-8 y y9-12 12-286 y13-17 5-45Adults 19-250 1-49 4-32 1-15 25-210 1-10 20-108 2-16 20-73 15-58 7-65 E, epinephrine;N,norephinephrine; D, dopamine. Interpretive Information Interpretive Reference Range Reference Clinical Use Catecholamines, Fractionated, Catecholamines, Urine Random • Central Diabetes Insipidus (CDI) 15035X Mutations
Clinical Use Clinical Background • Differentiate inherited CDI from CDI is an acquired or autosomal acquired CDI dominant inherited disorder character- • Screen for CDI carrier status in at- ized by polyuria, polydipsia, a low risk individuals urinary specific gravity, and high risk of severe dehydration. Arginine Reference Range vasopressin (AVP), also known as Negative (no mutations detected) antidiuretic hormone (ADH), is absent. CDI stems from the degenera- Interpretive Information tion or destruction of cells in the posterior pituitary, the site of AVP Mutation present production. Thus, it is also referred to • Central diabetes insipidus (affected as pituitary, neurohypophyseal, or or carrier) neurogenic diabetes insipidus. The disorder typically presents in infancy or early childhood, although late-onset cases have been reported. Although rare, inherited CDI can be caused by mutations in the AVP gene on chromosome 20. Prepro-AVP, the initial protein product of the AVP gene, undergoes several post-transla- tional steps to yield AVP, neurophysin, and glycoprotein. When mutations in the AVP gene are present, cytotoxic products that lead to destruction of the secretory neurons are generated. Since more than 30 relevant mutations have been identified, gene sequencing is the method of choice for diagnosis of the inherited form. After identifica- tion of a mutation in an affected indi- vidual, genetic testing can be used to evaluate other family members.
Method • Polymerase chain reaction (PCR) and DNA sequencing • Analytical specificity: mutations in 3 exons of the AVP gene
Specimen Requirements 5 mL room temperature whole blood 3 mL minimum Collect blood in a lavender-top (EDTA) or yellow-top (ACD solution B) tube.
46 Alphabetical Test Section 34468X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 1.5 ng/mL sensitivity: Analytical Clinical Background the major is (CGA) A Chromogranin catecholamine within the protein granules) (chromaffin vesicles storage cate- When adrenal medulla. of the exocytosis released by are cholamines or medulla normal adrenal from vesicle cells, all pheochromocytoma including are co-released, contents to be been shown CGA has also CGA. with polypeptide hormones co-released tissues: endocrine the following from cells, enteroendocrine islet pancreatic thyroid chief cells, parathyroid cells, cells, and anterior C parafollicular pituitary concentrations cells. CGA with in patients be elevated may eg, neoplasms, endocrine various pituitary para- anterior adenoma, medullary adenoma, thyroid thyroid and tumor, carcinoid carcinoma, In contrast, cell tumor. islet pancreatic neoplasia with non-endocrine patients have disease endocrine or benign concentrations. plasma CGA normal to has been reported CGA also Serum small cell with in patients be elevated carcinoma. lung Method • • Specimen Requirements 0.8 mL refrigerated serum (no additive mL minimum tube); 0.3 red top red top unacceptable SST 47 36.4 ng/mL ≤ Small cell lung carcinoma cell lung Small Pancreatic islet cell tumor islet Pancreatic Carcinoid tumor Carcinoid Medullary carcinoma thyroid Parathyroid adenoma Parathyroid Anterior pituitaryAnterior adenoma • • • • • • Assess multiple endocrine neoplasia endocrine multiple Assess Assess peptide-secreting endocrine endocrine peptide-secreting Assess neoplasms Adults Interpretive Information Interpretive Reference Range Reference Clinical Use Chromogranin A Chromogranin • • Collagen Cross-Linked 36167X N-Telopeptide (NTx), Urine 36421X
Clinical Use Clinical Background • Monitor therapeutic response in Healthy levels of bone mineral density patients with metabolic bone (BMD) are maintained by a balance disorders between bone resorption and bone formation. N-telopeptide (NTx), the • Predict future bone mineral density amino-terminal cross-linked peptide of (BMD) type I collagen, is released during bone • Predict therapeutic response prior to resorption and has been correlated with initiation of antiresorptive therapy BMD T-scores. Multiple studies have • Detect bone metastasis in patients shown that NTx not only correlates with various malignancies inversely with BMD response to therapy, but also is an early marker or predictor Reference Range of BMD response. Thus, therapeutic response can be determined within 3 to Men Units* 6 months of therapy rather than 1 to 2 2nd AM urine years. Studies have also demonstrated 18-29 y 12-99 that elevated pretreatment NTx values predict positive response to therapies 30-59 y 9-60 such as hormone replacement therapy 24-h urine 5-87 in postmenopausal women. In patients Women, premenopausal with malignancies, elevated levels of 2nd AM urine 4-64 NTx may indicate bone metastases. 24-h urine 5-79 Method Test results within the premenopausal • Immunochemiluminometric assay reference range do not rule out osteo- (ICMA) porosis nor the need for therapy. • Analytical sensitivity: 10 nmol BCE/L *nmol BCE/mmol creat; BCE, bone collagen equivalent. Specimen Requirements Urine, 2nd morning void (36167X) Interpretive Information Urine, 24-h collection (36421X) • Osteoporosis 2 mL refrigerated aliquot of urine • Osteopenia 1 mL minimum • Celiac disease Do not use preservatives. • Paget’s disease Record total 24-h volume on the vial and request form. • Primary hyperthyroidism • Rheumatoid arthritis • Growth hormone deficiency (non-adult onset)
• Therapeutic response (ie, decrease relative to baseline)
48 Alphabetical Test Section 17406X Electrochemiluminescent Electrochemiluminescent (ECLIA) immunoassay 30 pg/mL sensitivity: Analytical • • Clinical Background density bone mineral levels of Healthy balance a by are maintained (BMD) bone and bone resorption between a (CTx), C-telopeptide formation. peptide of type I collagen, cross-linked bone resorption during is released BMD with has been correlated and with inversely CTx correlates T-scores. an is therapy and response to BMD BMD predictor of marker or early response therapeutic Thus, response. to 6 within 3 determined can be to 2 1 rather than of therapy months that demonstrated Studies have years. values CTx pretreatment elevated therapies response to positive predict therapy replacement as hormone such patients In women. postmenopausal in levels of elevated malignancies, with bone metastases. may indicate CTx Method Specimen Requirements red (no additive frozen serum 1 mL minimum 0.5 mL top tube); 10 am 8 and sample between Collect hour fast. an 8 to 12 after 49 Therapeutic response (ie, response Therapeutic Growth hormone deficiency deficiency hormone Growth Rheumatoid arthritis Rheumatoid Primary hyperthyroidism Paget's disease Paget's Celiac disease Celiac Osteopenia Osteoporosis decrease relative to baseline) to baseline) relative decrease (non-adult onset) (non-adult • • • • • • • • Detect bone metastasis in patients patients in bone metastasis Detect various malignancies with Predict response prior to Predict therapeutic therapy of antiresorptive initiation y18-29 y30-39 y40-49 y50-68 y18-29 y30-39 87-1200 y40-49 70-780 60-700 87-345 64-640 60-650 40-465 Predict future bone mineral density density bone mineral future Predict (BMD) Monitor therapeutic response in response therapeutic Monitor bone with metabolic patients disorders Women Men pg/mL Interpretive Information Interpretive Reference Range Reference Clinical Use Collagen Type 1 Type Collagen (CTx) C-Telopeptide • • • • Corticosterone, LC/MS/MS 6547X
Clinical Use Clinical Background • Assess adrenal gland function Corticosterone is an intermediate in the adrenal corticosteroid biosynthetic • Marker for P450c17 enzyme deficiency pathway. It is the substrate for further 18-hydroxylation and dehydrogenation Reference Range to aldosterone. Corticosterone synthesis from 11-deoxycorticosterone Adults ng/dL ng/dL (DOC) is catalyzed by P450 . 8-10 AM 59-1293 c11 Measurement is useful in Cushing’s 4-6 PM ≤386 syndrome when exogenous steroid Children use is suspected. Rare cases of cortico- Premature infants ≤235 sterone-secreting adenomas have (31-35 wks)* been reported. Term infants* ≤170 Male Female Method <1 y 78-1750 89-1200 • Liquid chromatography, tandem 1-4 y 120-1290 160-2040 mass spectrometry (LC/MS/MS) 5-9 y ≤677 ≤677 • Analytical sensitivity: 20.0 ng/dL 10-13 y ≤1420 ≤1420 14-17 y ≤1238 ≤1238 Specimen Requirements Tanner stages 1 mL refrigerated serum (no additive II-III 115-1220 110-600 red top tube); 0.25 mL minimum IV-V 165-840 160-390 SST red top unacceptable *J Clin Endocrinol Metab. 1992;75:1491-1496.
Interpretive Information • Adrenal adenoma • Cushing’s disease
• P450c17 (17-hydroxylase) deficiency
• P450c11ase (aldosterone synthetase) deficiency
• Addison’s disease • Adrenal hypoplasia • ACTH deficiency
50 Alphabetical Test Section 36589X Extraction, radioimmunoassay (RIA) radioimmunoassay Extraction, 28 pg/mL sensitivity: Analytical no cross- specificity: Analytical LH releasing with ACTH, reactivity arginine PACAP-38, hormone, urocorin, urocortin, vasopressin, or BNP Clinical Background acid peptide 41-amino a CRH, hypo- in the produced normally of release the stimulates thalamus, CRH levels of Normal blood ACTH. by produced is also but CRH are low, are high levels and the placenta, and the second during measured CRH of pregnancy. trimesters third Cushing's in also are increased levels ectopic production. due to syndrome Method • • • Specimen Requirements plasma refrigerated 3.1 mL minimum 1.1 mL special Nichols blood in Collect Factor and Releasing PTH-RP Institute tube. collection in refrigerated immediately Centrifuge and plasma, remove centrifuge, refrigerate. 51 34 34 40 153 847 338
pg/mL J Clin EndocrinolMetab. Cushing's syndrome (adrenal) syndrome Cushing's Fetus Pregnancy Cushing’s syndrome (ectopic (ectopic syndrome Cushing’s CRH) • • • • Differential diagnosis of Cushing’s Cushing’s of diagnosis Differential syndrome Nonpregnant < 1st trimester pregnancy trimester pregnancy2nd trimester pregnancy3rd < < < Cord BloodCord < Pregnancy and cord blood reference reference blood Pregnancy and cord ranges from Men < Women Children 1986;63:1199-1203. Interpretive Information Interpretive Reference Range Reference Clinical Use Corticotropin Releasing Releasing Corticotropin (CRH) Hormone • Cortisol-Binding Globulin (CBG) 37371X
Clinical Use Clinical Background • Useful in evaluating unexplained Cortisol-binding globulin (CBG) is a cortisol concentrations serum alpha-2-globulin-binding protein with high affinity and limited Reference Range capacity for cortisol. It binds most mg/L (±92%) of the serum cortisol; the Adults 19-45 remaining cortisol (±8%) circulates in a free form or bound to albumin. Children Preterm infants (<8 d) 6-26 CBG is synthesized in the liver. Circulating levels increase in preg- Pediatric data from Pediatr Res.1994;37: 112-116. nancy or after administration of estrogen or oral contraceptives. Insulin Interpretive Information may inhibit synthesis, a fact that might explain the low CBG levels observed in • Pregnancy hyperinsulinemic states (eg, diabetes, • Estrogen therapy obesity, PCO). CBG levels are also • Chronic, active hepatitis reduced in nephrotic syndrome, • Inherited abnormality starvation, or severe chronic liver disease. • Hyperinsulinemic states The CBG assay is helpful in assessing • Nephrotic syndrome unexpected values of blood cortisol. Other assays useful in diagnosis and • Severe liver disease therapy of cortisol abnormalities • Malnutrition include plasma cortisol, ACTH, • Newborn β-lipotropin, urinary cortisol (by HPLC), or free cortisol in plasma or • Inherited abnormality serum.
Method • Radioimmunoassay (RIA) • Analytical sensitivity: 0.1 mg/L
Specimen Requirements 1 mL refrigerated serum 0.2 mL minimum No additive red top preferred SST red top acceptable
52 Alphabetical Test Section 37355X -DHE/DHF -DHE/DHF β decrease results; high concentrations concentrations results; high decrease interfereof prednisolone with measurement. also concentration Creatinine reported Liquid chromatography, tandem tandem chromatography, Liquid spectrometrymass (LC/MS/MS) 1 μg/L sensitivity: Analytical high specificity: Analytical 20 of concentrations • Specimen Requirements of a 24-h aliquot refrigerated 10 mL 2.6 mL minimum urine; or 10 g of boric acid urine with Collect during collection. refrigerated keep and on vial 24-h volume Record form. request Clinical Background Cushing's of diagnosis differential The ACTH on based has been syndrome or serum. in urine assays cortisol and for shown utility data have Recent urinary and combined free cortisol high by measurements cortisone performance chromatography liquid diagnostic HPLC, the Using (HPLC). is syndrome for Cushing's sensitivity cortisol of 17% over an increase 93%, cortisol Both 7% over cortisone. and in are elevated cortisone levels and and ectopic, with pituitary, patients while syndrome, Cushing's adrenal are levels cortisone cortisol and both receiving in patients decreased pred- (eg, glucocorticoid exogenous measure- the Additionally, nisolone). can be and cortisone of cortisol ment of apparent for the diagnosis used in excess (AME) mineralocorticoid is reduced, formation cortisone which mineral- to act as a cortisol allowing ocorticoid. Method • • • 53 g/24-h μg/24-h μg/24-h Adrenal insufficiency Adrenal Exogenous Cushing’s syndrome Cushing’s Exogenous Cushing’s syndrome Cushing’s • • •• 1-4.9 y1-4.9 y5-9.9 y10-13.9 y14-17.9 y1-4.9 0.9-8.2 1.0-30.0 1.0-45.0 y5-9.9 3.0-55.0 y10-13.9 y14-17.9 5-40 11-145 9-150 1-166 AdultsChildren Adults 4.0-50.0 Children 3-8 y 23-195 y9-12 y 13-17 Adults 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 Marker for apparent mineral- for apparent Marker (AME) syndrome excess ocorticoid Differentiate between exogenous exogenous between Differentiate Cushing’s endogenous and syndrome Diagnose Cushing’s syndrome Cushing’s Diagnose Cortisol, Free Cortisone Creatinine Interpretive Information Interpretive Reference Range Reference Clinical Use Cortisol, Free and Cortisone, and Free Cortisol, Urine 24-Hour • • • Cortisol, Free, 24-Hour Urine 11280X
Clinical Use Clinical Background • Screen for Cushing's syndrome and Cortisol primarily circulates bound to adrenal insufficiency cortisol-binding globulin (CBG). Only the unbound (free) cortisol appears to Reference Range be biologically active. The free fraction Cortisol, Free μg/24-h is 5% to 8% of total cortisol in normal Adults 4-50 individuals. Children Since the cortisol binding capacity of 1-4 y 0.9-8.2 blood proteins is limited, adrenal hyperfunction (Cushing's syndrome) is 5-9 y 1.0-30.0 invariably associated with a many-fold 10-13 y 1.0-45.0 increase in urinary cortisol levels. 14-17 y 3.0-55.0 Urinary free cortisol is not substantially Creatinine g/24-h increased in pregnancy, obesity, 3-8 y 0.11-0.68 diabetes, or hyperthyroidism, entities 9-12 y 0.17-1.41 that in the past could represent a diagnostic dilemma when older assays 13-17 y 0.29-1.87 of cortisol or metabolites were used. Adults 0.63-2.50 Exogenous Cushing's syndrome due to Interpretive Information surreptitious use of glucocorticoid can now be diagnosed by finding low • Adrenal hyperfunction, ie, cortisol and presence of synthetic Cushing’s disease steroids in the urine. • Adrenal insufficiency Urinary cortisol is the best screening test for Cushing's syndrome and can be used to rule out Addison's disease.
Method • Liquid chromatography, tandem mass spectrometry (LC/MS/MS) • Analytical sensitivity: 1 μg/L • Creatinine concentration also reported
Specimen Requirements 2 mL frozen aliquot of a 24-h urine 0.5 mL minimum No preservative (preferred) 25 mL 6N HCL acceptable 10 g boric acid acceptable 10 mL concentrated glacial acetic acid acceptable Record 24-h volume on vial and request form.
54 Alphabetical Test Section 16164X late night salivary night late n salivary cor- free tes after rinsing disruption. Measure- Liquid chromatography, tandem Liquid chromatography, mass spectrometry (LC/MS/MS) Provides best measurement of cortisol active biologically μg/dL 0.1 sensitivity: Analytical Method • • • Specimen Requirements refrigerated0.5 mL saliva minimum 0.2 mL foodPatient should refrain and from to sample cigarettes for 1 hour prior tube using Salivette Collect collection. (Sarstedt) 10 minu mouth with water. mouth with Cortisol or (hydrocortisone compound by the secreted glucocorticoid a F) is adrenalafter adrenocorticotropic cortex More stimulation. (ACTH) hormone is cortisol circulating of 90% than cortisol-bound to binding globulin in- are levels CBG or transcortin). (CBG estrogen therapy, pregnancy, creased in and decreased oral contraceptives, and starvation,in nephrotic syndrome, and Free cortisolchronic liver disease. the of cells acinar the through diffuses salivary glands. Salivary levels are about Measurements serum. of that two-thirds serum with of salivarycorrelate cortisol range, physiological the within levels thebutserum ratio of to salivary free The levels. higher at increases cortisol betwee (r) correlation cortisol concentrations a provides stress- in children and approach free screening measurements cortisol Salivary adults. re- assess the also have been used to dexamethasone.sponse to overnight Clinical Background tisol and urinary free cortisol0.63. is is salivaryof cortisol measurement The Cushing's for test screening as a useful episod- is secreted Cortisol syndrome. rhythm a circadian is there but ically, AM and at 7-8 peak nadir levels with soon aftervalues midnight. In addition in secretion cortisol to increased patients with Cushing's syndrome, dis- is circadian rhythm ruption of the from subtle altera- common, ranging tions to complete and morning ment of 55 N N CBG ↑↑ ↑↑ ↑↑ ↓↓ ↓↓ ↓↓ Total Cortisol ↓↓ ↑↑ N N N N Salivary Cortisol Screen for Cushing's syndrome for Cushing's Screen in free cortisol changes diurnal Assess CBG, cortisol binding globulin; N, normal. N, binding globulin; CBG, cortisol PregnancyOral contraceptives Estrogen N therapy Chronic liver disease StarvationNephrotic syndrome Adrenal N insufficiency Cushing's syndrome Interpretive Information Interpretive Reference Range Reference See report. Clinical Use Cortisol, Free, Saliva Free, Cortisol, • • Cortisol, Free, Serum 36423X
Clinical Use Clinical Background • Determine glucocorticoid status Cortisol (hydrocortisone or compound F) is a glucocorticoid secreted by the Reference Range adrenal cortex after adrenocortico- See report tropic hormone (ACTH) stimulation. More than 90% of circulating cortisol Interpretive Information is bound to cortisol-binding globulin (CBG or transcortin). CBG levels are Free Total increased in pregnancy, estrogen Cortisol Cortisol CBG therapy, and oral contraceptives, and Pregnancy N ↑↑decreased in nephrotic syndrome, Oral N ↑↑starvation, and chronic liver disease. contraceptives Thus, total cortisol levels can be Estrogen N ↑↑affected by these conditions. therapy Free cortisol, however, is the physio- Chronic liver N ↓↓logically active form. Free cortisol disease levels change independently and Starvation N ↓↓accurately reflect the clinical status; Nephrotic N ↓↓thus serum free cortisol is the most syndrome reliable test for glucocorticoid Adrenal ↓↓N status. It is markedly elevated in insufficiency Cushing's syndrome and falls to Cushing’s ↑↑N almost undetectable levels after syndrome dexamethasone (1 mg overnight) in normal individuals. CBG, cortisol binding globulin; N, normal. Method • Liquid chromatography, tandem mass spectrometry (LC/MS/MS) • Provides best measurement of biologically active cortisol • Analytical sensitivity: 0.1 μg/dL
Specimen Requirements 2 mL refrigerated serum 0.7 mL minimum No additive red top tube preferred SST red top acceptable
56 Alphabetical Test Section /dL 11281X μg -DHE/DHF -DHE/DHF β decrease results; high concentrations concentrations results; high decrease interfereof prednisolone with measurement. Analytical specificity: high specificity: Analytical 20 of concentrations Liquid chromatography, tandem tandem chromatography, Liquid spectrometrymass (LC/MS/MS) 0.1 sensitivity: Analytical Specimen Requirements additive serum (no refrigerated 1 mL mL minimum tube); 0.2 red top was specimen time of day Specify collected. • Clinical Background glucocorticoid is the major Cortisol gland. by the adrenal secreted in a ACTH by is regulated Secretion peaks secretion ACTH fashion. diurnal hours, stimulating in the early morning of serum cortisol peak a morning is therefore, Cortisol, concentration. AM) (8 the morning measured in best adrenal for possible evaluating when in the best measured and insufficiency to PM) (4-11 or evening afternoon and Cushing's normal differentiate post- and Baseline subjects. syndrome values suppression dexamethasone diagnosis. differential be useful in may be also may values ACTH-stimulated of measurements Combined useful. provide ACTH cortisol and serum discrimination diagnostic differential cases. in most Method • • 57
15 14 < J Clin Endocrinol Metab. Endocrinol J Clin J Clin Endocrinol Metab. Endocrinol J Clin Pituitary dysfunction (decreased Pituitary (decreased dysfunction Addison’s disease Addison’s Stress Adrenal macronodular hyper- macronodular Adrenal Adrenal micronodular dysplasia micronodular Adrenal Adrenal adenoma or carcinoma adenoma Adrenal Ectopic CRH syndrome Ectopic Ectopic ACTH syndrome ACTH Ectopic Cushing’s syndrome Cushing’s production of ACTH) production plasia MalesFemales 5-15 6-15 18-27 18-35 MalesFemales 4-13 4-16 15-45 16-32 • • • • • • • • • Tanner IV-V Tanner 1-12 mo1-12 1-5 y y6-12 II-III Tanner 3-23 32-60 6-25 3-15 22-40 17-28 Peak after IM injectionPeak >16 >20 Term infants (3 d) infants (3 Term <1 y y1-17 stimulationACTH Baseline min 60 < NA 2-17 Premature infants infants Premature wk) (31-35 8-10 AM8-10 4-6 PM stimulation ACTH Post 5-21 2-14 Direct assessment of adrenal function adrenal of assessment Direct Diurnal rhythmicity increased by increased rhythmicity Diurnal stress and other disease systemic Pediatric dataPediatric from Children 1991;73:674-686 and Adults μg/dL μg/dL 1989;69:1133-1136. Interpretive Information Interpretive Clinical Use Range Reference Cortisol, Total, LC/MS/MS Total, Cortisol, • Cortisone, 24-Hour Urine 37554X
Clinical Use Clinical Background • Evaluate adrenal function Cortisol, the active glucocorticoid • Marker for AME produced by the adrenal gland, is • Additional marker for exogenous inactivated to cortisone in peripheral steroid use tissues. Excretion of both cortisol and cortisone is increased in patients with Reference Range Cushing's syndrome but reduced in patients with adrenal insufficiency Cortisone μg/24-h and in those receiving exogenous Adults 23-195 glucocorticoid (eg, prednisone). In Children patients with apparent mineral- 1-4.9 y 5-40 ocorticoid excess (AME), cortisone 5-9.9 y 9-150 formation is reduced, allowing cortisol 10-13.9 y 11-145 to act as a mineralocorticoid. Patients 14-17.9 y 1-166 with AME have reduced cortisone and aldosterone excretion, while urinary Creatinine g/24-h free cortisol is normal or increased. 3-8 y 0.11-0.68 9-12 y 0.17-1.41 Method 13-17 y 0.29-1.87 • Liquid chromatography, tandem Adults 0.63-2.50 mass spectrometry (LC/MS/MS) • Analytical sensitivity: 1.0 μg/L Interpretive Information • Analytical specificity: high concentra- • Cushing’s syndrome tions of 20β-DHE/DHF decrease results; high concentrations of • Adrenal insufficiency prednisolone interfere with measure- ment. • Exogenous Cushing’s syndrome • Creatinine concentration also (prednisone) reported • AME Specimen Requirements 10 mL refrigerated aliquot of a 24-h urine; 2.1 mL minimum Collect urine with 10 g of boric acid or keep refrigerated during collection. Record 24-h volume on vial and request form.
58 Alphabetical Test Section 37098X -DHE/DHF decrease decrease -DHE/DHF β Liquid chromatography, tandem tandem chromatography, Liquid spectrometrymass (LC/MS/MS) 0.1 μg/dL sensitivity: Analytical high concentra- specificity: Analytical of 20 tions of high concentrations results; interfereprednisolone with measurement. Clinical Background glucocorticoid the active Cortisol, is gland, adrenal by the produced peripheral in to cortisone inactivated and cortisone Both cortisol tissues. with in patients are increased levels but reduced syndrome Cushing's exogenous receiving in patients In prednisone). (eg, glucocorticoid excess mineralocorticoid apparent is formation cortisone (AME), as act cortisol to allowing reduced, with Patients a mineralocorticoid. and cortisone have reduced AME cortisol but normal levels aldosterone levels. can inhibit ingestion Licorice to cortisone, of cortisol conversion circulating decreasing thereby levels. cortisone Method • • • Specimen Requirements serum refrigerated 1 mL minimum 0.3 mL top preferred red No additive red top acceptable SST preferred fasting Overnight was specimen time of day Specify collected. 59 1980;14:39-46. AME Exogenous Cushing’s Cushing’s Exogenous Adrenal insufficiency Adrenal Cushing’s syndrome Cushing’s (prednisone) • • • • Additional marker for exogenous for exogenous marker Additional use steroid Marker for AME Marker Evaluate adrenal function adrenal Evaluate AMPM (birth) infants Full-term 7 d 2.6-15.6 mo2 wk-3 y3 mo-1 y1-17 1.2-3.5 0.6-2.8 0.9-5.4 0.3-4.5 0.7-4.6 0.6-3.0 Pediatric data from Sippell WG, et al. et Sippell WG, from data Pediatric Pediatr Res. Children (AM) Children Adults μg/dL Interpretive Information Interpretive Reference Range Reference Clinical Use Cortisone, Serum Cortisone, • • • C-Peptide, Serum or Urine 372X. 4643X, 11182X
Clinical Use Clinical Background • Assess pancreatic islet cell function C-peptide is a 31-amino acid that connects insulin's 2 peptide chains; Reference Range it has no known biologic activity. Serum (372X) Because of differences in half-life and C-Peptide ng/mL hepatic clearance, peripheral blood Adults 0.8-3.1 levels of C-peptide and insulin are no longer equimolar but still remain Urine 24-h (4643X) highly correlated under resting and C-Peptide μg/g creat perturbatory conditions. Adults 10.8-107.9 Measurement of C-peptide is useful Creatinine g/24-h for distinguishing 1) insulin-secreting 3-8 y 0.11-0.68 tumors (insulinoma) from exogenous 9-12 y 0.17-1.41 insulin administration as a cause of 13-17 y 0.29-1.87 hypoglycemia (commercial insulin Adults 0.63-2.50 does not contain C-peptide); and 2) type 1 from type 2 diabetes mellitus Urine 2nd void (11182X) (type 2 is associated with abundant C-Peptide μg/g creat C-peptide secretion; type 1 has little Men 5-79 or no C-peptide secretion). Women 7-142 Creatinine mg/dL Method 0-6 mo 2-32 • Immunochemiluminometric assay 7-11 mo 2-36 (ICMA) 1-2 y 2-128 • Analytical sensitivity: 0.5 ng/mL 3-8 y 2-149 (serum) and 0.5 μg/mL (urine) 9-12 y 2-183 Specimen Requirements >12 y Serum (372X) Males 20-370 2 mL frozen serum Females 20-320 0.4 mL minimum Interpretive Information No additive red top preferred • Insulinoma SST red top acceptable • Type 2 diabetes Overnight fasting is required. Urine 24-h (4643X) • Type 1 diabetes 2 mL frozen aliquot of a 24-h urine • Exogenous insulin 0.5 mL minimum administration Do not use a preservative. Record 24-h volume on vial and request form. Urine 2nd Void (11182X) 2 mL frozen aliquot of a 2nd void urine; 1 mL minimum
60 Alphabetical Test Section 225X 37555X, 242X, 37555X, Extraction, radioimmunoassay Extraction, Clinical Background monophosphate adenosine 5´ cyclic 3´, second is an intracellular (cAMP) action of the by produced messenger Significant ATP. cyclase upon adenyl appear cAMP of intracellular amounts peak diurnal rhythm with a in plasma late a nadir in hours and at 1200 of urinary half Approximately evening. the while from plasma is filtered cAMP from kidney half is derived other kidney is generated cAMP in the tissue. (PTH) parathyroid hormone following PTH receptors. of renal stimulation Urinary generally elevated cAMP is hyperparathyroidism. with in patients cancer in also be increased It may Some with hypercalcemia. patients with pseudohypopara- patients characterized a condition thyroidism, to responsiveness renal by decreased respond fail to hormone, parathyroid a rise in urinary when with cAMP Measurement is given. PTH exogenous still be cAMP may of nephrogenous to diagnose on rare occasions used of pseudohypopara- subtypes thyroidism. Method • Specimen Requirements (37555X) Nephrogenous frozen and 5 mL frozen urine 25 mL and 1.2 mL 2.5 mL plasma; EDTA respectively minimum, to HCl of 6N 2.0 mL with urine Collect 3. pH below maintain (242X) Plasma plasma frozen EDTA 3.1 mL minimum 1 mL (225X) urine Random frozen urine 1 mL minimum 0.2 mL 6N HCl 2.0 mL of urine with Collect below 3. pH to maintain 61 mg/dL mg/dL 37555X) ( 0.8-7.5 μmol/L 3.9-13.1 nmol/L 1.4-5.0 nmol/dL 1990;27:99-101. (37555X) Renal insufficiency Renal Type I pseudohypopara- Type Hypoparathyroidism Hypercalcemia of malignancy Hypercalcemia Hyperparathyroidism thyroidism • • • • • MalesFemales 20--370 20-320 Assess severity of hypoparathyroidism severity Assess Diagnose pseudohypoparathyroidism pseudohypoparathyroidism Diagnose subtypes Diagnose hyperparathyroidism Diagnose 0-6 mo mo7-11 1-2 y3-8 y y9-12 y >12 2-32 2-36 2-128 2-149 2-183 0-2 d3-5 d6-7 d mo1 wk-1 y1 mo-1 2-9 y y10-17 0.7-1.4 0.4-1.1 0.3-0.8 0.3-0.9 0.2-0.7 0.3-0.7 0.5-1.0 *Values for children <2 y from Savory DJ, SavoryDJ, y from <2 children for *Values Ann Clin Biochem. Nephrogenous (242X, 37555X) Plasma 37555X) (225X, urine Random (37555X) Plasma Creatinine, Creatinine, random urine Creatinine, Adults infants* full-term Children, 0.6-1.4 Interpretive Information Interpretive Reference Range Reference Clinical Use Cyclic Adenosine Monophosphate Adenosine Cyclic AMP) (Cyclic • • • Cystatin C 10570X
Clinical Use Clinical Background • Assess glomerular filtration rate in Cystatin C is a low molecular weight type 1 and 2 diabetes mellitus (13,359 Da) protein belonging to the superfamily of cystine protease Reference Range inhibitors. It is produced by all Children & Adults 0.5-1.0 mg/L nucleated cells at a reasonably constant rate; production is minimally affected Interpretive Information by diet, inflammatory states, lean body • Impaired glomerular filtration mass, or circadian rhythm. Like creatinine, cystatin C is freely filtered by the glomeruli. It is reabsorbed and metabolized by renal tubular cells and does not appear in urine. Tan et al (Diabetes Care. 2002;25:2004-2009) showed serum cystatin C has good correlation with creatinine clearance (p = 0.74) and iohexol clearance (p = -0.80) and is a useful marker of renal dysfunction in diabetic patients with minimal tubular reabsorption. Because creatinine production is relatively variable, cystatin C has been proposed as a better marker of glomerular filtration.
Method • Nephelometry
Specimen Requirements 1.0 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable Overnight fasting is required.
62 Alphabetical Test Section 6559X Extraction, chromatography, chromatography, Extraction, (RIA) radioimmunoassay 1 ng/dL sensitivity: Analytical Clinical Background is a weak (DOC) Deoxycorticosterone from derived mineralocorticoid in of progesterone 21-hydroxylation is the adrenal cortex. DOC a precursor and aldosterone of corticosterone primary is under the like cortisol, and, used can be DOC of ACTH. control a replacement as therapeutically with in patients mineralocorticoid or primary insufficiency adrenal hypoaldosteronism. isolated Method • • Specimen Requirements serum refrigerated 3 mL minimum 1.1 mL top preferred red No additive red top acceptable SST 63 14-33 ng/dL ng/dL 60 min ng/dL Baseline J Clin EndocrinolMetab. deficiency) deficiency (aldosterone ase c11 c17 c11 Adrenal carcinoma (some cases) (some carcinoma Adrenal Adrenal insufficiency Adrenal hypoplasia Cushing’s syndrome Cushing’s Primary aldosteronism renin essential hypertension Low P-450 P-450 Congenital adrenal hyperplasia synthetase) deficiency (P-450 • • • • • • • • • First trimesterFirst trimesterSecond trimesterThird 10-75 5-25 30-110 MalesFemales 5-14 5-10 19-46 23-40 MalesFemales 4-30 2-12 12-74 13-63 MalesFemales 9-34 2-13 33-140 19-61 Diagnoseadrenal enzyme deficiencies functional adrenal Assess disorders Follicular phaseFollicular phaseLuteal Pregnancy 1.5-8.5 3.5-13.0 IV-V Tanner Tanner II-III Tanner Men and premeno- Men women pausal phase) (follicular <1 y1-5 y y 6-12 7-57 4-49 20-160 26-140 Pediatric dataPediatric from 1991;73:674-686. ACTH ACTH stimulation MenWomen 3.5-11.5 Interpretive Information Interpretive Reference Range Reference Clinical Use Deoxycorticosterone (DOC) Deoxycorticosterone • • 11-Deoxycortisol, LC/MS/MS 30543X
Clinical Use Clinical Background • Marker for adrenal P-450c11 (11β- Cortisol is formed in the adrenal hydroxylase) enzyme deficiency gland by the enzymatic action of • Assess pituitary-adrenal reserve with 11β-hydroxylase (P-450c11) on the metyrapone test 11-deoxycortisol. Deficiency of this enzyme causes congenital adrenal Reference Range hyperplasia (CAH) characterized by Men ng/dL hypertension and virilization. ACTH, 18-29 y <119 11-deoxycortisol, and androgen levels 30-39 y <135 are all increased. This defect accounts 40-49 y <76 for about 5% of CAH cases. 50-59 y <42 The drug metyrapone blocks Women 11β-hydroxylase, which in normal 18-29 y <107 individuals results in increased ACTH 30-39 y <51 and 11-deoxycortisol production. Thus, the drug is used to test adrenal 40-49 y <62 and pituitary reserve. In patients 50-66 y <37 with primary and secondary adrenal Children insufficiency, there is little or no Premature infants <235 increase in 11-deoxycortisol in (31-35 wk)* response to metyrapone administra- Term infants* <170 tion. In patients with Cushing's 1-12 mo 10-200 syndrome caused by ectopic ACTH or 1-4 y 7-210 adrenal tumors, metyrapone does not 5-9 y <122 change ACTH or 11-deoxycortisol 10-13 y <245 levels (see Dynamic Test section). 14-17 y <302 Measurements of cortisol and ACTH Tanner II-III have generally replaced the metyrapone test. Males 11-150 Females 15-130 Method Tanner IV-V • Liquid chromatography, tandem Males 14-120 mass spectrometry (LC/MS/MS) Females 17-120 • Analytical sensitivity: 20.0 ng/dL *Data from J Clin Endocrinol Metab. 1991; 73:674-686 and J Clin Endocrinol Metab. 1989; 69:1133-1136. Specimen Requirements 1 mL frozen serum (no additive red Marked elevation of 21-deoxycortisol, top tube); 0.25 mL minimum as may occur in 21-hydroxylase defi- ciency, can increase apparent levels of SST red top unacceptable. 11-deoxycortisol. An early-morning specimen is Interpretive Information preferred. • Congenital adrenal hyperplasia (P-450c11 deficiency) • After metyrapone in normal subjects • Adrenal insufficiency
64 Alphabetical Test Section 29497X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 20 nmol/L sensitivity: Analytical also concentration Creatinine reported Clinical Background by together are linked fibers Collagen to as referred molecules interchain Collagen cross-links. pyridinium been have cross-links pyridinium tissue connective in all identified is broken skin. As collagen except breakdown small collagenase, by down with the urine in excreted are products including cross-links, attached deoxypyridinoline and pyridinoline is constantly Bone collagen (DPD). Thus, is rich in DPD. over and turning bone for marker DPD is a useful urine and bone degradation matrix resorption. Method • • • Specimen Requirements urine refrigerated 3 mL minimum 1 mL prior to preferred, morning void 2nd 10 AM. use preservatives.Do not 65 mg/dL Relative to baseline indicates indicates to baseline Relative Paget's disease Paget's Primary hyperparathyroidism Osteoporosis therapeutic response therapeutic • • • • Monitor therapy Monitor Assess rate of bone collagen collagen of bone rate Assess degradation MalesFemales 20-370 20-320 MenWomen3-9 y y10-13 y14-19 3.3-13.5 2.1-8.1 13.0-41.9 11.8-35.1 6.8-27.7 *Test results within the premenopausal premenopausal the within results *Test out rule not do range reference osteoporosis or the need for therapy. 0-6 mo mo7-11 1-2 y3-8 y y9-12 y >12 2-32 2-36 2-128 2-149 2-183 Children Creatinine DPD, Free DPD, y)*Adults(20-58 creat nmol/mmol Interpretive Information Interpretive Reference Range Reference Clinical Use Deoxypyridinoline (DPD, DPYD), (DPD, Deoxypyridinoline Free • • Dexamethasone 29391X
Clinical Use Clinical Background • Monitor therapy or compliance Dexamethasone is a potent synthetic • Evaluate hypothalamic-pituitary- glucocorticoid widely used in adrenal axis medicine. The dexamethasone suppression test (DST) is a standard Reference Range diagnostic tool for Cushing’s disease ng/dL and has been applied to the evaluation of hypothalamic-pituitary-adrenal axis Baseline <20 function in patients with psychiatric 8-10 AM, after 1 mg 180-550 disease. The measurement of overnight dexamethasone dexamethasone in serum allows the physician to assess compliance Interpretive Information during therapy or when evaluating • Dexamethasone administration DST results.
Method • Liquid chromatography, tandem mass spectrometry (LC/MS/MS) • Analytical sensitivity: 20 ng/dL
Specimen Requirements 0.85 mL refrigerated serum (no additive red top tube); 0.25 mL minimum SST red top unacceptable Collect specimen between 8 and 10 AM.
66 Alphabetical Test Section 410X 38954X Extraction, chromatography, chromatography, Extraction, (RIA) radioimmunoassay 3 ng/dL sensitivity: Analytical mass Gas chromatography, spectrophotometry (GC/MS) Clinical Background is a (DHEA) Dehydroepiandrosterone by the synthesized androgen weak half-life It has a short cortex. adrenal dehydro- to is usually converted and sulfate. epiandrosterone secretion can produce Excessive DHEA via and virilization hirsutism, acne, to testosterone. conversion Method Serum (410X) • • (38954X) Urine • Specimen Requirements Serum (410X) serum refrigerated 1 mL minimum 0.3 mL top preferred red No additive red top acceptable SST is preferred. fasting Overnight request sex on test age and Specify form. (38954X) Urine urine of a 24-h frozen aliquot 5 mL minimum 2.1 mL do not collection; during Refrigerate use a preservative. volume Record 24-h form. and request on vial 67 761 3343 < < ng/dL ng/dL 60 min 60 ng/dL J Clin Endocrinol Metab. μg/24-h μg/g/creat Baseline Anorexia nervosaAnorexia Addison’s disease Addison’s Premature adrenarche Premature Congenital adrenal hyperplasia adrenal Congenital Cushing’s disease Cushing’s Adrenal tumors Adrenal • • • • • • MalesFemales 100-400 165-690 325-1460 195-510 MalesFemales 25-300 69-605 62-390 95-885 <1 y1-5 y y6-12 26-585 11-155 9-42 18-1455 34-320 21-98 ACTH ACTH stimulation Assess adrenal reserve after ACTH reserve adrenal Assess ACTH after stimulation Marker of adrenal androgen androgen of adrenal Marker production wk infants (1st Term of life) Pregnancy and womenMen infants Premature wk) (31-35 545-1845 135-810 3-8 y y9-12 y13-17 Adults 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 Tanner IV-V Tanner Tanner II-III Tanner *Pediatric data from 1991;73:674-686. Post-ACTH stimulationPost-ACTH Children* min 60 Serum (410X) MenWomen 180-1250 130-980 Urine (38954X) Urine AdultsDHEA, MenDHEA, 21-2710 WomenDHEA, Creatinine 24-1640 13-730 g/24-h Interpretive Information Interpretive Reference Range Reference Clinical Use DHEA (Dehydroepiandrosterone) • • DHEA Sulfate 402X
Clinical Use Clinical Background • Marker for adrenal cortical function Dehydroepiandrosterone sulfate and disease (DHEA-S), which is synthesized almost Reference Range exclusively by the adrenals, is a weak androgen, the most abundant C19 Men μg/dL μg/dL plasma steroid, and the major source 18-29 y 110-510 of urinary 17-ketosteroids. Measuring 30-39 y 110-370 serum DHEA-S, therefore, can replace 40-49 y 45-345 older urinary 17-ketosteroid deter- 50-59 y 25-240 minations. 60-69 y 25-95 DHEA-S levels are markedly increased 70-90 y <75 in patients with congenital adrenal Women hyperplasia or adrenal carcinoma. 18-29 y 45-320 Moderate increases are seen in the 30-39 y 40-325 majority of patients with pituitary- 40-49 y 25-220 dependent Cushing's disease, whereas 50-59 y 15-170 low or normal levels are usually exhibited by patients with Cushing's 60-69 y <185 syndrome due to an adrenal adenoma. 70-90 y <90 Adrenal cancer is typically associated Children Male Female with very high levels of DHEA-S. Since 0-1 mo* <316 15-261 DHEA-S levels reflect adrenal 1-6 mo* <58 <74 androgen production, the measure- 7-12 mo* <26 <26 ment of serum concentrations serves 1-3 y* <15 <22 as an early indicator for the onset of 4-6 y* <27 <34 adrenarche. 7-9 y <91 <92 Serum DHEA-S measurements are 10-13 y <138 <148 most commonly employed in the 14-17 y 38-340 37-307 differential diagnosis of a virilized patient. In patients with virilizing Tanner stages tumors, DHEA-S levels usually exceed I<89 <46 7,000 μg/dL. II <81 15-113 III 22-126 42-162 Method IV 33-177 42-241 • Immunochemiluminometric assay V† 110-510 45-320 (ICMA) *From Clin Chem. 1993;39(6):1171 [abstract • Analytical sensitivity: 15 μg/dL 0243]. † Same as adults aged 18-29 years. Specimen Requirements Interpretive Information 1 mL refrigerated serum • Congenital adrenal hyperplasia 0.5 mL minimum • Adrenal carcinoma No additive red top preferred • Virilizing tumors of the adrenals SST red top acceptable • Cushing’s disease, pituitary dependent Specify age and sex on test request form. • Addison’s disease • Adrenal hypoplasia
68 Alphabetical Test Section 204X - reductase. Small amounts of DHT of DHT Small amounts - reductase. Extraction, chromatography, chromatography, Extraction, (RIA) radioimmunoassay 3 ng/dL sensitivity: Analytical α Clinical Background is a potent (DHT) Dihydrotestosterone the from derived largely androgen of testos- conversion tissue peripheral by the enzyme catalyzed terone, 5 has testes. DHT by the are secreted DHT properties. androgenic potent from in females is produced also androstenediol. and testosterone activity exerts its androgenic DHT receptors to testosterone by binding is an tissues. It target in selective for androgen bioactive important development, external genitalia male and skin metabolism. growth, prostate agent in prostate is a causal DHT in measurements and hyperplasia, com- to assess can be used blood of to inhibitors and response pliance conversion. testosterone-to-DHT Method • • Specimen Requirements serum refrigerated 4 mL minimum 1.1 mL top preferred red No additive red top acceptable SST request sex on test age and Specify form. 69 -reductase -reductase α ng/dL ng/dL J Clin Endocrinol Metab. -reductase activity -reductase -reductase deficiency -reductase α α -reductase deficiency -reductase α High 5 High Chronic anovulatoryChronic syndrome Hirsutism 5 5 that reduce Drugs Hypogonadism activity (finasteride, etc) (finasteride, activity • • • • • • -reductase α Diagnose 5 Diagnose inhibit of drugs that efficacy Assess 5 Assess pseudohermaphroditism Assess Includes data from data Includes 1979;48:821-826. MenWomenChildren bloodCord 1-6 moPrepubertal II-IIITanner Males IV-VTanner <2-8 Females 12-85 <2-5 <5 3-33 25-75 5-30 22-75 5-19 <5 <5 3-30 Interpretive Information Interpretive Reference Range Reference Clinical Use Dihydrotestosterone (DHT) Dihydrotestosterone • • • Dihydrotestosterone, Free 36168X
Clinical Use Clinical Background • Assess pseudohermaphroditism Most dihydrotestosterone (DHT) is • Diagnose 5α-reductase deficiency transported in the circulation bound • Assess efficacy of drugs that inhibit to sex hormone binding globulin 5α-reductase (SHBG). Approximately 1%, however, circulates unbound. Blood levels are Reference Range dependent on rates of production, metabolic clearance, and binding DHT, Total ng/dL protein concentration. Because SHBG Men 25-75 levels are altered by medications, Women 5-30 disease, and sex steroids, as well as DHT, Free pg/mL insulin, measurement of free DHT Men 1.0-6.2 more accurately reflects the level of Women 0.3-1.9 bioactive DHT than measurements of total serum DHT. % DHT, Free % Men 0.62-1.10 Method Women 0.47-0.68 • Equilibrium dialysis, extraction, chromatography, radioimmunoassay Interpretive Information (RIA) • Hirsutism Specimen Requirements • Chronic anovulation 5 mL refrigerated serum (no additive • High 5α--reductase activity red top tube); 2.5 mL minimum • Hypogonadism • 5α-reductase deficiency • Drugs inhibiting 5α--reductase
70 Alphabetical Test Section 36169X Liquid chromatography tandem tandem chromatography Liquid spectrometrymass (LC/MS/MS) 2 pg/mL sensitivity: Analytical dialysis Equilibrium Clinical Background of circulating 95% than More are estradiol, including estrogens, such proteins by serum binding bound binding protein hormone as sex binding corticosteroid (SHBG), The and albumin. (CGB), protein is is SHBG, which important most and binds steroid by the liver produced Estrogens affinity. high with hormones SHBG increase thyroid hormones and while androgens concentrations, SHBG hepatic production. reduce and by disease also are altered levels also concentrations, CBG medications. by are influenced origin, of hepatic and medications. disease, hormones, concentration estradiol Dialyzable true hormone reliably reflects most status. Method Estradiol • • Free Estradiol, • Specimen Requirements 1.0 mL refrigerated serum (no additive mL minimum tube); 0.5 red top red top unacceptable SST 71 % 29 10 0.38 0.45 pg/mL Postmenopause Ovarian failure Ovarian Liver disease Liver Male hypogonadism hypogonadism Male Estrogen secreting tumors secreting Estrogen (gynecomastia) • • • • • Assess estrogen status in males and males status in estrogen Assess females Follicular peakMidcycle LutealPostmenopausal 0.36-2.34 0.65-1.79 0.79-1.64 0.56-1.87 Follicular peakMidcycle LutealPostmenopausal 0.72-5.89 0.43-5.03 < 0.40-5.55 Follicular peakMidcycle LutealPostmenopausal 94-762 39-375 < 48-440 Estradiol Men < Estradiol, Free Estradiol, MenWomen 1.25-1.85 Women Women Free Estradiol, Men < Interpretive Information Interpretive Reference Range Reference Clinical Use Estradiol, Free Estradiol, • Estradiol, Ultra Sensitive 30289X
Clinical Use Clinical Background • Determine estrogen status in women Estradiol-17β (E2) is the major • Monitor follicular development bioactive estrogen produced in the during induction of ovulation ovary. E2 is also produced by the • Assess estrogen production in males adrenal glands, and in males by the testes, as well as by peripheral Reference Range conversion from testosterone. pg/mL pg/mL Serum E2 is measured to determine Men <29 the estrogen status of women, such as Women in some cases of amenorrhea, and as a guide to monitoring follicular Follicular phase 39-375 development during induction of Midcycle peak 94-762 ovulation. Luteal phase 48-440 The assay has high sensitivity and is Postmenopausal <10 therefore well suited for measurements Children Males Females in children and for diagnosing Pre-pubertal (1-9 y) <4<16 menopause. 10-11 y <12 <65 Method 12-14 y <24 <142 15-17 y <31 <283 • Liquid chromatography tandem mass spectrometry (LC/MS/MS) Interpretive Information • Analytical sensitivity: 2 pg/mL • Ovarian tumors Specimen Requirements • Adrenal feminizing tumors 0.5 mL refrigerated serum(no additive • Precocious puberty (female) red top tube); 0.2 mL minimum • Liver disease SST red top unacceptable • Male gynecomastia Specify age and sex on test request form. • Ovarian failure • Oral contraceptives
72 Alphabetical Test Section 439X Extraction, radioimmunoassay (RIA) radioimmunoassay Extraction, 25 pg/mL sensitivity: Analytical Clinical Background steroids are 18-carbon Estrogens glands, adrenal by the gonads, secreted estrogens principal The placenta. and and estrone (E1), (E2), are estradiol estrogen is the major (E3). E2 estriol is derived ovaries. E1 by the produced peripheral and ovarian secretion from adrenal gonadal and from conversion in E3 is produced androstenedione. E2. from by metabolism the liver these all of placenta produces The E1 measures This assay estrogens. and E2. Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST 73 130 130 pg/mL Oral contraceptives Oral Ovarian failure Ovarian Male gynecomastia Male Precocious puberty (female) puberty Precocious Adrenal feminizing tumors feminizing Adrenal Ovarian tumors Ovarian • • • • • • Screen for estrogen-producing for estrogen-producing Screen tumors Assess estrogen production in males production estrogen Assess Determine estrogen status in women status estrogen Determine Early follicularEarly follicularLate LutealPostmenopausal 70-400 100-900 < 70-700 Males < Females for recommended assay is not This children. in pre-pubertal use Interpretive Information Interpretive Reference Range Reference Clinical Use Estrogen, Total, Serum Total, Estrogen, • • • Estrogens, Fractionated, Serum 36742X
Clinical Use Clinical Background • Assess estrogen status Estrogens are 18-carbon steroids Reference Range secreted by the gonads, adrenal glands, and placenta. The principle estrogens pg/mL pg/mL are estradiol (E2), estrone (E1), and Estrone (E1) estriol (E3). E2 is the major estrogen Men <68 Women produced by the ovaries. E1 is derived Follicular 10-138 from peripheral conversion from Midcycle 49-268 gonadal and adrenal androstenedione. Luteal 16-173 E3 is the major placental estrogen. The Postmenopausal <65 major circulating estrogens in non- Children Males Females pregnant women are E2 and E1. Pre-pubertal (1-9 y) <10 <34 10-11 y <12 <72 Method 12-14 y <28 <75 Estrone, Estradiol 15-17 y <64 <188 • Liquid chromatography tandem Estradiol (E2) mass spectrometry (LC/MS/MS) Men <29 Estriol Women Follicular 39-375 • Extraction, chromatography, Midcycle 94-762 radioimmunoassay (RIA) Luteal 48-440 Postmenopausal <10 Specimen Requirements Children Males Females 3 mL refrigerated serum (no additive Pre-pubertal (1-9 y) <4<16 red top tube); 0.8 mL minimum 10-11 y <12 <65 SST red top unacceptable 12-14 y <24 <142 15-17 y <31 <283 Estriol (E3) ng/mL Men <0.2 Women Non-pregnant <0.1 Weeks gestation 6-16.5 0.1-1.60 17.0-24.0 1.5-5.8 25.0-32.5 2.7-9.0 33.0-40.0 7.2-28.0
Interpretive Information • Ovarian tumors • Feminizing adrenal tumors • Precocious puberty
• Ovarian failure • Postmenopausal • Gonadotropin deficiency
74 Alphabetical Test Section 23244X Liquid chromatography tandem tandem chromatography Liquid spectrometrymass (LC/MS/MS) 10 pg/mL sensitivity: Analytical Clinical Background ovarian of 2 major (E1) is 1 Estrone women in nonpregnant estrogens by produced estrogen a major and primary The the placenta. ovarian (E2). Circulating is estradiol estrogen ovarian secretion derived from E1 is from and peripheral conversion androstenedione. adrenal and gonadal than potency lower estrogenic E1 has to glucuronide is conjugated E2. E1 in liver and excreted by the or sulfate extragonadal and bile. The urine to E1, of androstenedione conversion occurs aromatase, by P-450 catalyzed E1 tends thus, in lipid tissue; largely in obese subjects. increased to be the major is conversion Extragonadal in estrogens of circulating source change women. A postmenopausal is a ratio estradiol/estrone in the and senescence of ovarian reflection menopause. of development Method • • Specimen Requirements 0.5 mL refrigerated serum (no additive mL minimum tube); 0.25 red top red top unacceptable SST request sex on test age and Specify form. 75 34 72 75 68 65 < 188 Females 1228 < 64 < < pg/mL pg/mL Hypogonadism Luteal phase of menstrual cycle menstrual phase of Luteal Aging and obesity Aging Postmenopause (by peripheral (by peripheral Postmenopause conversion) Pregnancy • • • • • 12-14 y12-14 y15-17 < < FollicularMidcycleLutealPostmenopausal 10-138 49-268 < 16-173 Pre-pubertal (1-9 y) (1-9 Pre-pubertal y10-11 <10 < Assess placental estrogen production placental Assess Assess estrogen status in post- status in estrogen Assess women menopausal Women Children Males Men < Interpretive Information Interpretive Reference Range Reference Clinical Use Estrone, Serum Estrone, • • Estrone Sulfate 37104X
Clinical Use Clinical Background • Monitor estrogen replacement Estrone sulfate (E1S) is the most therapy abundant estrogen in peripheral blood. The primary source is the Reference Range liver, where it is formed as the pg/mL primary product of estradiol and Men 230-2200 estrone metabolism. E1S also is the Women major estrogen secreted by the Follicular phase 300-2600 adrenal cortex. It is a weak estrogen Luteal phase 100-3200 but can be converted in the body to Postmenopausal 100-1300 estradiol. E1S is a major component of the drug Premarin®’. Interpretive Information • Pregnancy Method • Ovarian tumors • Hydrolysis, extraction, chroma- tography, radioimmunoassay (RIA) • Adrenal tumors • Analytical sensitivity: 40 pg/mL • Hypogonadism Specimen Requirements 2 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable
76 Alphabetical Test Section 8340X Colorimetry 5.0 μmol/L sensitivity: Analytical Clinical Background glycoproteins forms stable Glucose by a proteins plasma many with glyco- The reaction. nonenzymatic primarily serum proteins, sylated 1 to of short half-life have a albumin, determination Fructosamine 2 weeks. glyco- of on measurement is based therefore is and proteins serum sylated short-term for monitoring useful the (eg, determining control glycemic less in in therapy of a change success with glycated than is possible time used can also be It hemoglobin). with conjunction or in separately glycosylated glucose and/or blood management, in diabetes hemoglobin in pregnancy-associated especially need for there is a in which diabetes sugar of blood monitoring careful control. do hemoglobins abnormal Since not interfere the fructosamine with used can also be it determination, with abnormal patients to monitor anemia) sickle cell (eg, hemoglobin, and gestational diabetes. Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST 77
1986;
1988;34:
Ann Clin Biochem. Arch Dis Child. Arch Clin Chem. Clin Relatively, with improved improved with Relatively, Hyperglycemia Uncontrolled diabetes Uncontrolled diabetic control diabetic •• • • Manage patients with gestational with gestational patients Manage diabetes Monitor glucose control (short-term, control (short-term, glucose Monitor diabetes with in patients weeks) 1-2 >5 y adults Similar to 1989;26:328-331; 2444-2447. 61:113-117; and Pediatric data from AdultsChildren 190-270 μmol/L Interpretive Information Interpretive Reference Range Reference Clinical Use Fructosamine • • FSH (Follicle Stimulating Hormone) 470X
Clinical Use Clinical Background • Diagnose gonadal function disorders Follicle stimulating hormone (FSH, follitropin) is a glycoprotein produced Reference Range by the anterior pituitary gland. Pro- IU/L duction is regulated by hypothalamic Men 1.6-8.0 gonadotropin releasing hormone Women (GnRH) and feedback from gonadal Follicular phase 2.5-10.2 steroid hormones. In the female, FSH stimulates follicular growth, prepares Midcycle 3.1-17.7 ovarian follicles for luteinizing Luteal phase 1.5-9.1 hormone (LH) activity, and enhances Postmenopausal 23.0-116.3 the LH-induced release of estrogen. Pediatric After menopause, decreased ovarian Use test code 36087X estradiol secretion results in increased FSH and LH levels. In the male, FSH Interpretive Information stimulates seminiferous tubule and • Primary hypogonadism testicular growth and is involved in • Gonadotropin-secreting the early stages of spermatogenesis. pituitary tumors Primary testicular failure results in increased FSH and LH levels. • Menopause In patients with testicular or ovarian • Hypothalamic GnRH deficiency disorders, low levels of serum FSH are indicative of pituitary or hypothalamic • Pituitary FSH deficiency dysfunction. Assays for both FSH and • Ectopic steroid hormone LH are useful in the diagnosis, production management, and treatment of infertility in both genders.
Method • Immunochemiluminometric assay (ICMA), Centaur • Analytical sensitivity: 0.07 IU/L
Specimen Requirements 1 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable
78 Alphabetical Test Section 36087X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 0.05 IU/L sensitivity: Analytical Clinical Background (FSH, hormone stimulating Follicle anterior the by is produced follitropin) by pituitary stimulation gland after releasing gonadotropin hypothalamic FSH stimulates (GnRH). hormone produc- and sperm inhibin testicular and ovarian inhibin in males and tion in females. GnRH estrogen production by are regulated FSH production and whereby low feedback systems, negative stimulate hormones of gonadal levels FSH circulating high levels inhibit and levels. FSH is secreted transiently during pulse the GnRH after which infancy, secretion controlling generator is Secretion quiescent. becomes the pubescence, during reactivated FSH is increased sign of which first Puberty at night. LH secretion and secretion as gonadotropin progresses gonadal and increasing increases, with associated is responsiveness sex in circulating increases sustained levels. hormone Method • • Specimen Requirements serum refrigerated 0.5 mL minimum 0.3 mL top preferred red No additive red top acceptable SST 79 Pediatric Pediatric 2nd ed. Baltimore, MD: Baltimore, 2nd ed. Hyperprolactinemia Isolated gonadotropin gonadotropin Isolated Isolated FSH deficiency Isolated Pituitary insufficiency Hypothalamic GnRH Hypothalamic deficiency Delayed puberty Delayed Germinal cell aplasia Germinal Menopause Gonadotropin-secreting Gonadotropin-secreting Primary hypogonadism Precocious puberty Precocious deficiency pituitary tumors • • • • • • • • • • • Monitor FSH suppressive therapy FSH suppressive Monitor Diagnose (including differential differential (including Diagnose dysfunction gonadal diagnosis) Diagnose and manage pubertal pubertal and manage Diagnose disorders 0-9 y y10-13 y14-17 0-8 y y9-13 y14-17 0.3-4.0 <3.0 0.4-7.4 0.5-4.5 0.4-6.5 0.8-8.5 In male infants, FSH peaks (typically FSH peaks infants, In male at this assay) IU/L using 3.0-6.0 pre- falling to of age, 4 months age. In by 1 year of levels pubertal (as high FSH peaks infants, female at this assay) IU/L using as 30.0 to falling slowly of age, 3 months of by 1-2 years levels prepubertal JR. Ducharme (Forest MG, age gonadal hormones. and Gonadotropic In: Bertrand J, Rappaport R, eds. PC, Sizonenko Endocrinology. Williams & Wilkins; 1993:100-120). & Wilkins; Williams Females Males IU/L Interpretive Information Interpretive Reference Range Reference Clinical Use FSH (Follicle Stimulating Hormone), (Follicle FSH Pediatrics • • • Gastric Parietal Cell Antibody 15114X
Clinical Use Clinical Background • Differential diagnosis of anemia Gastric parietal cell antibodies are • Assess atrophic gastritis directed primarily against the gastric • Evaluate polyglandular autoimmune proton pump, H/K ATPase. The syndromes antibody is detected in approximately 90% of patients with pernicious Reference Range anemia and the underlying type A chronic atrophic gastritis, in 20% to Units 30% of patients with varied auto- Negative <20 immune endocrine disorders, and in Equivocal 20.1-24.9 2.5% to 10% (varies with age) of the Positive >25 healthy population. Interpretive Information Approximately 50% of patients with pernicious anemia have thyroid auto- • Atrophic gastritis antibodies, and 30% of patients with • Pernicious anemia autoimmune thyroid disease have PCA. • Polyglandular autoimmune disease Method • Enzyme linked immunosorbent assay (ELISA) • Analytical sensitivity: 20 Units
Specimen Requirements 0.5 mL refrigerated serum 0.1 mL minimum No additive red top preferred SST red top acceptable
80 Alphabetical Test Section 478X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 10 pg/mL sensitivity: Analytical gastrin detects specificity: Analytical big reactivity with 7% cross (G17); react not cross (G34); does gastrin with G13. Clinical Background hormone is a polypeptide Gastrin gastric the cells of by the G secreted duodenal proximal and mucosa antral of potent stimulator Gastrin is a cells. has and also acid secretion, antral cells, the parietal actions on trophic cells, and enterochromaffin-like chief Gastrin mucosa. in the oxyntic cells food intake increase with levels age. acids) and amino (protein, acid with gastric decrease Levels secretion. (ZE syndrome In Zollinger-Ellison neoplasm a pancreatic syndrome), gastrin, of large amounts produces gastric excess in turn stimulates which ulcer disease. peptic and acid secretion non- but of pathological, A variety gastric in which disorders tumorous, to low also lead secretion is acid these gastrin; in blood elevations anemia pernicious include disorders with anacidity. atrophic gastritis and tests or calcium-stimulation Secretin- tumors in differentiating can assist antrum by the gastrin secreted from slight or moderate with in patients hypergastrinemia. Method • • • Specimen Requirements frozen serum 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST is required. fasting Overnight 81 100 pg/mL , and pepsid and pepsid , ®’ , Prilosec . 2000;79:379-411.) ®’ Atrophic gastritis with anacidity gastritis Atrophic Pernicious anemia Pernicious Gastrinoma • • • Differential diagnosis of peptic ulcer peptic of diagnosis Differential tumors and gastrin-secreting disease Diagnose gastrinoma Diagnose 5-7 y 13-64 interfere production. with gastrin Adults < Zollinger-Ellison syndrome: gastrin gastrin syndrome: Zollinger-Ellison et al. (Roy PK, >200 pg/mL levels Medicine Zantac Children Interpretive Information Interpretive Reference Range Reference Clinical Use Gastrin • • Glucagon 519X
Clinical Use Clinical Background • Diagnose glucagonoma Glucagon is produced by alpha cells • Diagnose glucagon deficiency in of the pancreatic islets. Like insulin, patients with hypoglycemia glucagon secretion is regulated by blood glucose concentrations; Reference Range however, regulation is reciprocal in pg/mL that high glucose levels stimulate Adults <60 insulin and inhibit glucagon release. Conversely, low insulin levels stimulate Children glucagon secretion. Glucagon secre- Cord blood <215 tion also is stimulated by a high- Day 1 <240 protein meal. Day 2 <400 Glucagon acts to increase blood sugar Day 3 <420 by stimulating hepatic glycogenolysis Days 4-14 <148 and gluconeogenesis, augmenting Pediatric data from J of Clin Invest. 1974; ketogenesis, and inhibiting lipo- 53:1159-1166 and Pediatr Res. 1981;15: genesis. Thus, glucagon and insulin 912-915. provide a bihormonal regulation of blood glucose concentration. Interpretive Information The plasma glucagon assay is useful • Newborn primarily when considering a • Glucagonoma glucagon-secreting tumor of the • Diabetes mellitus (relative or pancreas. Glucagonomas cause an actual) unusual but characteristic syndrome consisting of a rash (migratory • Diabetes mellitus (occasional) necrolytic dermatitis located in the • Hypoglycemia (rare) groin, around the mouth and nose, and in intertriginous areas), mild diabetes, weight loss, and hypo- aminoacidemia. Measurement of plasma glucagon confirms the diagnosis; glucagon levels are very high (>1,000 pg/mL) in the setting of a glucagonoma.
Method • Extraction, radioimmunoassay (RIA) • Analytical sensitivity: 50 pg/mL
Specimen Requirements 3 mL room temperature EDTA plasma 1.1 mL minimum Overnight fasting is required.
82 Alphabetical Test Section 483X Spectrophotometry (hexokinase) 2 mg/dL sensitivity: Analytical Clinical Background are concentrations glucose Blood balancing by maintained narrowly promotes Insulin factors. hormonal and storage, utilization glucose levels. Carbohydrate blood lowering blood glucose raises ingestion peak levels are concentrations; insulin by stimulated modulated counter regulatory Several secretion. growth (glucagon, hormones catecholamines) cortisol, hormone, glucose to increase function stimulating by concentrations and gluconeogenesis glycogenolysis, glucose output, hepatic increased expense at the blood glucose raising and other substrate of carbohydrate stores. Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.2 mL top preferred red No additive red top acceptable SST is preferred. fasting Overnight 83 Hypoglycemic syndromes Hypoglycemic Addison’s disease Addison’s Glucagon deficiency Glucagon Islet cell tumor Islet Pheochromocytoma Cushing’s syndrome Cushing’s Diabetes mellitus Diabetes • • • • • • • Diagnose hypoglycemia Diagnose NormalImpairedDiabetes 100-125 65-99 >125 Diagnose diabetes mellitus diabetes Diagnose Assess carbohydrate metabolic status metabolic carbohydrate Assess *Fasting Adolescents and adults* and Adolescents mg/dL Interpretive Information Interpretive Reference Range Reference Clinical Use Glucose, Serum Glucose, • • • Glutamic Acid Decarboxylase-65 34878X Autoantibodies (GAD-65)
Clinical Use Clinical Background • Assess risk of type 1 diabetes Type 1 diabetes mellitus is charac- • Predict onset of type 1 diabetes terized by lymphocytic infiltration • Diagnose new onset type 1 diabetes of islet cells and circulating auto- antibodies against a variety of islet Reference Range cell antigens (ICA). These include glutamic acid decarboxylase (GAD), <1 U/mL insulin, IA-2, GM2-1, and others. Direct Interpretive Information assays for autoantibodies to GAD-65, IA-2, and insulin are now available for • Type 1 diabetic patients detecting individuals with a genetic • Patients at risk for developing predisposition to type 1 diabetes. type 1 diabetes GAD-65 autoantibodies are observed • Stiff-man syndrome (SMS) in 70% to 80% of prediabetic and type 1 diabetic subjects including 7% to 8% of adult onset diabetics with type 1 diabetes. When a positive GAD-65 antibody test is coupled with positive autoantibody tests for IA-2 and insulin, the risk of diabetes increases to >90% (see Test Application and Interpretation section).
Method • Radiobinding assay • Analytical sensitivity: 0.2 U/mL
Specimen Requirements 1 mL frozen serum 0.2 mL minimum No additive red top preferred SST red top acceptable
84 Alphabetical Test Section 5032X Affinity column chromatography column Affinity on is based albumin % glycated relative of microalbumin percentage albumin. of total to amount Clinical Background glycoproteins forms stable Glucose by a proteins plasma many with Among reaction. nonenzymatic is albumin proteins, the plasma most important the quantitatively of Concentrations protein. glycated diabetic are high in albumin glycated be to been shown and have patients of hyper- the degree to proportional over albumin turns Since glycemia. glycated albumin to 2 weeks, in 1 moni- are useful for determinations in glucose control short-term toring glycated In contrast, patients. diabetic of has a half-life which hemoglobin, of for assessment is best 120 days, in such patients. control long-term Method • • Specimen Requirements plasma EDTA refrigerated 3 mL minimum 2 mL required fasting Overnight 85 Relatively, with improved improved with Relatively, Microvascular disease/diabetic disease/diabetic Microvascular Hyperglycemia Uncontrolled diabetes Uncontrolled diabetic control diabetic complications • • • • Assess glucose control (short-term, control (short-term, glucose Assess diabetes, with in patients weeks) 1-2 with pregnancy- those especially diabetes associated Adults and childrenAdults 0.8-1.4% Interpretive Information Interpretive Reference Range Reference Clinical Use Glycated Albumin Glycated • Growth Hormone (GH) 521X
Clinical Use Clinical Background • Assess pituitary growth hormone GH is a polypeptide (~ 20,000 MW) disorders secreted by the anterior pituitary gland under the control of hypo- Reference Range thalamic growth hormone releasing Adults ng/mL hormone (GHRH) and somatostatin, Fasting <10 which stimulate and inhibit release, After 75 g oral glucose <1 respectively. Children <13 Excess GH production can be caused by pituitary adenoma. In adults, excess Interpretive Information GH production produces acromegaly; in children, it produces gigantism. • Gigantism Insufficient GH production in children • Acromegaly results in growth retardation and, in • Selected pituitary tumors adults, decreased muscle mass. • Pregnancy, due to HPL cross- Measuring GH concentration in reactivity children is useful for clinically • Laron dwarfism (GH resistance) evaluating short stature and helping to differentiate between abnormal GH • Pituitary GH deficiency production and other causes of growth failure. In adults a GH deficiency • Hypopituitarism, congenital or acquired syndrome has been described that is associated with osteoporosis, dys- • GH secretory dysfunction lipidemia, increased visceral obesity, Because of diurnal variability, and poor quality of life. Because random growth hormone values are random GH measurements are not not reliable for diagnosis of growth usually meaningful, inhibitory proto- hormone deficiency, acromegaly cols, provocative tests, and prolonged (adults), or gigantism (pediatrics). sampling usually are employed. See Disorders of Growth Hormone in the Test Application and Interpretation section.
Method • Immunochemiluminometric assay (ICMA) • Analytical sensitivity: 0.1 ng/mL
Specimen Requirements 1 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable
86 Alphabetical Test Section 37072X Radiobinding assay (RBA) assay Radiobinding is antibody when reported Titer present Clinical Background replace- a routine GH is now Human patients. in GH-deficient therapy ment is biosynthetically used the GH of Most DNA with recombinant produced and to minimize technology is purified some Nonetheless, antigenicity. autoantibodies; will develop patients and may be neutralizing may not these About response. the growth not inhibit neutral- produce patients will 5% of with thus, all patients antibodies; izing do who deficiency GH documented to GH therapy not respond as expected testing. antibody receive GH should Method • • Specimen Requirements serum refrigerated 0.5 mL minimum 0.2 mL top preferred red No additive red top acceptable SST 87 Possibly in patients with auto- in patients Possibly Selected patients on GH therapy patients Selected immune hypophysitis immune • • Monitor patients receiving GH receiving patients Monitor therapy Adults and childrenAdults Negative Interpretive Information Interpretive Reference Range Reference Clinical Use Growth Hormone (GH) Antibody (GH) Hormone Growth • Growth Hormone-Binding Protein 39573X (GHBP)
Clinical Use Clinical Background • Diagnose Laron dwarfism Human growth hormone (GH) exists • Differential diagnosis of growth in several isometric forms in serum – hormone resistance either free or bound to a specific • Indicate GH responsiveness binding protein (GHBP). This protein is identical to the extracellular domain Reference Range of the GH receptor and appears to be derived from the GH receptor gene by pmol/L alternative splicing. Levels vary with Adults 400-4260 age, being low in neonates and highest Children in young adults. Normal or elevated 3-8 y 320-3820 levels in patients with classical Laron 9-13 y 240-2890 syndrome indicate a defect in the 14-17 y 290-3140 transmembrane, intracellular, or post GH receptor domains. Very low or Interpretive Information absent GHBP denotes a defect in the • Estrogen treatment extracellular domain. A low serum GHBP in relatives of patients with • Familial short stature with high Laron syndrome helps identify GHBP (J Clin Endocrinol Metab. heterozygous carriers of patients with 2004;89:1031-1044.) defects in the extracellular domain. • Laron dwarfism Method • Enzyme-linked immunosorbent assay (ELISA) • Analytical sensitivity: 10 pmol/L
Specimen Requirements 1 mL frozen serum (no additive red top tube); 0.1 mL minimum SST red top unacceptable
88 Alphabetical Test Section 37557X Extraction, radioimmunoassay (RIA) radioimmunoassay Extraction, 16 pg/mL sensitivity: Analytical Clinical Background hormone releasing hormone Growth hypo- by the is secreted (GHRH) pituitarythe and stimulates thalamus GHRH hormone. growth to release the most is probably deficiency growth hormone cause of common in man. deficiency from hyper- results Acromegaly is and hormone of growth secretion cases, to a primarydue, in over 99% of cases remaining The pituitary tumor. due hypersecretion from GHRH result tumor or peripheral to hypothalamic islet tumors, (pancreatic tumors tumors, carcinoid or thymic bronchial tumors). rarely neuroendocrine and due to acromegaly with In patients peripheral GHRH secretion, ectopic elevated. levels are GHRH diagnosis between differential The GHRH a pituitary and ectopic tumor measur- achieved by is hypersecretion Circula- levels. GHRH ing peripheral in GHRH is nonhypothalamic ting origin. Method • • Specimen Requirements frozen plasma 4 mL minimum 1.1 mL Protein PTH-Related in special Collect tube Factors and Releasing (PTH-RP) Nichols Diagnostics by Quest provided in immediately Centrifuge Institute. separate centrifuge, refrigerated and freeze. plasma, 89 49 pg/mL GHRH) Horm Metabol Res. Horm Metabol ( Pregnancy (maternal and fetal and (maternal Pregnancy Ectopic GHRH production GHRH Ectopic samples) • • Differential diagnosis of acromegaly of diagnosis Differential Pediatric data from 1987;19:434-436. Adults < Children (4-14 y) (4-14 Children 6.8-19.0 Interpretive Information Interpretive Reference Range Reference Clinical Use Growth Hormone Releasing Hormone Growth Hormone • hCG, Total with HAMA Treatment 19720X
Clinical Use Clinical Background • Diagnose pregnancy Heterophilic antibodies, defined as • Diagnose chorionic tumors and antibodies against animal immuno- ectopic hCG-producing tumors globulins, can be a source of inter- ference in many immunoassays. Reference Range The most frequently characterized interference comes from endogenous hCG, Total, HAMA <5 mIU/mL human anti-mouse antibodies hCG, Total <5 mIU/mL (HAMA), which can react with the mouse monoclonal antibodies utilized Interpretive Information in the assay. HAMA presence in the • Pregnancy patient sample can lead to over- or • Trophoblastic tumors underestimation of human chorionic • Gonadal germ cell tumors gonadotropin (hCG). In this test, HAMA is removed by precipitation • Midline trophoblastic tumors prior to hCG determination.
• Ectopic pregnancy (relative to Method uterine pregnancy) • HAMA precipitation • Immunochemiluminometric assay (ICMA) • Analytical sensitivity: 2 mIU/mL • Analytical specificity: detects intact and free beta hCG
Specimen Requirements 2 mL room temperature serum 1.0 mL minimum No additive red top preferred SST red top acceptable
90 Alphabetical Test Section 8396X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 2 mIU/mL sensitivity: Analytical intact detects specificity: Analytical free beta hCG and Clinical Background gonadotropin chorionic Human developing by the is secreted (hCG) days after 6 early as often as placenta and production maximal conception; the occur during serum levels peak remain significant trimester but first immuno- The gestation. throughout serum assay for chemiluminometric test fast and accurate provides a hCG the assay is Because for pregnancy. for early it is useful sensitive, highly when pregnancy of determination low. levels are relatively hCG capable tissues are of other A variety and increased production, of hCG in nonpregnant in men or levels this neoplasia. In suggest women mIU/mL >10,000 hCG levels context, in cell tumors, only in germ occur differentia- with trophoblastic patients gastric primaryof a lung or tion with gestational in women or cancer, are Levels disease. trophoblastic cell tumors: in germ prognostic indicates good mIU/mL <5,000 to 50,000 mIU/mL 5,000 prognosis; prognosis; intermediate indicates poor indicates >50,000 mIU/mL and prognosis. also is appropriate measurement hCG gyneco- of men with evaluation in the pre- with isosexual and boys mastia testicular to diagnose puberty cocious malignancies. Method • • • Specimen Requirements serum refrigerated 1 mL minimum 0.4 mL top preferred red No additive red top acceptable SST 91 <5 mIU/mL Ectopic pregnancy (relative to (relative pregnancy Ectopic Midline trophoblastic tumors trophoblastic Midline Gonadal germ cell tumors germ Gonadal Trophoblastic tumors Trophoblastic Pregnancy uterine pregnancy) uterine • • • • • <1 wk1-2 wk2-3 wk3-4 wk4-5 wk5-6 wk6-8 wk2-3 mo 100-5,000 50-500 5-50 500-10,000 1,000-50,000 10,000-100,000 15,000-200,000 10,000-100,000 Diagnose chorionic tumors and tumors chorionic Diagnose tumors hCG-producing ectopic Diagnose pregnancy Diagnose PostmenopausalPregnant Expected* <10 Non-pregnant, Non-pregnant, premenopausal *Bayer Centaur product insert methods assay different from Values vary. may MenWomen <5 Interpretive Information Interpretive Reference Range Reference Clinical Use hCG, Total, Quantitative Quantitative Total, hCG, • • Hemoglobin A1c 496X
Clinical Use Clinical Background • Monitor glucose control (long-term, Hemoglobin A1 (HbA1 or glycated 2-3 months) in patients with diabetes hemoglobin) is structurally related to adult hemoglobin (HbA) but has a Reference Range glucose molecule attached to the Non-diabetic <6.0% terminal valine of the beta chain. Glycosylation is a nonenzymatic, Interpretive Information irreversible process dependent on • Diabetes mellitus the glucose concentration and the • Chronic hyperglycemia duration of exposure of the erythro- cyte to glucose. HbA1 is continuously • Presence of hemoglobin S formed during the 120-day life of the (sickle cell) erythrocyte, and a single measurement • Presence of hemoglobin C of HbA reflects the average blood variant 1 glucose level during the preceding • Relatively decreased by months. HbA1 can be separated into improved diabetic control HbA1a, HbA1b, and HbA1c fractions. • High levels of hemoglobin F HbA1c correlates best with high glucose concentrations.
Method • Colorimetry, immunoturbidimetric • Analytical sensitivity: 3% at a hemoglobin concentration of 8.2 mmol/L (13.2 g/dL)
Specimen Requirements 2 mL refrigerated whole blood 0.3 mL minimum Collect in EDTA (lavender-top) tube.
92 Alphabetical Test Section 1648X 39625X or random urine urine or random (39625X) ; 5 mL minimum High-performance liquid chroma- electrochemical (HPLC), tography detection 0.2 mg/L sensitivity: Analytical reported concentration Creatinine Clinical Background 5-HIAA yields metabolism Serotonin urinary as a major Because metabolite. large secrete tumors carcinoid and 5-HIAA, of serotonin amounts their are useful in for 5-HIAA tests (see many foods However, diagnosis. large also contain and drugs below) be and should of serotonin amounts 48 hours before at least avoided Drugs to avoid urine. collecting glyceryl phenothiazines, include inhib- MAO reserpine, guaiacolate, and imipramine. itors, Method • • • Specimen Requirements of aliquot room temperature 10 mL 24-h urine (1648X) of 6N with 25 mL 24-h urine Collect urine to to random Add 6N HCl HCl. urine Record 24-h pH <3. maintain form. vial and request volume on total tea, tobacco, avoid preparation: Patient avoid food days prior; coffee for 3 and banana, eg, avocado, in indoles, high and pineapple, plum, walnut, tomato, eggplant. 93 8 6 12 10 g/24-h mg/dL mg/24-h mg/g creat mg/g Carcinoid tumor Carcinoid • Diagnose carcinoid tumors carcinoid Diagnose MaleFemale 20-370 20-320 0-6 mo mo7-11 1-2 y3-8 y y9-12 y >12 2-32 2-36 2-128 2-149 2-183 >10 y>10 (39625X ) Creatinine 3-8 y y9-12 y13-17 Adults (1648X) 5-HIAA y2-10 y>10 (1648X) Creatinine < 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 < < 5-HIAA (39625X) 5-HIAA 2-10 y2-10 < Interpretive Information Interpretive Reference Range Reference Clinical Use 5-HIAA (5-Hydroxyindoleacetic Acid), (5-Hydroxyindoleacetic 5-HIAA Urine • Homovanillic Acid (HVA), Urine 39527X 6346X
Clinical Use Clinical Background • Diagnose catecholamine-producing Measurements of urinary vanillyl- tumors mandelic acid (VMA) and homo- vanillic acid (HVA) are valuable in Reference Range identifying patients with neuro- 24-H urine (39527X) blastoma. About 80% of patients with Homovanillic acid mg/24-h neuroblastoma will have elevated 3-8 y 0.5-6.7 urinary VMA. Other metabolites of dopamine, such as urinary dopamine, 9-12 y 1.1-6.8 HVA, and 3-methoxy-4-hydroxy- 13-17 y 1.4-7.2 phenylglycol (MHPG), are also Adults 1.6-7.5 usually elevated. Creatinine g/24-h Neuroblastomas are malignant tumors 3-8 y 0.11-0.68 occurring in children. About half of 9-12 y 0.17-1.41 the cases occur before 2 years of age. 13-17 y 0.29-1.87 Neuroblastomas secrete mostly pre- Adults 0.63-2.50 cursors of norepinephrine (dopamine and its metabolites) and seldom Random urine (6346X) produce hypertension. Patients usually Homovanillic acid mg/g creat present with an abdominal mass. Birth to 6 mo 9.1-36 Surgery and radiation therapies offer 7-11 mo 11.2-33 significant hope for survival. Early 1-2 y 8.5-38 diagnosis is therefore essential. 3-8 y 2.1-23 Method 9-12 y 1.1-12 • High-performance liquid chroma- Adults 1.4-5.3 tography (HPLC), electrochemical Creatinine mg/dL detection 0-6 mo 2-32 • Analytical sensitivity: 0.5 mg/L 7-11 mo 2-36 • Creatinine concentration also 1-2 y 2-128 reported 3-8 y 2-149 Specimen Requirements 9-12 y 2-183 10 mL room temperature aliquot of a >12 y 24-h urine (39527X) or random urine Male 20-370 (6346X); 5 mL minimum Female 20-320 Collect 24-h urine in 25 mL 6N HCl; add 6N HCl to random urine to Interpretive Information maintain pH <3. Record 24-h total • Neuroblastoma volume on vial and request form. • Ganglioblastoma Patient preparation: avoid alcohol, coffee, tea, tobacco, and strenuous exercise prior to collection; preferably abstain from medications for 3 days, but common antihypertensives (diuretics, ACE inhibitors, calcium channel blockers, alpha and beta blockers) cause minimal or no inter- ference.
94 Alphabetical Test Section 15202X Modified Porter-Silber reaction Porter-Silber Modified 0.1 mg/L sensitivity: Analytical also concentration Creatinine reported Clinical Background 17-hydroxycorticosteroids Urine 17-hydroxycorti- include (17-OHCS) F from compound derived costerone and 17-hydroxy-11- (hydrocortisone) from (derived dehydrocorticosterone parent The E, cortisone). compound and protein regulate hormones in- are and metabolism carbohydrate of ACTH control in feedback volved 17-OHCS levels Urine secretion. activity. adrenal cortical reflect Method • • • Specimen Requirements of a 24-h aliquot refrigerated 20 mL 10 mL minimum urine; (pre- of boric acid with 10 g Collect ferred) or 25 mL 6N HCl or 50% acetic vial and volume on Record total acid. form. request 95 g/24-h mg/24-h Infection Surgery etc Burns, Renal failure Renal Liver disease Liver Starvation ACTH deficiency ACTH Addison’s disease Addison’s Marked stress Marked Adrenal tumors Adrenal Cushing’s syndrome Cushing’s • • • • • • • • MalesFemales 3.0-10.0 2.0-7.0 MalesFemales 2.0-6.0 2.8-6.8 Reflect cortisol secretion cortisol Reflect Assess adrenocortical function adrenocortical Assess 17-20 y 17-20 1-2 y3-4 y5-6 y7-8 y y9-10 y11-12 y 13-16 0.5-2.5 1.0-4.0 1.0-4.8 1.0-5.6 1.0-7.0 1.5-8.0 Prescription and over-the-counter over-the-counter and Prescription affect results. may medications 3-8 y y9-12 y13-17 Adults 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 Creatinine 17-Hydroxycorticosteroid MenWomenChildren 3.0-10.0 2.0-6.0 Interpretive Information Interpretive Reference Range Reference Clinical Use 17-Hydroxycorticosteroids, 17-Hydroxycorticosteroids, Urine 24-Hour • • 18-Hydroxycorticosterone 6595X
Clinical Use Clinical Background • Differential diagnosis of primary Primary aldosteronism should be hyperaldosteronism due to adrenal suspected whenever a patient has the adenoma triad of hypertension, hypokalemia, and inappropriate renal potassium Reference Range wasting. The biggest challenge is the Adults (8-10 AM) ng/dL ng/dL differential diagnosis between the Upright 5-80 more common adrenal adenoma and Supine 4-37 bilateral hyperplasia. Because adrenal adenomas increase the circulating Children levels of 18-hydroxycorticosterone Premature infants (31-35 wk) <380 (18-OH B), serum levels of 18-OH B Term infants (3 d) <942 are useful to differentiate them from ACTH stimulation Baseline 60 min bilateral adrenal hyperplasia. The 1-12 mo 5-310 67-470 cutoff point appears to be about 1-5 y 7-155 49-370 50 ng/dL of serum. Most values are 6-12 y 10-74 79-360 around 100 ng/dL. The higher the Tanner II-III level of this precursor, the greater the Males 5-73 91-1475 possibility that an adrenal adenoma is present. Females 11-82 69-195 Tanner IV-V Method Males 14-62 73-205 • Extraction, chromatography, radio- Females 11-68 82-320 immunoassay (RIA) Pediatric data from J Clin Endocrinol Metab. 1992;75:1491-1496 and J Clin Endocrinol. • Analytical sensitivity: 7 ng/dL 1989;69:1133-1136. Specimen Requirements Interpretive Information 3 mL refrigerated serum • Adrenal adenoma 1.1 mL minimum No additive red top preferred • Adrenal P-450c11ase deficiency SST red top acceptable • Addison’s disease
96 Alphabetical Test Section 38071X -Hydroxycortisol, a metabolite of a metabolite -Hydroxycortisol, urinary is the major -Hydroxycortisol Liquid chromatography, tandem tandem chromatography, Liquid spectrometrymass (LC/MS/MS) 1 μg/L sensitivity: Analytical also concentration Creatinine reported β β Clinical Background 6 P-450 hepatic reflects cortisol, adrenal useful as has been activity and enzyme It drug toxicity. of hepatic a marker assessing adrenal is useful for also in the newborn. function cortical 6 in the neonatal metabolite cortisol period. Method • • • Specimen Requirements urine of a 24-h frozen aliquot 20 mL minimum 2 mL acid 10 g of boric urine with Collect collection. during or keep refrigerated and on vial total volume Record form. request 97 g/24-h μg/24-h . J Clin . J Clin 1991;73:674-686. Hepatic drug toxicity Hepatic • Hydroxycortisol -Hydroxycortisol, 24-Hour Urine 24-Hour -Hydroxycortisol, β Marker for hepatic microsmal microsmal for hepatic Marker induction enzyme 0-2 mo y2 mo-3 3-8 y y9-12 y13-17 3-6 3-49 15-95 24-197 44-350 β− Endocrinol Metab. *Pediatric ranges from Lashansky G, Fishman K, et al Saenger P, 6 Creatinine AdultsChildren* 3-8 y 18-460 y9-12 y13-17 Adults 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 Interpretive Information Interpretive Reference Range Reference Clinical Use 6 • 18-Hydroxycortisol, Free, 11178X 24-Hour Urine
Clinical Use Clinical Background • Diagnose glucocorticoid remediable GRA is a rare autosomal dominant aldosteronism (GRA) disease usually characterized by hypokalemia and early onset of Reference Range severe hypertension refractory to 18-Hydroxycortisol μg/24-h conventional antihypertensive Men 51-515 therapies. It is caused by a chimeric Women 43-295 duplication involving the 11β-hydrox- ylase (CYP11B1) and aldosterone Children synthase (CYP11B2) genes such that 1-4.9 y 6-193 aldosterone secretion is solely 5-9.9 y 16-240 regulated by ACTH. Urinary levels of 10-13.9 y 19-480 18-hydroxycortisol and 18-oxocortisol 14-17.9 y 17-500 levels are uniquely elevated in GRA. Dexamethasone suppression and Creatinine g/24-h genetic testing also are useful for 3-8 y 0.11-0.68 diagnosis. Some affected individuals 9-12 y 0.17-1.41 have mild hypertension without 13-17 y 0.29-1.87 hypokalemia but a suppressed plasma Adults 0.63-2.50 renin.
Interpretive Information Method • Glucocorticoid remediable • Liquid chromatography, tandem aldosteronism mass spectrometry (LC/MS/MS) • Analytical sensitivity: 1 μg/L • Adrenal insufficiency Specimen Requirements 10 mL refrigerated aliquot of a 24-h urine collection; 2.1 mL minimum Collect urine in 10 g boric acid or refrigerate during collection.
98 Alphabetical Test Section 37916X as one of these as one of c21 Radiobinding assay (RBA) assay Radiobinding 1.0 U/mL sensitivity: Analytical Clinical Background is disease Addison's Autoimmune of the presence by characterized cortical adrenal to autoantibodies have studies Recent cell antigens. biosynthetic the adrenal identified P-450 enzyme antigens. Autoimmune Addison's Addison's Autoimmune antigens. 2 polyglandular is part of disease type I (PAS), syndromes autoimmune auto- II, including type II. Type and is and diabetes, thyroiditis immune frequent. the more for assay direct autoantibody This sensitive is more anti-21-hydroxylase than the anti- quantifiable and (IFA) immunofluorescence adrenal of other Measurement method. provides autoantibodies glandular and manage- diagnostic additional information. ment Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.2 mL top preferred red No additive red top acceptable SST 99 Isolated autoimmune Addison’s Addison’s autoimmune Isolated PAS, type II type PAS, PAS, type I type PAS, disease • • • Identify adrenal gland involvement gland involvement adrenal Identify in PAS Confirm autoimmune Addison’s Addison’s autoimmune Confirm disease Adults and childrenAdults U/mL <1.0 Interpretive Information Interpretive Reference Range Reference Clinical Use 21-Hydroxylase Antibody 21-Hydroxylase • • 17-Hydroxypregnenolone 8352X
Clinical Use Clinical Background • Marker for adrenal 3β-hydroxy- 17-Hydroxypregnenolone, a Δ5 steroid, steroid dehydrogenase enzyme is an intermediate in the steroid deficiency hormone biosynthetic pathways. It is formed by the hydroxylation of Reference Range pregnenolone and metabolized to ng/dL ng/dL 17-hydroxyprogesterone or Adults 20-450 dehydroepiandrosterone (DHEA). Post ACTH stimulation, 290-910 17-hydroxypregnenolone production is 60 min, men and stimulated by ACTH and suppressed by premenopausal women dexamethasone. (follicular phase) Production is increased in patients Children with congenital adrenal hyperplasia Premature infants (31-35 wk) (CAH) associated with deficiency of <2409 the 3β-hydroxysteroid dehydrogenase Term infants (3 d) <830 enzyme. ACTH Baseline 60 min stimulation Method 1-12 mo 14-830 395-3290 • Extraction, chromatography, 1-5 y 10-100 45-740 radioimmunoassay (RIA) 6-12 y 11-190 70-660 • Analytical sensitivity: 10 ng/dL Tanner II-III Males 20-360 88-675 Specimen Requirements Females 58-450 250-800 2 mL refrigerated serum Tanner IV-V 0.4 mL minimum Males 32-300 220-860 No additive red top preferred Females 53-540 500-1600 SST red top acceptable Pediatric data from J Clin Endocrinol Metab. 1991;73:674-686 and J Clin Endocrinol Metab. 1989;69:1133-1136.
Interpretive Information • CAH due to 3β-hydroxysteroid dehydrogenase enzyme deficiency • Hirsutism (polycystic ovary or idiopathic) • Adrenal tumors
• Adrenal insufficiency
100 Alphabetical Test Section 17180X 17654X is appropriate ol or androstene- ssical 21-hydroxyl- it is then further then further it is deficiency) c17; c17 Late-onset CAHLate-onset disease Cushing’s (cortisone, treatment Steroid disease Addison’s Congenital adrenal hyperplasia hyperplasia adrenal Congenital pseudohermaphroditism Male hydrocortisone) (CAH), 21-hydroxylase deficiency (P450 • • • • • • Liquid chromatography, tandem tandem chromatography, Liquid spectrometrymass (LC/MS/MS) 8 ng/dL sensitivity: Analytical Interpretive Information Interpretive metabolized to cortis dione. 17-OHP measurement 21-hydroxyl- partial or complete when ase deficiency is suspected in 1) infants with features of adrenal insufficiency (hypotension, vomiting, hyper- hypoglycemia, and fever, and genitaliakalemia) or ambiguous 2) women of with clinical evidence possible androgen excess, particularly Ashkenazi Jews who have a high prevalence of noncla Some authorities ase deficiency. consider measurement of ACTH- essential levels 17-OHP stimulated Cushing's diagnosis. the exclude to disease may also cause 17-OHPelevated levels and should be excluded before treatmentglucocorticoid of presumed deficiency 21-hydroxylase nonclassical is considered. Method • • Specimen Requirements red (no additive frozen serum 0.5 mL minimum 0.25 mL top tube); red top unacceptable SST 17-Hydroxyprogesterone is (17-OHP) steroidan adrenal intermediate in the biosynthesis of cortisol. Its synthesis from progesterone is the catalyzed by enzyme P450 Clinical Background 101 90 45 360 420 169 185 225 285 1989; 1991;
c21 * ng/dL ng/dL Baseline 60 min J Clin Metab. J Clin Endocrinol Metab. Metab. Endocrinol J Clin (17654X)
J Clin Endocrinol Metab. Midcycle phaseMidcycle phaseLuteal Postmenopausal < < < 1-12 mo1-12 1-5 y y6-12 Males 11-170Females 85-465 Males 4-115Females 50-350 7-69 75-220 12-130 18-220 69-310 80-420 51-190 36-200 105-230 80-225 (21-hydroxylase) enzyme deficiency enzyme (21-hydroxylase) y18-30 y31-40 y41-50 y51-60 phaseFollicular 32-307 42-196 33-195 < 37-129 Marker for adrenal P450 for adrenal Marker 10-13 y10-13 < Neonatal/Infant d) infants (3 Term mo1-12 1-4 y y5--9 y14-17 < ACTH stimulation 11-170 4-115 < 16-283 Tanner II-III Tanner IV-V Tanner *Includes data from from data *Includes 73:674-686; 69:1133-1136; and 1994;78:266-270. Women Adults (17180X) Adults Men Premature infants (31-35 wk) infants Premature < Reference Range Reference Clinical Use 17-Hydroxyprogesterone, LC/MS/MS 17-Hydroxyprogesterone, • Hydroxyproline, Free, Plasma 37558X
Clinical Use Clinical Background • Assess bone matrix turnover Collagen metabolism releases a variety of collagen constituents into the Reference Range circulation. These include amino- and μmol/L carboxy-terminal extension peptides, 0-30 d 0-91 pyridinoline, and deoxypyridinoline 31 d-24 mo 0-63 cross-links, and hydroxyproline. 2-18 y 3-45 Hydroxyproline in plasma appears as both peptide bound and free forms. >18 y 0-53 Bone resorption is the major con- Interpretive Information tributor to collagen turnover, and the constituent molecules, including • Paget's disease hydroxyproline, are useful as markers • Bone metastatic cancer of the rate of bone matrix resorption. • Hyperparathyroidism Hydroxyproline is most useful in conditions associated with markedly • Osteoporosis increased bone resorption (Paget's • Glucocorticoid excess disease, malignancy, hyperpara- thyroidism). • Fluoride excess • Bisphosphonate therapy Method • Anabolic steroid therapy • High performance liquid chroma- tography (HPLC) • Analytical sensitivity: 1 μmol/L
Specimen Requirements 2.0 mL frozen sodium heparin plasma 0.5 mL minimum Separate plasma within 30 minutes of collection and freeze. Provide patient age, sex, clinical history, tentative diagnosis, and therapy during the last 3 days (eg, drugs, X-ray, infant formula, diet).
102 Alphabetical Test Section 535X 17190X, 685X, 17190X, ) 17190X ); 2.1 mL minimum for total for ); 2.1 mL minimum free and for ); 4.2 mL minimum Analytical sensitivity: 0.5 μg/mL and respectively 2 μg/mL, free and total, also concentration Creatinine reported Colorimetry 685X 535X Collect with 25 mL of 6N HCl. Record of 6N HCl. with 25 mL Collect form. vial and request volume on total and collagen-derived meat Avoid prior 24 to 48 hours for therapeutics to collection. • Specimen Requirements of a 24-h aliquot refrigerated 25 mL free for 2.1 mL minimum urine; ( ( ( total Clinical Background acid is an amino Hydroxyproline in quantities in appreciable present urine the in and excreted collagen Because collagen breakdown. after urinary is derived hydroxyproline it collagen, entirely from almost catabolism. reflects the rate of collagen urinary amounts of Small hydroxy- acids, amino free as present are proline and small with most is associated while This peptides. nondialyzable large referred is collectively combination total hydroxyproline. to as are levels hydroxyproline Urine and Paget's disease in increased greatly in hyperpara- extent to a lesser (primarythyroidism and secondary). also observed levels are Elevated in and psoriasis, with burns, patients Urinary hydroxyproline acromegaly. marker as a tumor suggested has been mammary for lymphomas, carcinoma, prostate bone metastatic and carcinoma. are in- of free hydroxyproline Levels errors with inborn in patients creased hydroxyprolinemia, of metabolism, and familial aminoglycinuria. Method • • 103 2.7
g/24-h mg/24-h J Clin Invest. 1970; 1967;1:266-270; J Pediatr Gastroent Nutr. J Pediatr Gastroent Nutr. J Clin Invest.
Pediatr Res. Pediatr 1966;69:266-273; and Familial aminoglycinuria Familial Hydroxyprolinemia Inborn errors of metabolism errors of Inborn Acromegaly Psoriasis Burns Hyperparathyroidism (primary Hyperparathyroidism Paget’s disease Paget’s and secondary)and • • • • • • • • 3 d1 mo y1-10 y11-14 32-63 8-20 15-150 68-169 Assess efficacy of therapy for bone bone of therapy for efficacy Assess disease Index for the rate of bone the rate of bone formation for Index turnover and Premature infants, 3 infants, Premature wk old infants Full-term 6-35 *Pediatric data from Hydroxyproline, Free Free Hydroxyproline, 17190X) (685X, Adults < 1991;13:176-181; J Pediatr. 49:716-729; Creatinine Creatinine 535X, 17190X) (685X, Hydroxyproline, Total Total Hydroxyproline, 17190X) (535X, MenWomenChildren* 7-49 9-73 3-8 y y9-12 y13-17 Adults 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 1962;41:1928-1935. Interpretive Information Interpretive Reference Range Reference Clinical Use Hydroxyproline, Free and Total, Total, and Free Hydroxyproline, Urine 24-Hour • • Hydroxyproline, Free and Total, 11258X Random Urine 37407X
Clinical Use Clinical Background • Index for the rate of bone formation Hydroxyproline is an amino acid and turnover present in appreciable quantities in • Assess efficacy of therapy for bone collagen and excreted in the urine disease after collagen breakdown. Because urinary hydroxyproline is derived Reference Range almost entirely from collagen, it Hydroxyproline, Free (11258X) μg/mL reflects the rate of collagen catabolism. Adults <1.6 Small amounts of urinary hydroxy- proline are present as free amino acids, Hydroxyproline, Total (37407X) while most is associated with small and Adults <60 large nondialyzable peptides. This Creatinine mg/dL combination is collectively referred to 0-6 mo 2-32 as total hydroxyproline. 7-11 mo 2-36 Urine hydroxyproline levels are greatly 1-2 y 2-128 increased in Paget's disease and to a 3-8 y 2-149 lesser extent in hyperparathyroidism (primary and secondary). Elevated 9-12 y 2-183 levels are also observed in patients with >12 y burns, psoriasis, and acromegaly. Male 20-370 Urinary hydroxyproline has been Female 20-320 suggested as a tumor marker for lymphomas, mammary carcinoma, and Interpretive Information bone metastatic prostate carcinoma. • Paget's disease Levels of free hydroxyproline are in- • Hyperparathyroidism (primary creased in patients with inborn errors and secondary) of metabolism, hydroxyprolinemia, • Burns and familial aminoglycinuria. • Psoriasis Method • Acromegaly • Colorimetry • Inborn errors of metabolism • Analytical sensitivity: 0.5 μg/mL and • Hydroxyprolinemia 2 μg/mL, free and total, respectively • Familial aminoglycinuria • Creatinine concentration also reported
Specimen Requirements 25 mL refrigerated aliquot of a random or 2nd void urine; 2.1 mL minimum Patient should be on a diet sufficiently low in collagen for 24 hours prior.
104 Alphabetical Test Section
15026X manifests with manifests Gas chromatography/mass Gas chromatography/mass spectrometry (GC/MS) and gonadal 31 adrenal Includes metabolites steroid 1.0 μg/L sensitivity: Analytical is an autosomal recessive, severe form recessive, severe autosomal is an in usually detected of hypertension with low manifests AME childhood. is and and aldosterone renin levels of urinary by low diagnosed cortisone/ cortisol metabolites, total ratio corresponding ratio (or cortisol and tetra- metabolites), of their hydroaldosterone. syndrome) (Liddle's Hypoaldosteronism and with hypertension manifests and Renin hypokalemia. severe un- are often levels aldosterone on is based Diagnosis detectable. (cortisol) corticosteroid normal low urinary with tetra- metabolism excretion. hydroaldosterone Method • • • Specimen Requirements urine of a 24-h frozen aliquot 7 mL minimum 2.1 mL collection. during Refrigerate use preservatives.Do not and on vial total volume Record form. request hypertension, increased aldosterone aldosterone increased hypertension, suppression loss, and potassium levels, system. renin-angiotensin of the on increased is based Diagnosis and urinary aldosterone serum Cortisol tetrahydroaldosterone. urinary including metabolism, normal. is generally metabolites, aldosteronism Glucocorticoid-suppressible of low- dominant form autosomal is an detected usually hypertension renin by elevated diagnosed It is in children. and urinary aldosterone excre- serum tion of tetrahydroaldosterone, and 18-oxo-tetra- 18-hydroxycortisol, hydrocortisol. (AME) mineralocorticoid excess Apparent Clinical Background Primary aldosteronism 105 10 6-79 <3000 - and α Females μg/24-h μg/24-h* † 42 < -THF/THE and and -THF/THE -cortolone, Males α β μg/24-h - and - and α -THF)/THE >3 α -THF 100-2660 100-1250 -THF, THE, -THF, -Cortol 30-490-Cortolone 25-215 47-1400 90-1100 Tetrahydroaldosterone18-Oxo-THF >80 Tetrahydroaldosterone18-Oxo-THF >50 Tetrahydroaldosterone <10 metabolites cortisol Total <10 >10 Detect treatable causes of low-renin causes of treatable Detect adults and children in hypertension (THF + 5 (THF Cortisol/cortisoneTetrahydroaldosterone >1 <10 -Cortol-Cortolone 8-340 17-215 8-560 10-220 α -cortol, cortisol, and cortisone. cortisol, -cortol, α Total cortisone metabolites Total include THF, cortisol/cortisone ratios. cortisol/cortisone β Glucocorticoid-suppressible Glucocorticoid-suppressible aldosteronism mineralocorticoid Apparent (AME) excess Primary aldosteronism *Forexcept all THF+5 † THF, tetrahydrocortisol; THE, tetrahydro- tetrahydrocortisol; THF, cortisone. 5 α β CortisoneTHEα β 19-245 6- Tetrahydroaldosterone 79 24-160 445-5960 330-3430 18-Oxo-THF < CortisolTHF5 7-185 280-2820 13-115 210-1680 Hypoaldosteronism Interpretive Information Interpretive Reference Range Reference Clinical Use Hypertension, Endocrine Hypertension, • IA-2 Antibody 36177X
Clinical Use Clinical Background • Assess risk of type 1 diabetes Type 1 diabetes mellitus is charac- • Predict onset of type 1 diabetes terized by lymphocytic infiltration • Diagnose new onset type 1 diabetes of islet cells and circulating auto- antibodies against a variety of islet Reference Range cell antigens (ICA). These include glutamic acid decarboxylase (GAD), Adults and children <0.8 U/mL insulin, IA-2, GM2-1, and others. Direct Interpretive Information assays for autoantibodies to GAD-65, IA-2, and insulin are now available for • Patients at risk for developing detecting individuals with a genetic type 1 diabetes predisposition to type 1 diabetes. • Type 1 diabetic patients A positive correlation exists between the IA-2 antibody concentration and the severity of the autoimmune process as well as between the number of positive autoantibodies (IA-2, GAD-65, and insulin) and the risk for type 1 diabetes. See Test Application and Interpretation section (Disorders of Carbohydrate Metabolism).
Method • Radiobinding assay (RBA) • Analytical sensitivity: 0.8 U/mL
Specimen Requirements 1 mL refrigerated serum (no additive red top tube); 0.5 mL minimum
106 Alphabetical Test Section or lc 36590X Radioimmunoassay (RIA) Radioimmunoassay 5 ng/mL sensitivity: Analytical no cross- specificity: Analytical or IGFBP-3 with IGFBP-2 reaction other indicators of poor long-term of poor long-term indicators other marker and 5) as a control, diabetic with associated resistance for insulin syndrome. the metabolic Method • • • Specimen Requirements frozen serum 1 mL minimum 0.2 mL top preferred red No additive red top acceptable SST is preferred. fasting Overnight Clinical Background and (IGF-I factors growth Insulin-like serum in are transported IGF-II) proteins. to 1 binding of several bound IGFBP-1, proteins, of the binding One When conditions. with glycemic varies serum low (fasting), insulin is serum rise quickly IGFBP-1 of concentrations of infusion 6-fold. Conversely, 4- to suppresses subjects in normal glucose secretion. IGFBP-1 for 1) is useful IGFBP-1 Measuring respon- insulin monitoring directly insulin- marker for 2) as a siveness, in 3) (insulinomas), tumors producing acute for monitoring mellitus diabetes 4) in action, in insulin fluctuations for determining mellitus diabetes to in- is due hyperglycemia whether to poor dosage or insulin adequate of dietarypatients control intake in elevated HbA chronically with 107 1995; ng/mL J Clin Endocrinol Metab. Insulinoma Increased insulin effect insulin Increased Decreased insulin effect insulin Decreased • • • 5-9 y y10-14 y15-18 20-105 10-70 10-40 Marker for insulinoma Marker Directly monitor insulin monitor Directly responsiveness AdultsChildren* 13-73 *Data from: 80:2534-2542. Interpretive Information Interpretive Reference Range Reference Clinical Use IGF Binding Protein-1 (IGFBP-1) Protein-1 Binding IGF • • IGF Binding Protein-2 (IGFBP-2) 37102X
Clinical Use Clinical Background • Marker of growth hormone action Insulin-like growth factors (IGF-I, • Tumor marker for prostatic IGF-II) are transported in serum carcinoma bound to several IGF binding proteins (IGFBP). These proteins bind both Reference Range IGF-I and II with high affinity but do ng/mL not bind insulin. The major function 1-9 y 69-480 of these proteins is to transport IGFs within the circulation and out of the 10-17 y 50-326 vascular compartment. 18-49 y 55-240 >49 y 28-444 One of 6 IGF binding proteins, IGFBP-2 is present in very high Interpretive Information concentrations during fetal develop- ment, progressively falling after birth • Chronic renal failure to its lowest level between 18 and • GH deficiency 35 years. The concentration of • Leukemia IGFBP-2 correlates inversely with changes in growth hormone (GH) • Mesenchymal tumors (with hypoglycemia) secretion. GH administration will sig- nificantly suppress IGFBP-2 concentra- • Prostatic carcinoma tions while increasing IGFBP-3 levels. Combined measurement of IGF-I, • Acromegaly IGFBP-2, and IGFBP-3 allows direct • Severe liver disease assessment of GH action. IGFBP-2 is also used as a tumor marker for prostatic carcinoma. Adult men with significant widespread prostatic carcinoma have IGFBP-2 levels 2 to 3 times greater than normal. In contrast, men with early-stage disease and normal prostate specific antigen (PSA) values showed an increase in IGFBP-2 levels of only 30%. This suggests that the test may be useful as an adjunct to PSA in the management of selected patients with prostatic carcinoma.
Method • Radioimmunoassay (RIA) • Analytical sensitivity: 0.56 ng/mL
Specimen Requirements 1 mL frozen serum 0.2 mL minimum No additive red top preferred SST red top acceptable
108 Alphabetical Test Section 34458X Acromegaly Hypopituitarism GH deficiency Isolated diabetes controlled Poorly restriction and protein Caloric • • • • • Immunochemiluminometric assay Immunochemiluminometric (ICMA) 0.5 mg/L sensitivity: Analytical Interpretive Information Interpretive Clinical Background binding factor growth Insulin-like with IGF-II and bind IGF-I proteins insulin. do not bind but affinity high proteins IGF binding 6 distinct Of the time, this at characterized structurally be the shown to has been IGFBP-3 the IGFs, transporting carrier of major IGF-I of circulating 95% approximately and IGF-II. res- (GH) hormone is growth IGFBP-3 acro- in are high Thus, levels ponsive. in hypopituitarism, and low megaly in GH-deficient levels increase and administration. after GH children decrease contrast, levels, in IGFBP-2 both Thus, GH administration. after of IGF-I/IGFBP-2 and the ratios assays as are useful IGFBP-2/IGFBP-3 and dis- and for of GH action markers hormone growth between criminating due to stature and short deficiency Other in children. etiologies other result in that of short stature causes poorly levels include IGFBP-3 reduced IGFBP-3 assay The diabetes. controlled status, nutritional in assessing is useful both during IGFBP-3 decreases since restriction. and protein caloric Method • • Specimen Requirements additive serum (no refrigerated 1 mL mL minimum tube); 0.5 red top red top unacceptable SST 109 No primary data mg/L mg/L range as a guideline available; use 81-85 y/o use 81-85 available;
IIIIIIIVV 1.4-5.2 2.3-6.3 3.1-8.9 1.2-6.4 3.7-8.7 2.8-6.9 3.9-9.4 2.6-8.6 3.3-8.1 2.7-9.1 Assess nutritional status nutritional Assess Differential diagnosis of GH GH of diagnosis Differential non-GH-deficient and deficiency children short Tanner stageTanner Male Female 1-7 d d8-15 y16 d-1 2 y3 y4 y5 y6 y7 y8 y9 y 0.5-1.4 10 y <0.7 0.7-3.6 11 y12 y13 y 0.8-3.9 14 y 0.9-4.3 15 y 1.0-4.7 16 y 1.1-5.2 17 y 1.3-5.6 18 y 1.4-6.1 19 y 1.6-6.5 20 y 1.8-7.1 2.1-7.7 y21-30 2.4-8.4 y31-40 2.7-8.9 y41-50 3.1-9.5 y51-60 3.3-10.0 y61-70 3.5-10.0 y71-80 3.4-9.5 y81-85 3.2-8.7 y >85 3.1-7.9 2.9-7.3 3.4-7.8 2.9-7.2 3.4-7.0 3.3-6.7 3.4-6.9 3.0-6.6 2.5-5.7 2.2-4.5 Reference Range Reference Clinical Use IGF Binding Protein-3 (IGFBP-3) Protein-3 Binding IGF • • IGF-I 839X
Clinical Use Clinical Background • Assess growth-related disorders Insulin-like growth factor I (IGF-I, • Monitor growth hormone replace- somatomedin-C) is a protein involved ment therapy in adults with GH in stimulating somatic growth; it is deficiency regulated principally by growth Reference Range hormone (GH) and nutritional intake. Male Female IGF-I is transported in serum by several ng/mL ng/mL IGF-binding proteins. The most im- Adults portant are IGFBP-2 and IGFBP-3. 19-30 y 126-382 138-410 These proteins help minimize fluctua- tions and maintain relatively high IGF-I 31-40 y 106-255 126-291 serum levels. 41-50 y 86-220 88-249 51-60 y 87-225 92-190 Measuring IGF-I is useful in several growth-related disorders. Dwarfism 61-70 y 75-228 87-178 caused by deficiency of growth 71-80 y 31-187 25-171 hormone (hypopituitarism) results 81-88 y 68-157 31-162 in decreased serum levels of IGF-I. >88 y Not Not In adults, a low IGF-I level in the established established presence of more than 3 pituitary Children hormone deficiencies obviates the 1-7 d <31 <31 need for a GH stimulation test. Acro- 8-14 d <43 <43 megaly (growth hormone excess) 15 d-1 y 25-265 25-265 results in elevated levels of IGF-I. IGF-I 1-2 y 45-222 99-254 measurements are also helpful in assessing nutritional status; levels are 3-4 y 36-202 36-202 reduced in undernutrition and 5-6 y 32-259 57-260 restored with a proper diet. IGF-I has 7-8 y 65-278 97-352 been utilized for dose titration in GH 9-10 y 52-330 49-461 replacement therapy in adults with GH 11-12 y 80-723 101-580 deficiency. The Endocrine Society 13-14 y 142-855 199-658 consensus statement recommends 15-16 y 176-845 236-808 achieving an IGF-I level that is within 17-18 y 152-668 165-526 the sex and age adjusted reference range. Tanner stages I 59-296 45-358 Method II 56-432 111-426 • Immunochemiluminometric assay III 135-778 169-644 (ICMA) IV 230-855 297-627 • Analytical sensitivity: 25 ng/mL V 181-789 142-868 Interpretive Information Specimen Requirements • Acromegaly 1 mL frozen serum • Gigantism 0.5 mL minimum No additive red top preferred • Isolated GH deficiency SST red top acceptable • Hypopituitarism • Laron dwarfism • Undernutrition 110 Alphabetical Test Section 20707X Immunoradiometric assay (IRMA) Immunoradiometric 150 ng/mL sensitivity: Analytical Clinical Background growth insulin-like The somatomedins, are IGF-II), and I and II (IGF-I factors and to proinsulin related structurally cell role in regulating an essential play Their function. and proliferation IGF and of by way mediated are effects neutral IGF-II is a receptors. insulin 75% with polypeptide 7500-dalton IGF-I, and like with IGF-I, homology It is multiple tissues. in it is produced than in its action insulin-like more on dependent are less and levels IGF-I, Plasma concentra- hormone. growth approximately of IGF-II are tions IGF-I. higher than 3 times hypoglycemia tumor Non-islet-cell associated is a syndrome (NICTH) and insulin, IGF-I, low levels of with of secretion and increased IGFBP-3 IGF-I/ The IGF-II (pro-IGF-II). "big" markedly increased. ratio is IGFBP-3 tumor is preferred treatment The hormone but growth removal, IGF-I/ the can normalize treatment the ameliorate ratio and IGFBP-3 hypoglycemia. Method • • Specimen Requirements additive serum (no refrigerated 1 mL mL minimum tube); 0.1 red top red top unacceptable SST is preferred. fasting Overnight 111 Growth hormone deficiency hormone Growth Infants under 1 year under 1 Infants Wilms tumor Wilms Acromegaly (variable) Acromegaly Hepatoma Mesenchymal tumors (with (with tumors Mesenchymal hypoglycemia) • • • • • • Assess growth factor status growth factor Assess Evaluate non-islet-cell tumor tumor non-islet-cell Evaluate (NICTH) hypoglycemia 18-30 y18-30 y31-40 y41-50 y51-60 0-4 y5-9 y 546-1260 y10-13 460-1240 y14-17 414-1230 414-1248 Not established 754-1216 610-1217 649-1225 Children Adults ng/mL Interpretive Information Interpretive Reference Range Reference Clinical Use IGF-II IGF-II II) Factor Growth (Insulin-Like • • Inhibin A 34472X
Clinical Use Clinical Background • Prenatal risk assessment for Down Inhibin A is a glycoprotein produced syndrome by the placenta and by granulosa cells of the ovary, particularly during the Reference Range late follicular and luteal phases of the Women pg/mL menstrual cycle. The active form is a Premenopausal <98 dimer consisting of an alpha subunit Postmenopausal <10 linked to a betaA subunit. Inhibin A Men <21 has both endocrine (suppression of FSH by the pituitary) and paracrine Interpretive Information (stimulation of thecal cell androgen synthesis) functions. • Granulosa cell tumors Measurement of inhibin B may be • Down syndrome-affected preferable to that of inhibin A for pregnancies detection of recurrent granulosa cell • Menopause tumors, studies of reproductive aging in females, and studies in males.
Method • Enzyme-linked immunosorbent assay (ELISA)
Specimen Requirements 2 mL frozen serum 0.5 mL minimum No additive red top preferred SST red top acceptable
112 Alphabetical Test Section
B 34445X Enzyme-linked immunosorbent assay immunosorbent Enzyme-linked (ELISA) subunit. Inhibin B suppresses pituitary B suppresses Inhibin subunit. have and may also of FSH synthesis the roles within paracrine additional ovary Measurement of inhibin or testis. of inhibin to that be preferable B may granulosa of recurrent detection A for of reproductive studies cell tumors, males. in and studies in females, aging Method • Specimen Requirements red (no additive frozen serum 1 mL minimum 0.5 mL top tube); Clinical Background produced B is a glycoprotein Inhibin and testis of the cells Sertoli by the ovary, of the cells the granulosa follicular the early during particularly The cycle. of the menstrual phase of consisting form is a dimer active beta linked to a subunit an alpha 113 92 139 161 <30 pg/mL pg/mL Anorchia Decreased ovarian reserve/ ovarian Decreased Granulosa cell tumors cell Granulosa menopause • • • PremenopausalPostmenopausal3-9 y <255 <30 < 10-13 y10-13 y14-17 42-339 68-300 < < Differential diagnosis of diagnosis Differential cryptorchidism versus anorchia Marker of testicular function of testicular Marker Predict ovarian reservePredict outcome and vitro fertilization of in Detect recurrence of granulosa cell of granulosa recurrence Detect tumors Children Males Females MenWomen <305 Interpretive Information Interpretive Reference Range Reference Clinical Use Inhibin B Inhibin • • • • Insulin Antibodies 36178X
Clinical Use Clinical Background • Assess risk for type 1 diabetes Type 1 diabetes is characterized by • Predict onset of type 1 diabetes lymphocytic infiltration of pancreatic • Diagnose new onset type 1 diabetes islet cells and autoantibodies against several islet cell antigens including Reference Range glutamic acid decarboxylase (GAD-65), IA-2, and insulin. This assay is designed Adults and children <1 U/mL to assess the low titers of autoantibody Interpretive Information in prediabetic subjects and other patients with autoimmune disorders • Prediabetic (type 1) subjects who have not received exogenous • Polyendocrine autoimmune insulin. For type 1 diabetes risk syndromes assessment, a combination of IA-2, • Other autoimmune disease GAD-65, and insulin autoantibodies increases the sensitivity.
Method • Radiobinding assay (RBA)
Specimen Requirements 1 mL frozen serum (no additive red top tube); 0.2 mL minimum SST red top unacceptable
114 Alphabetical Test Section 36700X PEG precipitation, precipitation, PEG (RIA) radioimmunoassay 2.5 μU/mL sensitivity: Analytical free, detects specificity: Analytical detect does not insulin; bioavailable cross- insulin; no antibody-bound cross-reacts with C-peptide; reactivity with proinsulin Clinical Background is measurement insulin free The glucose blood in interpreting helpful to its relationship and concentration in insulin-treated injections insulin insulin antibodies. who have patients diabetic patient if a For instance, at hypoglycemic becomes frequently concomitantly day, the of times certain suggest levels would free insulin high dosage. the insulin to reduce the need concen- glucose elevated In contrast, insulin with low free together trations for insulin indicate insufficient levels sequential, Multiple, control. adequate for are also helpful insulin levels free of and quantity the rate evaluating from subcutaneous absorption insulin injections. and free of C-peptide availability The assess- permits measurements insulin secretion insulin of endogenous ment of circu- as the concentrations as well (from active insulin biologically lating have circu- who in patients all sources) insulin antibodies. lating Method • • •• Specimen Requirements serum refrigerated 1.8 mL minimum 0.6 mL top preferred red No additive red top acceptable SST required fasting Overnight 115 injection Untreated type 1 diabetes type Untreated Insulin resistance Insulin Some type 2 diabetic patients type 2 diabetic Some Neisidioblastosis Insulinoma Following exogenous insulin insulin exogenous Following mellitus • • • • • • Monitor therapy in patients with in patients therapy Monitor antibodies insulin Adults μU/mL 4.0-20.0 Interpretive Information Interpretive Reference Range Reference Clinical Use Insulin, Free (Bioactive) Free Insulin, • Insulin, Total (Free and Bound) 36702X
Clinical Use Clinical Background • Determine insulin dosage in patients The total insulin concentration with insulin-dependent diabetes and includes free plus antibody-bound insulin antibodies insulin. In patients without insulin antibody, total and free insulin Reference Range concentrations are similar. In patients <20.0 μU/mL with insulin antibodies, total insulin levels are dependent on the binding Interpretive Information capacity of the circulating endogenous • (With circulating insulin insulin antibody and the availability of antibodies) insulin to bind to the antibody sites. Total insulin is measured by eluting • Insulinoma antibody-bound insulin with acid, • Some type 2 diabetic patients precipitating the endogenous anti- • Infantile hypoglycemia body, and measuring the total insulin • Hyperinsulinism by direct radioimmunoassay. Total insulin levels are useful in assessing the • Untreated type 1 diabetes available insulin in serum, including mellitus antibody-bound insulin. Serum insulin levels are useful in the Individuals with circulating insulin differential diagnosis of fasting hypo- antibodies may demonstrate variable glycemia (see Dynamic Test section) concentrations depending upon the but must always be evaluated in the level and binding capacity of the light of a simultaneous plasma glucose. antibodies. With any cause of hyperglycemia, such as a meal, insulin is stimulated; with fasting or hypoglycemia, insulin is normally suppressed.
Method • Acid treatment, PEG precipitation, radioimmunoassay (RIA) • Analytical sensitivity: 2.5 μU/mL • Analytical specificity: detects free and antibody-bound insulin; cross-reacts with proinsulin; no cross-reactivity with C-peptide
Specimen Requirements 1.8 mL refrigerated serum 0.6 mL minimum No additive red top preferred SST red top acceptable Overnight fasting is required.
116 Alphabetical Test Section 11303X Electrochemiluminescence assay Electrochemiluminescence (ECLA) 0.5 μg/L sensitivity: Analytical cross- <3.5% specificity: Analytical <1% cross- with hCG; reactivity glycoprotein with other reactivity hormones Clinical Background of form a hyperglycosylated is ITA (hCG) gonadotropin chorionic human embryonic during implanta- secreted the of invasion and trophoblast tion in early levels rise wall. ITA uterine almost which time during pregnancy, molecules is ITA. 100% of hCG-related than less weeks of gestation, By 12 is ITA. molecules of hCG-related 2.9% than rather ITA of Measurement more allows hCG therefore regular in of pregnancy detection dependable missed menstrual after a the week period. Method • • • Specimen Requirements serum room temperature 2 mL minimum 0.2 mL top preferred red No additive red top acceptable SST 117 Trophoblastic tumors Trophoblastic Pregnancy • • Early detection of pregnancy detection Early <1 μg/L Interpretive Information Interpretive Reference Range Reference Clinical Use Invasive Trophoblast Antigen Trophoblast Invasive (Pregnancy) (ITA) • Islet Cell Antibody (ICA) 36741X
Clinical Use Clinical Background • Assess risk of type 1 diabetes Autoantibodies directed against • Predict onset of type 1 diabetes pancreatic islet cells (islet cell anti- • Diagnose new onset type 1 diabetes bodies, ICA) include glutamic acid decarboxylase (GAD), IA-2, insulin, Reference Range GM2-1, and other cell surface proteins. Although ICA attacks all pancreatic Antibody Negative islet cells, cell destruction appears Titer <1.25 JDF Units specific for insulin-producing islet cells NOTE: End point titers are compared to a (beta cells), leading to insulin defi- single international reference standard ciency. ICA are present in the serum of and values are reported in JDF (Juvenile Diabetes Foundation) units. patients during the prediabetic phase and predict development of type 1 Interpretive Information disease. • Patients at risk for developing Radiobinding assays are available for type 1 diabetes assessment of GAD-65, IA-2, and • Type 1 diabetes insulin autoantibodies and are more reliable as markers for the prediabetic state. This IFA assay measures a variety of antibodies, has limited sensitivity, and is only semi-quantitative.
Method • Immunofluorescence assay • Titer provided (at an additional charge) if antibody is present
Specimen Requirements 2.0 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable
118 Alphabetical Test Section 15201X Modified Zimmerman reaction Zimmerman Modified 0.1 mg/L sensitivity: Analytical also concentration Creatinine reported Clinical Background with react (17-KS) 17-Ketosteroids yield a to acid para-aminobenzoic absorption with an product colored (the Zimmerman μM 520 at maximum androgens Adrenal reaction). dehydro- include as 17-KS measured androstenedione, epiandrosterone, and androsterone, etiocholanolone, In men, reduced metabolites. their are of the 17-KS two-thirds about while cortex, by the adrenal secreted by all are secreted almost in women, both Thus, in cortex. the adrenal adrenal reflects 17-KS excretion sexes production. androgen Method • • • Specimen Requirements of a 24-h aliquot refrigerated 20 mL 10 mL minimum urine; acid 10 g of boric urine with Collect use 30 mL alternatively (preferred); 6N acetic acid. or 25 mL 50% HCL and on vial total volume Record form. request 119 g/24-h mg/24-h Corticosteroids, estrogen, oral estrogen, Corticosteroids, ACTH deficiency ACTH Addison’s disease Addison’s ACTH or gonadotropin or gonadotropin ACTH Physiological stress (surgery, (surgery, stress Physiological Precocious puberty caused by caused puberty Precocious Cushing’s syndrome Cushing’s contraceptives stimulation burns, infectious diseases) infectious burns, adrenal hyperplasia adrenal • • • • • • • Assess adrenocortical function adrenocortical Assess 17-20 y17-20 y>20 y>16 <1 y1-4 y5-8 y y9-12 y13-16 9-22 8-20 6-15 <1.0 <2.0 <3.0 3-10 5-12 Some medications may interfere medications Some with test. this 3-8 y y9-12 y13-17 Adults 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 Women Children Creatinine 17-Ketosteroids Men Interpretive Information Interpretive Reference Range Reference Clinical Use 17-Ketosteroids (17-KS), Urine (17-KS), 17-Ketosteroids • 17-Ketosteroids, Fractionated, 4932X 24-Hour Urine
Clinical Use Clinical Background • Assess adrenal and gonadal Androgens secreted by the adrenal androgen status gland or gonads are metabolized and excreted in the urine as 17-ketosteroid Reference Range compounds. Fractionation of specific See next page metabolites allows more specific assessment of androgen status than the Interpretive Information older modified Zimmerman reaction • Hyperandrogenism assay. Detection by mass spectrometry further increases specificity as com- • Cushing’s syndrome pared to capillary gas chromatography. • Adrenal hyperplasia Androstenedione is metabolized to androsterone by 5α-reductase activity • Adrenal insufficiency and to etiocholanolone by 5β-reduct- • Chronic illness ase activity. Both androsterone and • 5α-Reductase deficiency etiocholanolone reflect overall levels of (decreased androsterone/ androstenedione (and, less directly, etiocholanolone ratio) the levels of DHEA, testosterone, and 17-hydroxyprogesterone). The ratio of androsterone to etiocholanolone can be used as a relative index of 5α- reductase to 5β-reductase activity. The 11-oxo and 11β-hydroxy metabolites of androsterone and etiocholanolone tend to reflect the patterns of the parent compounds. Urinary DHEA reflects overall serum DHEA levels. Pregnanetriol is a metabolite of 17-hydroxyprogesterone.
Method • Gas chromatography/mass spectrometry (GC/MS) • Creatinine concentration also reported
Specimen Requirements 5 mL frozen aliquot of a 24-h urine 2.6 mL minimum Refrigerate during collection; do not use preservatives.
Continued 120 Alphabetical Test Section † g/24-h 4932X μg/24-h μg/g creat Not determined; use random urine range † 5-9.9 y5-9.9 y10-13.9 y14-17.9 MenWomen Ratio Androsterone/Etiocholanolone <1 y y1-4.9 71-1000 47-790 y5-9.9 27-214 19-563 y10-13.9 53-530 53-530 15-817 y14-17.9 Creatinine 0.60-15.50 0.25-5.37 0.45-3.43 0.55-3.12 0.74-3.18 3-8 y9-12 y y13-17 Adults 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 11-Oxo-androsterone <6 mo mo6-11.9 y1-4.9 y5-9.9 y10-13.9 y14-17.9 MenWomen11-Oxo-etiocholanolone 13-578 1-200 <1 y y1-4.9 15-111 y5-9.9 8-87 3-69 6-118 y10-13.9 4-104 y14-17.9 8-106 13-84 13-84 AdultsPregnanetriol <1 y y1-4.9 78-1165 16-296 3-493 18-786 68-870 6-765 16-765 ND* ND (16-233creat). μg/g *Notdetermined; use random urine range (49-345 μg/g creat). 121 ) 105 403 740 100 100 100 continued ( μg/24-h creat μg/g -Hydroxyandrosterone -Hydroxyetiocholanolone β β 1-4.9 y1-4.9 y5-9.9 y10-13.9 y14-17.9 Adults 14-680 < 10-363 7-716 8-580 < <1 y y1-4.9 y5-9.9 y10-13.9 y14-17.9 MenWomenDHEA <1 y 240-2300 320-5400 y1-4.9 y 3-225 5-9.9 102-1510 150-2100 4-378 430-2500 16-830 y10-13.9 y14-17.9 <2750 MenWomenEtiocholanolone <1 y y1-4.9 21-2170 21-2170 y5-9.9 < < y10-13.9 24-1640 5-316 < 13-730 15-732 y14-17.9 MenWomen11 <1 y 245-2300 430-3300 y1-4.9 y5-9.9 280-2100 2-261 280-2100 6-29 98-1746 18-465 y10-13.9 104-2006 y14-17.9 MenWomen11 <1 y 195-1500 143-1180 130-900 23-673 100-802 180-850 44-677 180-850 68-1152 < Androsterone Reference Range Reference 17-Ketosteroids, Fractionated, Fractionated, 17-Ketosteroids, Urine 24-Hour Leptin 37109X
Clinical Use Clinical Background • Assess leptin control pathway Leptin is an adipocyte-derived hormone essential for normal body Reference Range weight regulation. Production is under Males Females neuroendocrine control such that ng/mL ng/mL blood levels vary directly with the Adults 1.2-9.5 4.1-25.0 amount of triglyceride stored in (BMI* 18-25) adipose tissue depots; there is a Children† negative correlation between leptin Prepubertal 1.6-10.8 1.7-10.6 and testosterone blood levels. Tanner II-III 2.1-11.6 2.6-11.5 Leptin acts through specific receptors Tanner IV-V 3.4-10.2 3.4-13.0 in the hypothalamus to modulate appetite and thermogenesis; increased *BMI = body mass index = wt (kg)/ht (m2) †Pediatric reference ranges from Clin levels suppress appetite and increase Endocrinol. 1997;46:727-733. thermogenesis. Leptin deficiency leads to hypopituitarism including gonado- Interpretive Information tropin, growth hormone, and thyroid • Obesity deficiencies. Gene mutations resulting in leptin deficiency or leptin receptor • Leptin receptor defect defects lead to massive obesity.
• Under nutrition Method • Leptin deficiency • Radioimmunoassay (RIA) • Analytical sensitivity: 0.5 ng/mL
Specimen Requirements 1.0 mL refrigerated serum 0.2 mL minimum No additive red top preferred SST red top acceptable
122 Alphabetical Test Section 615X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 0.7 IU/L sensitivity: Analytical This assay is appropriate for most for assay is appropriate This "third generation" The LH purposes. sensitive 36086X) is (test code assay and is more mIU/mL to 0.03 use in children. for appropriate Clinical Background lutropin) (LH, hormone Luteinizing by the produced is a glycoprotein pituitary is anterior gland. Production gonado- by hypothalamic regulated hormone (GnRH) releasing tropin steroid gonadal feedback from and ovulation LH stimulates hormones. production ovarian steroid and the in and progesterone) (estrogen LH controls In the male, female. of testosterone. cell secretion Leydig low are LH concentrations In females, of the phase the follicular during peak midcycle rise to a cycle, menstrual and, following ovulation, to cause than levels lower fall to ovulation, After phase. the follicular during rise to concentrations LH menopause, those or higher than as high as levels similar peak; in the midcycle found men. in castrated levels are seen high Method • • Specimen Requirements serum refrigerated 1.0 mL minimum 0.5 mL top preferred red No additive red top acceptable SST 123 mIU/mL GnRH analog treatment GnRH Ectopic steroid hormone steroid Ectopic Pituitary LH deficiency Hypothalamic GnRH GnRH Hypothalamic deficiency Menopause Gonadotropin-secreting Gonadotropin-secreting Primary hypogonadism production pituitary tumors • • • • • • •• Diagnose gonadal function and function gonadal Diagnose pituitary disorders Follicular phaseFollicular Mid-cycle phaseLuteal Postmenopausal 1.9-12.5 5.0-52.3 0.5-16.9 8.7-76.3 MenWomen 1.5-9.3 Interpretive Information Interpretive Reference Range Reference Clinical Use LH (Luteinizing Hormone) (Luteinizing LH • LH (Luteinizing Hormone), 36086X Pediatrics
Clinical Use Clinical Background • Diagnose (and differential diagnosis Luteinizing hormone (LH, lutropin) is of) gonadal dysfunction produced by the anterior pituitary • Monitor LH suppressive (Lupron®’) gland. Production is regulated by therapy hypothalamic gonadotropin releasing hormone (GnRH). LH stimulates • Applicable to pediatric samples ovulation and ovarian steroid hormone Reference Range production in the female and testosterone secretion by the testes in Males Females the male. Children mIU/mL mIU/mL GnRH and LH secretion are regulated 1-7 y <0.10 <0.45 by negative feedback systems, whereby 8-9 y <0.44 <3.36 reduced levels of gonadal hormones 10-11 y <2.28 <5.65 stimulate and increased levels inhibit 12-14 y 0.31-5.29 <11.00 circulating LH concentrations. 15-17 y 0.15-5.33 <15.80 LH levels are useful in assessing disorders of gonadal function. Interpretive Information GnRH stimulation of LH is a useful • Primary hypogonadism provocative test in selected patients • Gonadotropin-secreting with delayed puberty and in pituitary tumors confirming pituitary hypofunction in secondary hypogonadism. • Hypothalamic GnRH deficiency • Pituitary LH deficiency Method • Electrochemiluminescence assay (ECL) • Analytical sensitivity: 0.03 mIU/mL • Analytical specificity: 1.1% cross- reactivity with TSH; no cross- reactivity with FSH, GH, hCG, or prolactin
Specimen Requirements 0.5 mL refrigerated serum 0.2 mL minimum No additive red top preferred SST red top acceptable
124 Alphabetical Test Section rmal rmal 16122X L and/or big-big and/or big-big L n a monomeric, n a monomeric, Immunochemiluminometric assay Immunochemiluminometric without PEG with and (ICMA) precipitation 1.0 ng/mL sensitivity: Analytical emove highemove molecular weight iso- biologically active, free form. Two other biologically active, freeform. Two identified:havebeen forms big PRL (40-60big-big kd) and kd). (>100 PRL Big-bigPRL (macroprolactin) ismost IgG anti-PRL of complex a frequently and is believedand PRL tobebio- hyper- Unlike inactive. logically macroprolactinemia prolactinemia, comprised of big PR forms, including macroprolactin,and such1 with treatment. no If the PRL obtained the treated from (ie, sample normalizes is PRL) monomeric and ofthat<50% in the untreated sample, macroprolactin ispresent. mono- A no above still is that PRL meric Clinical Background se- peptide a 23 kd (PRL), Prolactin pituitary the anterior by gland, creted i circulates mostly associated to be does not appear PRL pituitary of increased incidence with Since most PRL cross- assays adenomas. varying in with macroprolactin react high give artificially they may degrees, of macro- presence results in the PRL incorrect to an leading prolactinemia, of hyperprolactinemia. diagnosis In this test, the PRL level is determined was treated 1 of which 2 aliquots, from to glycol (PEG) polyethylene with r may be due to co-existence of macro- be due to co-existence may hyperprolactinemia with prolactinemia a pituitary ruling out necessitates and adenoma. Method • • Specimen Requirements frozen serum 1.0 mL minimum 0.4 mL top preferred red No additive red top acceptable SST preferred fasting Overnight 125 † † * 50% <50% > 10 3.6-12.0 6.1 2.6-18.0 ng/mL ng/mL tin <50% (macro- meric prolactin still above prolactin still meric 50%) indicates presence of Dopamine agonists (eg, agonists Dopamine Breast-feeding Antidepressants Stress (physical and emotional) (physical Stress Hypothalamic or pituitary tumor Hypothalamic Macroprolactinemia bromocriptine, cabergoline, cabergoline, bromocriptine, pergolide) • • • • • • Tanner II-IIITanner IV-VTanner 2.8-11.0 < 3.2-20.0 Tanner ITanner < Rule out macroprolactinemia as a macroprolactinemia out Rule circulating increased of cause concentration prolactin Non-pregnantPregnantPost-menopausal 3.0-30.0 2.0-20.0 10.0-209.0 % monomeric prolac % monomeric Monomeric Prolactin Monomeric Interpretive Information Interpretive Macroprolactin Reference Range Reference Clinical Use total prolactin reference range suggests range suggests reference prolactin total hyperprolactinemia. *ng/mL mono † Macroprolactin Prolactin, % Monomeric Prolactin, Prolactin, Monomeric Prolactin, Macroprolactin prolactin > macroprolactinemia. Children Males Females Prolactin, Total Prolactin, MenWomen 2.0-18.0 • MEN 2 and FMTC Mutations, 36587X Exons 10, 11, and 13-16
Clinical Use Clinical Background • Screen at-risk individuals for MEN 2 Multiple endocrine neoplasia type 2 and FMTC (MEN 2) is an autosomal dominant syndrome associated with a high risk of • Diagnose MEN 2 and FMTC medullary thyroid carcinoma (MTC). • Differentiate between familial and The MEN 2A subtype is associated with sporadic MTC hyperparathyroidism, and both MEN 2A and MEN 2B subtypes are associated Reference Range with an increased risk for pheochromo- Negative (no mutations detected) cytoma. MEN 2B is further character- ized by mucosal neuromas on the lips and tongue and in the gastrointestinal Interpretive Information tract. Mutation present Over 95% of MEN 2A cases and over • FMTC and/or MEN 2A 85% of familial MTC (FMTC) cases have a germline mutation in 1 of 5 • MEN 2B conserved cysteine residues encoded in exon 10 or 11 of the RET proto- oncogene. A point mutation in exon 16 is present in over 95% of MEN 2B cases. Other mutations in these exons and in exons 13, 14, and 15 have also been associated with MEN 2. Once the mutation in an affected family member has been identified, testing of first-degree relatives allows presympto- matic identification of those at risk for MEN 2 and prophylactic treatment. Correlation between specific mutations and aggressiveness of MTC may aid in appropriately timing prophylactic thyroidectomy and pheochromocytoma screening. Genetic testing is recom- mended for all individuals with MTC since RET mutations have been detect- ed in 1% to 20% of those who appear to have sporadic MTC (ie, those without a known family history of MEN 2). Method • Polymerase chain reaction (PCR) and semi-automated DNA sequencing • Analytical specificity: mutations in exons 10, 11, 13, 14, 15, and 16 of the RET proto-oncogene Specimen Requirements 5 mL room temperature whole blood 3 mL minimum Collect blood in a lavender-top (EDTA) or yellow-top (ACD solution B) tube. Alternatively, submit amniotic fluid, bone marrow, tissue biopsy (eg, dissected CVS) or extracted DNA.
126 Alphabetical Test Section 19548X Liquid chromatography, tandem tandem chromatography, Liquid spectrometry mass (LC/MS/MS) 25 pg/mL sensitivity: Analytical cross-reacts specificity: Analytical and with acetaminophen 0.08% tyramine with 3-hydroxy 0.03% (dopamine) Clinical Background and metanephrine Normetanephrine catechol-o- extra-neuronal are the (COMT) transferase methyl catecholamines of the metabolites and epinephrine, norepinephrine of plasma Measurement respectively. for is more sensitive metanephrines pheochromocytoma, of the detection measure- specific, than be less but may Proper of catecholamines. ment results requires of interpretation medication/drug of recent awareness agents, history (eg, antihypertensive pre- and other cocaine) alcohol, severe (eg, stress, factors analytical failure, myocardial heart congestive of influence release that infarction) and catecholamines metanephrines. Method • • • Specimen Requirements plasma EDTA refrigerated 2.5 mL minimum 1.5 mL from abstain preparation: Patient strenuous tobacco, coffee, tea, alcohol, possible, medication and, if exercise, Over- to collection. days prior for 3 Patient fasting is preferred. night condi- in a relaxed be seated should of sample. before collection tion 127 57 148 205 pg/mL Stress Neuroblastoma Pheochromocytoma • • • Diagnose pheochromocytoma Diagnose Normetanephrine < Metanephrines, TotalMetanephrines, < Metanephrine < Interpretive Information Interpretive Reference Range Reference Clinical Use Metanephrines, Fractionated, Plasma Fractionated, Metanephrines, • Metanephrines, Fractionated, Urine 14962X 14961X
Clinical Use Clinical Background • Diagnose pheochromocytoma A diagnosis of pheochromocytoma can be confirmed by measuring urinary Reference Range levels of the catecholamines and the 14962X 14961X catecholamine metabolite vanillyl- Metanephrine μg/24-h μg/g creat mandelic acid. Urinary metanephrine 3 mo-4 y 25-117 Not established determinations are considered the 5-9 y 11-139 106-527 most accurate single screening method for pheochromocytoma. Quantitative 10-13 y 51-275 34-357 assessment using 24-hour urine collec- 14-17 y 40-189 24-302 tion or semiquantitation relative to 18-29 y 25-222 39-146 creatinine excretion is recommended. 30-39 y 36-190 32-134 40-49 y 58-203 33-192 Method >50 y 90-315 21-153 • Liquid chromatography tandem Normetanephrine mass spectrometry (LC/MS/MS) 3 mo-4 y 54-249 Not established • Analytical sensitivity: 5 μg/L 5-9 y 31-398 149-781 Specimen Requirements 10-13 y 67-503 38-523 14-17 y 69-531 14-302 24-Hour urine (14962X) 18-29 y 40-412 91-365 Random urine (14961X) 30-39 y 35-482 67-390 5 mL room temperature aliquot of 40-49 y 88-649 85-514 urine; 1.5 mL minimum >50 y 122-676 108-524 After urine collection, add 6N HCl to Metanephrines, Total maintain a pH below 3. Record patient's age and total volume (24-h 3 mo-4 y 79-345 Not established sample only) on vial and request form. 5-9 y 49-408 255-1167 10-13 y 110-714 86-845 Patient preparation: abstain from medication, tobacco, tea, and coffee 14-17 y 107-741 39-578 for 3 days prior to collection. 18-29 y 94-604 156-442 30-39 y 115-695 94-445 40-49 y 182-739 155-608 >50 y 224-832 149-603 Creatinine mg/dL 0-6 mo 2-32 7-11 mo 2-36 1-2 y 2-128 3-8 y 2-149 9-12 y 2-183 >12 y Males 20-370 Interpretive Information Females 20-320 • Pheochromocytoma • Neuroblastoma • Stress
128 Alphabetical Test Section 17105X 17102X 300 mg/ HPLC 3 mg/mL sensitivity: Analytical y children <18 in Not evaluated those with in Not evaluated disease vascular peripheral also concentration Creatinine reported Clinical Background (nephropathy) impairment Renal type 2 1 and type in both is common urinary in increases Slight diabetes. (micro- of albumin excretion hour) to 299 mg/24 = 30 albuminuria in nephropathy clinical can predict In contrast, with diabetes. patients (> albuminuria clinical 24 hour) usually indicates failing renal failing renal 24 hour) usually indicates early is an Microalbuminuria function. failure, renal of impending indication strict by reversible is potentially which and antihypertensive control glycemic therapy. high performanceThis chroma- liquid detects both method (HPLC) tography albumin reactive immunochemically albumin. unreactive intact and intact unreactive Immunochemically immuno- by is not detected albumin method can The HPLC assays. of more cases detect therefore or than dipstick microalbuminuria methods. immunoassay Method • • • • • Specimen Requirements (17105X) urine 24-Hour (17102X) urine Random urine of a 24-h frozen aliquot 1 mL minimum 0.4 mL use a preservative; Do not avoid glass on Record total vial volume containers. only). (24-h sample request form and 129 300 g/24 h g/24 mg/dL 300 > <30 <30 > agnostic only after agnostic only 30-299 30-299 2006; 29(Suppl 2006; 1): mg/24-h mg/g creat Diabetes Care. Diabetes Routine management of patients of patients management Routine diabetes (ADA-recommended) with Therapeutic monitoring of patients patients of monitoring Therapeutic nephropathy with Early detection of diabetic nephro- of diabetic detection Early pathy Normal Normal (non-diabetic) Micro- albuminuria Clinical albuminuria S4-S42. Reference: American Diabetes Association Association Diabetes American Reference: (ADA). confirmation on 2 to 3 specimens collected collected 3 specimens 2 to on confirmation period.within a 3-6 month Elevated levels are di Microalbumin (adults) Microalbumin urine24-Hour urineRandom Creatinine 3-8 y <30 mg/24-h mg/g creat <30 y9-12 y13-17 Adults0-6 mo mo7-11 1-2 y3-8 y 0.11-0.68 y9-12 0.17-1.41 0.29-1.87 y >12 Males 0.63-2.50 Females 2-32 2-36 2-128 2-149 2-183 20-370 20-320 Interpretive Information Interpretive Reference Range Reference Clinical Use Microalbumin, Intact, Urine Intact, Microalbumin, • • • Nephrogenic Diabetes Insipidus 15028X (Autosomal) Mutations
Clinical Use Clinical Background • Differentiate inherited NDI from Nephrogenic diabetes insipidus (NDI) acquired NDI is characterized by inability of the kidneys to concentrate urine despite • Screen for autosomal NDI carrier the presence of arginine vasopressin status in at-risk individuals (AVP), also known as antidiuretic hormone (ADH). This insensitivity to Reference Range AVP results in polyuria, polydipsia, low Negative (no mutations detected) urinary specific gravity, and high risk of severe dehydration, especially in Interpretive Information affected infants. Mutation present Although acquired NDI is more common than inherited disease, • Nephrogenic diabetes insipidus genetic forms also occur. They can be (affected or carrier) transmitted in X-linked recessive, autosomal recessive, or autosomal dominant patterns. X-linked NDI, caused by mutations in the AVPR2, is the most common genetic cause of NDI. Most cases of autosomal NDI have a recessive mode of inheritance, but about 10% of them (1% of all in- herited NDI) result from mutations with dominant expression. Insertion of the aquaporin 2 (AQP2) protein into the luminal membrane of collecting duct cells is the final step in the antidiuretic action of AVP, provid- ing specific channels that increase water permeability of the membrane. Mutations disrupting the AQP2 gene on chromosome 12 alter the amino acid sequence of the AQP2 protein and cause about 10% of familial NDI. Since about 25 different mutations have been identified in the AQP2 gene, gene sequencing is the method of choice for detecting AQP2 mutations. After identi- fication of a mutation in an affected individual, genetic testing can be used to evaluate other family members. Method • Polymerase chain reaction (PCR) and DNA sequencing • Analytical specificity: mutations in 4 exons of the AQP2 gene Specimen Requirements 5 mL room temperature whole blood 3 mL minimum Collect blood in a lavender-top (EDTA) or yellow-top (ACD solution B) tube
130 Alphabetical Test Section
AVPR2 gene, 15034X mutations. mutations. AVPR2 AVPR2 gene AVPR2 gene is on the X chromo- X on the gene is ) protein is not present on the AVPR2 Polymerase chain reaction (PCR) and sequencing DNA the in mutations specificity: Analytical X-linked AVPR2 After identification of a mutation in an in a mutation of identification After can testing genetic individual, affected be used to evaluateotherfamily members. gene sequencing is the method of method of the gene sequencing is choice for detecting Method • • Specimen Requirements 5 mL room temperatureblood whole 3 mL minimum (EDTA) Collect blood in a lavender-top tube. solution B) or yellow-top (ACD some, thedisorderis most common in males, but somefemale hetero- zygotesalso have symptoms.Since 180 different mutations have about been identified in the Nephrogenic diabetes insipidus (NDI) diabetes insipidus Nephrogenic is characterized by inability of the urine despite to concentrate kidneys vasopressin the presence of arginine known as antidiuretic also (AVP), hormone (ADH). This insensitivity to in polyuria, polydipsia, results low AVP and high risk urinary specific gravity, in especiallyof severe dehydration, affected infants. Although acquired inherited than common is more NDI disease, genetic forms also occur. They X-linked in can be transmitted recessive, autosomal recessive, or autosomal dominant patterns. The X-linked form is the most common cause of inherited NDI. receptor 2 When functional AVP ( gene cause the X on chromosome about 90% of inherited NDI. Because the Clinical Background collecting of membrane basolateral to cells, the kidney is not able duct of action to the antidiuretic respond the Mutations disrupting AVP. 131 Nephrogenic diabetes insipidus insipidus diabetes Nephrogenic or carrier) (affected Screen for X-linked NDI carrier NDI carrier for X-linked Screen females in at-risk status Differentiate inherited NDI from NDI inherited Differentiate NDI acquired Negative (no mutations detected) (no mutations Negative Interpretive Information Interpretive present Mutation • Reference Range Reference Clinical Use Nephrogenic Diabetes Insipidus Diabetes Nephrogenic Mutations (X-Linked) • • Osmolality, Random Urine 678X
Clinical Use Clinical Background • Diagnose and manage fluid and Osmolality is a measurement of the electrolyte disorders amount of nonelectrolyte substances dissolved per kilogram of pure water. Reference Range Urine osmolality is a measure of solute 50-1200 mOsm/kg concentration in the urine and reflects the concentrating ability of the kidney. Interpretive Information The major factors regulating urine • Dehydration osmolality are vasopressin (antidiuretic hormone) and the urine-concen- • Syndrome of inappropriate trating apparatus of the kidney, which ADH secretion (SIADH) responds to vasopressin. Consequently, • Liver disease pituitary and renal disorders, as well as • Heart disease cardiovascular dysfunction impairing renal blood flow, can impair physio- • Overhydration logical regulation of urine osmolality. • Hypokalemia Method • Diabetes insipidus • Freezing point depression
Specimen Requirements 1 mL refrigerated, random urine 0.2 mL minimum Do not use a preservative.
132 Alphabetical Test Section 677X Freezing point depression Freezing Clinical Background of the is a measurement Osmolality substances of nonelectrolyte amount water. pure of per kilogram dissolved solute of is an index osmolality Serum It is the blood. in concentration by hypothalamic- controlled precisely pituitary of posterior regulation to leading secretion, vasopressin by excretion of water modulation serum of Measurement the kidneys. in assessing is helpful osmolality of this dysfunction or effectiveness system. Method • Specimen Requirements serum room temperature 1 mL minimum 0.2 mL top preferred red No additive red top acceptable SST 133 Syndrome of inappropriate of inappropriate Syndrome Hyponatremia Overhydration Diabetes insipidus Diabetes Alcohol poisoning Alcohol Hypernatremia Hyperglycemia Dehydration ADH secretion (SIADH) secretion ADH • • • • • • • • Assess fluid and electrolyte balance electrolyte and fluid Assess 278-305 mOsm/kg mOsm/kg 278-305 Interpretive Information Interpretive Reference Range Reference Clinical Use Osmolality, Serum Osmolality, • Osteocalcin (Bone Gla Protein, BGP) 5586X
Clinical Use Clinical Background • Determine efficacy of therapy in Osteocalcin is a 6-kd protein consti- osteoporosis and metastatic bone tuting 1% to 2% of total bone protein. disease It contains 3 gamma-carboxyglutamic • Useful marker of bone formation acid (Gla) residues that bind hydroxy- and remodeling lapatite. Osteocalcin is produced exclusively by bone osteoblasts, and Reference Range production is dependent on 1,25- dihydroxyvitamin D, vitamin K, and ng/mL vitamin C. Measurement provides a Men 11.3-35.4 specific biochemical index of bone Women 7.2-27.9 activity. As assessed by bone histo- Children morphometry, serum osteocalcin 6-9 y 40.2-108.0 levels correlate with bone formation 10-13 y 35.8-165.5 and not directly with bone resorption. 14-17 y Serum osteocalcin is an important bone formation marker. Males 27.8-194.1 Females 16.3-68.7 Method Interpretive Information • Immunoradiometric assay (IRMA) • Primary hyperparathyroidism • Analytical sensitivity: 2.0 ng/mL • Secondary osteosarcoma Specimen Requirements • Healing bone fractures 1 mL frozen serum • Hyperthyroidism 0.2 mL minimum • Paget’s disease No additive red top preferred • Therapeutic agents such as SST red top acceptable phenytoin and 1,25-dihydroxy- Overnight fasting is preferred. vitamin D Avoid hemolysis and lipemia. • Hypoparathyroidism • Cushing’s syndrome
134 Alphabetical Test Section 4789X Extraction, radioimmunoassay (RIA) radioimmunoassay Extraction, 50 pg/mL sensitivity: Analytical no cross- specificity: Analytical or insulin with glucagon reaction Clinical Background is a 32- (PP) polypeptide Pancreatic is produced that peptide acid amino cells of the endocrine by the primarily extracts present in it is also pancreas; The physiological mucosa. of gastric clearly defined. of PP is not role by hypo- is influenced Secretion and injection, pentagastrin glycemia, Plasma meals. of protein ingestion with change normally concentrations time and ingestion, type of food age, collection. of blood in the are used PP concentrations tumors. PP-producing of management observed have been plasma levels High with pancreatic patients in some and (VIPomas tumors endocrine endocrine multiple insulinomas), (MEN 1), type 1 syndrome neoplasia and medullary carcinoma, thyroid syndrome. Zollinger-Ellison level MEN 1, a PP with In patients the age-matched than 3 times greater an islet suggestive of mean is normal cell tumor. Method • • • Specimen Requirements plasma EDTA refrigerated 2 mL minimum 0.6 mL is preferred. fasting Overnight 135 163 180 276 644 519 361 297 480 1990;30:65-70, 1990;30:65-70, 1989;21:38-42, and 1980;69:211-214. Reprod Nutr Dev. Nutr Dev. Reprod Zollinger-Ellison syndrome Zollinger-Ellison Medullary carcinoma thyroid Insulinoma VIPoma • • • • Preterm infants*Preterm < Term infants*Term < Term infants-6 d* infants-6 Term y*1 mo-2 3-9 y y10-13 y14-17 < < < < < 30-39 y30-39 y40-49 y50-62 y>62 blood Cord 70-400 established Not 70-430 100-780 Predict recurrence of pancreatic of pancreatic recurrence Predict tumors Monitor pancreatic carcinoma carcinoma pancreatic Monitor therapy Assist in early diagnosis of pancreatic diagnosis of pancreatic Assist in early tumors Assess pancreatic tumor burden tumor pancreatic Assess 18-29 y18-29 < Children *Data from *Data from Adults pg/mL Materia Medica Polona. Acta Paediatr Scand. Interpretive Information Interpretive Reference Range Reference Clinical Use Pancreatic Polypeptide Pancreatic • • • • Pheochromocytoma Gene 19568X, 19567X, Mutations 19571X
Clinical Use Clinical Background • Diagnose or exclude pheochromo- Pheochromocytomas are catechola- cytoma or paraganglioma caused by mine-producing tumors that arise from mutations in the SDHB (test code chromaffin cells, the largest compact 19568X), SDHD (test code 19567X), collection of which is in the adrenal and VHL (test code 19571X) genes medulla. Tumors that arise within the • Screen individuals who have a family adrenal medulla are referred to as history of pheochromocytoma and/ pheochromocytomas, whereas those or paraganglioma arising outside the adrenal are called • Assist in treatment selection for paragangliomas or extraadrenal patients with a gene mutation pheochromocytomas. These tumors are characterized by hypertension Reference Range and elevated levels of catecholamines and should be differentiated from Negative (no mutations detected) ganglioneuromas. Although affecting Interpretive Information only a small percentage of patients with hypertension, pheochromo- SDHB mutation present cytomas are usually curable; however, • Abdominal or thoracic they are often fatal if left untreated. pheochromocytoma Pheochromocytoma is familial in up • Paraganglioma type 4 to 25% of affected individuals; age of SDHD mutation present onset is <30 years. The genes involved • Head or neck pheochromocytoma include NF-1; RET (see MEN and FMTC Mutations, test code 36587X); • Paraganglioma type 1 succinate dehydrogenase subunits B VHL mutation present (SDHB), C (SDHC), and D (SDHD); • Adrenal or thoracic and VHL. Pheochromocytoma causing pheochromocytoma NF-1 and SDHC mutations are rare. • von Hippel-Lindau syndrome Method • Polymerase chain reaction (PCR) and DNA sequencing • Analytical specificity: SDHB (8 exons) (test code 19568X), SDHD (4 exons) (test code 19567X), and VHL (3 exons) (test code 19571X); large deletions and deep intronic mutations affecting mRNA splicing are not detected
Specimen Requirements 5 mL room temperature whole blood 3 mL minimum Collect in lavender-top (EDTA) tube
136 Alphabetical Test Section 10537X Low Low High eful in evaluating erization studies, Analytical sensitivity: 0.37 ng/mL/h Angiotensin I generation, radio- immunoassay (RIA) • Specimen Requirements plasma EDTA 3 mL frozen 0.4 mL minimum refrigerated temperatures.Avoid When submitting cathet 1°aldosteronism Low Hyporeninemic High Low hypoaldosteronism Renin AldosteronePotassium Low Method • the at sample each of a portion retain referring laboratory. Patient preparation: moderate sodium no ambulatory 30 minutes; for diet; medications,3 weeks preferably for collection. sample to prior Renin is a proteolytic produced enzyme is modulated Secretion by the kidney. Erect blood flow. renal in by changes hemor- posture, sodium depletion, cardiacand low output all rhage, increase renin reducing secretion by flow. of plasma renin measurement The activity (PRA) us is Clinical Background hypertension. Primary hyperaldos- teronism is associated with sodium retention, increased blood volume, and low renal blood flow, increased PRA rules high or Thus, a normal PRA. out primary aldosteronism. Conversely, a normal or low PRArule helps out an Additionally, renal hypertension. elevated PRA may indicate renovascular hypertension due to renal artery stenosis. The interpretation of PRA values is facilitated by measurementsof aldosterone and serum potassium levels. 137 – –
0.65 >1.5 15.0 17.0 16.0 16.0 <0.65 >10.0 > <0.65 rd 1/3 1976;89:256; 1976;89:256;
Eur J Pediatr. J Eur †† 5.95.34.4 < < < 7.5 < 15.0 10.0 sis of hypertension egnant individuals egnant individuals J Pediatr. Supine Upright § † 1979;13:817; and and 1979;13:817; Essential hypertension ( hypertension Essential Primary aldosteronism Hyporeninemic hypo- Hyporeninemic Medications (eg, diuretics, diuretics, (eg, Medications Secondary hypoaldosteronism Addison’s disease Addison’s Renal hypertension Renal aldosteronism ACE inhibitors) ACE of cases; PRA <0.65 ng/mL/h) PRA <0.65 of cases; • • • • • • • possible tension likely tension possible tension more likely tension tension likely tension In ambulatory, briefly seated hypertensive briefly seated In ambulatory, 7-9 y y10-12 y13-15 < < < Primary aldosteronism Sodium/volume hyper- Sodium/volume hyper- Renovascular hyper- Renovascular SupineUprightUpright/sitting 0.65-5.0 0.3-3.0 0.4-8.8 Assess renin-aldosterone axis Differentialdiagno Renin-mediated hyper- Renin-mediated 3-12 mo3-12 < 1-3 y4-6 y < < To be used in conjunction with aldosterone be used in To Pediatricfrom data Clinical Cut-off Values Cut-off Clinical concentration; this concentration; condition. is a rare 1994;153:284. Pediat Res. § † †† *In apparently healthy, non-hypertensive, non-hypertensive, *In apparently healthy, non-pr non-medicated, on unrestricteddiets.on Adults* ng/mL/h ng/mL/h Children patients. Interpretive Information Interpretive Reference Range Reference Clinical Use Plasma Renin Activity (PRA) Activity Renin Plasma • • Pregnanetriol, Urine 738X
Clinical Use Clinical Background • Diagnose congenital adrenal hyper- Pregnanetriol is a metabolite of plasia (CAH) (21-hydroxylase the adrenal steroid precursor, deficiency) 17-hydroxyprogesterone. 21-Hydroxyl- • Therapeutic monitoring of patients ase catalyzes the conversion of with 21-hydroxylase deficiency 17-hydroxyprogesterone to 11-deoxy- cortisol. However, when there is Reference Range decreased 21-hydroxylase activity, Pregnanetriol μg/24-h 17-hydroxyprogesterone accumulates Males 71-1000 in the blood and its breakdown product, pregnanetriol, accumulates Females 47-790 in urine. μg/g creat 17-Hydroxyprogesterone is also a Males and females 53-530 normal secretory product of the Creatinine g/24-h ovary. There is a significant increase in 3-8 y 0.11-68 secretion at midcycle, coinciding with the LH peak, and an increase during 9-12 y 0.17-1.41 the luteal phase, correlating with 13-17 y 0.29-1.87 increases in estradiol and progesterone >18 y 0.63-2.50 secretion. Pregnanetriol subsequently increases. Thus, ovarian disorders can Interpretive Information be associated with abnormal preg- • CAH caused by 21-hydroxylase nanetriol levels as well. enzyme deficiency • Stein-Levinthal syndrome Method • Adrenal and ovarian tumors • Gas chromatography/mass spectrometry (GC/MS) • 17-hydroxylase deficiency • Creatinine concentration also syndrome (rare) reported • Ovarian failure post puberty Specimen Requirements 5.0 mL frozen aliquot of a 24-h urine collection; 2.1 mL minimum Refrigerate during collection; do not use preservatives.
138 Alphabetical Test Section
5 U -HSD -HSD β 31493X C-21 steroid, C-21 steroid, 5 U -HSD) deficiency, an unusual form an unusual deficiency, -HSD) -hydroxysteroid dehydrogenase dehydrogenase -hydroxysteroid β Extraction, chromatography, chromatography, Extraction, (RIA) radioimmunoassay 10 ng/dL sensitivity: Analytical β deficiency, the test can evaluate gluco- test can evaluate the deficiency, The therapy. replacement corticoid the in suggesting is also helpful assay tumor, virilizing an adrenal of presence secrete these tumors all of since almost and DHEA-sulfate amounts of large pregnenolone. are moderately levels Pregnenolone due to disease in Cushing’s increased pituitary or adenoma ACTH-secreting causes. ectopic Method • • Specimen Requirements serum refrigerated 4 mL minimum 1.1 mL top preferred red No additive red top acceptable SST steroids. After diagnosis of 3 of After diagnosis steroids. Clinical Background of the cells steroid-producing Normal synthesize gonads glands and adrenal from circulating steroids various small in results This ester. cholesterol as such of precursors amounts a pregnenolone, leaking into the circulation. Most of Most circulation. into the leaking from is pregnenolone the circulating are and levels cortex, the adrenal after ACTH increased modestly and children in normal administration adults. in diagnosing assay is useful This 3 (3 hyperplasia adrenal of congenital cortisol with blocked associated of levels and increased synthesis 139 ng/dL ng/dL J Clin Endocrinol Metab. J Clin -hydroxysteroid -hydroxysteroid β Normal children and adults and adults children Normal Cushing’s disease Cushing’s Adrenal virilizing tumor virilizing Adrenal Congenital adrenal hyperplasia adrenal Congenital after ACTH administration ACTH after MalesFemales 11-50 20-77 37-150 91-220 MalesFemales 10-45 15-84 58-110 33-140 • • • • Tanner IV-V Tanner <1 y1-5 y y6-12 II-III Tanner 10-140 49-360 10-48 15-45 34-135 39-105 ACTH stimulation Baseline stimulation ACTH 60 min DHEA-sulfate DHEA 17-Hydroxypregnenolone Diagnose 3 Diagnose tumors virilizing adrenal Diagnose dehydrogenase deficiency dehydrogenase MenWomenChildren 10-230 10-200 1992;75:1491-1496. Pediatric data from Related Assays Related Interpretive Information Interpretive Reference Range Reference Clinical Use Pregnenolone • • • • • Progesterone 17183X
Clinical Use Clinical Background • Assess ovarian function In the cycling female, the primary • Assess abnormal pregnancy source of progesterone is the corpus • Detect progesterone-secreting luteum. After ovulation, a corpus tumors luteum is formed, and there is a dramatic rise in progesterone levels Reference Range that persists for about 2 weeks. If preg- nancy occurs, corpus luteum survival Men ng/mL ng/mL is prolonged, and progesterone is 18-29 <0.3 necessary to maintain pregnancy. As 30-39 <0.2 pregnancy advances, progesterone is 40-49 <0.2 secreted by the placenta. Decreased 50-59 <0.2 levels of progesterone are seen in the Women short and inadequate luteal phase and Early follicular phase <0.6 in the first trimester of abnormal Late follicular phase <14.5 pregnancies. Mid-cycle <16.1 Progesterone is secreted by the Luteal phase <31.4 adrenal gland in adult males and in Postmenopausal <0.2 children. In addition, high levels of progesterone can indicate tumors of Children Males Females the adrenals or ovaries. 5-9 y <0.7 <0.6 10-13 y <1.2 <10.2 Method 14-17 y <0.8 <11.9 • Liquid chromatography, tandem mass spectrometry (LC/MS/MS) Interpretive Information • Includes online extraction, HPLC • Ovarian tumor separation, and MS/MS • Pregnancy • Analytical sensitivity: 0.1 ng/mL • Adrenal tumor • Selected steroid hormone Specimen Requirements biosynthetic defects 1 mL refrigerated serum (no additive red top tube); 0.25 mL minimum • Amenorrhea SST red top unacceptable • Fetal death An early-morning specimen is • Threatened abortion preferred. • Gonadal agenesis Specify age, sex, and menopausal status on test request form.
140 Alphabetical Test Section 760X Radioimmunoassay (RIA) Radioimmunoassay 5.0 pmol/L sensitivity: Analytical Clinical Background islet the beta in is produced Proinsulin into is cleaved and the pancreas of cells to release prior and C-peptide insulin a small Normally, the circulation. into 3%) (2% to of proinsulin amount and is secreted conversion escapes cell beta during with the insulin along a slight has Proinsulin stimulation. and severely action, hypoglycemic hyperinsulinemic hypoglycemic of to 40% that up have shown patients is insulin serum immunoreactive their material. proinsulin-like that most clinical disorder The in elevated results consistently is the insulinoma, proinsulin serum cell tumor islet or malignant a benign of Increased levels pancreas. of the in also been reported have proinsulin failure, renal with chronic patients hyper- and familial hyperthyroidism, insulinemia. Method • • Specimen Requirements frozen serum 1 mL minimum 0.8 mL top preferred red No additive red top acceptable SST is required. fasting Overnight 141 18.8 pmol/L Familial hyperproinsulinemia Familial Hyperthyroidism Chronic renal failure renal Chronic Severe hypoglycemic hypo- hypoglycemic Severe Insulinoma insulinemia • • • • • Monitor therapy in patients with in patients therapy Monitor insulinomas Diagnose insulinomas Diagnose Adults < Interpretive Information Interpretive Reference Range Reference Clinical Use Proinsulin • • Prolactin 746X
Clinical Use Clinical Background • Diagnose and manage pituitary Prolactin is a protein hormone adenomas secreted by the anterior pituitary • Differential diagnosis of male and gland and the placenta. It may female hypogonadism modulate the number of follicles developing in the follicular phase of Reference Range each menstrual cycle. During and ng/mL following pregnancy, prolactin, in Men 2.0-18.0 association with other hormones, stimulates breast development and Women milk production. Nonpregnant 3.0-30.0 Pregnant 10.0-209.0 Prolactin secretion is stimulated by sleep, stress (physical and emotional), Postmenopausal 2.0-20.0 and the hypothalamic hormone, thyro- Children tropin releasing hormone (TRH). Males Prolactin secretion is decreased by Tanner I <10 dopamine and dopamine analogs Tanner II-III <6.1 such as bromocriptine. Tanner IV-V 2.8-11.0 Hypersecretion of prolactin can be Females caused by pituitary tumors, hypo- Tanner I 3.6-12.0 thalamic disease, breast or chest wall Tanner II-III 2.6-18.0 stimulation, hypothyroidism, renal Tanner IV-V 3.2-20.0 failure, acute exercise, stress, eating, and several medications (eg, pheno- Interpretive Information thiazines, metoclopramide). Hyper- prolactinemia inhibits gonadotropin • Hypothalamic or pituitary tumor secretion and can produce hypo- • Stress (physical and emotional) gonadism in men and women with • Antidepressants accompanying low or inappropriately • Breast-feeding "low normal" LH and FSH levels.
• Bromocriptine (dopamine Method agonist) • Immunochemiluminometric assay (ICMA), ADVIA Centaur®’ • Analytical sensitivity: 1.0 ng/mL
Specimen Requirements 1 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable Overnight fasting is preferred.
142 Alphabetical Test Section 36578X Radiobinding assay (RBA) assay Radiobinding Clinical Background auto- (PTH) hormone Parathyroid to misleading may lead antibodies by PTH measuring when results auto- Such methods. immunoassay be seen but may are rare antibodies in and disease states in autoimmune receiving PTH who are patients for osteoporosis. therapy Method •• Specimen Requirements serum refrigerated 1 mL minimum 0.2 mL top preferred red No additive red top acceptable SST 143 PTH therapy for osteoporosis therapy for PTH Autoimmune disease Autoimmune • • Assess possibility of antibody of antibody possibility Assess interference PTH measure- with ments Negative Interpretive Information Interpretive Reference Range Reference Clinical Use PTH Antibody PTH • PTH, Intact and Calcium 8837X 36736X
Clinical Use Clinical Background • Discriminate primary hyper- Parathyroid hormone (PTH) acts to parathyroidism from tumor hyper- increase calcium absorption from the calcemia gut and to mobilize calcium from bone. • Diagnose hypoparathyroidism The net effect is to increase the extra- cellular concentration of calcium and • Monitor severity of secondary prevent hypocalcemia. PTH secretion hyperparathyroidism in chronic by the parathyroid gland is modulated renal failure by serum calcium concentration. Low calcium stimulates and high calcium Reference Range inhibits PTH secretion. PTH, Intact pg/mL PTH levels are used to assess disorders Adults 10-65 of calcium metabolism, including primary and secondary hyperpara- Children thyroidism, tumor hypercalcemia, and 6-9 y 9-59 hypoparathyroidism. 10-13 y 11-74 Method 14-17 y 9-69 PTH (8837X, 36736X) Calcium mg/dL • Immunochemiluminometric assay Adults 8.8-10.1 (ICMA) Ionized Calcium mg/dL • Analytical sensitivity: 3 pg/mL Adults 4.6-5.4 • Analytical specificity: 100% cross- Children reactivity with intact PTH (amino 8 mo-10 y 4.9-5.4 acids 1-84) and 45% with the 7-84 C-terminal fragment; no detectable 11-17 y 4.8-5.3 cross-reactivity with other PTH Pediatric data from J Pediatr. 1989;114:952- fragments or with calcitonin 956 and Eur J Pediatr. 1991;150:464-467. Calcium, total (8837X) Interpretive Information • Spectrophotometry • Primary hyperparathyroidism • Analytical sensitivity: 0.2 mg/dL • Secondary hyperparathyroidism Calcium, ionized (36736X) • Renal failure • Ion specific electrode • Analytical sensitivity: 0.4 mg/dL • Pseudohypoparathyroidism Specimen Requirements • Hypoparathyroidism PTH and total calcium (8837X) • Hypercalcemia of malignancy 2 mL frozen serum 1 mL minimum (infants, 0.5 mL) PTH and ionized calcium (36736X) PTH 2 mL frozen serum (no additive red top or SST red top tube); 1 mL minimum Calcium, ionized 2 mL refrigerated serum (SST red-top tube, centrifuged, unopened; 0.6 mL minimum
144 Alphabetical Test Section 34478X Immunoradiometric assay (IRMA) Immunoradiometric 2.5 pmol/mL sensitivity: Analytical PTH-RP; intact specificity: Analytical with cross-reactivity no detectable mid-molecule, (C-terminal, PTH or intact) acids 39-84, amino Clinical Background protein hormone-related Parathyroid amino-terminal has an (PTH-RP) to parathyroid similar structure to bind PTH-RP can (PTH). hormone is and receptors stimulate PTH and It is by solid tumors. mainly produced normal lactating found in the also mammary keratinocytes, gland, gland. and parathyroid placenta, high in approxi- levels are PTH-RP tumor- with patients 70% of the mately be may It hypercalcemia. associated of diagnosis in the differential useful specifically differen- hypercalcemia, primary tiating hyperparathyroidism to cancer. related hypercalcemia from primary in chronic or elevated is not It failure. renal Method • • • Specimen Requirements frozen plasma 1.5 mL minimum 1.0 mL red-top syringe or blood in Collect to a transfer and immediately tube PTH-RP Institute special Nichols cold, mix thoroughly. tube; collection centrifuge. in refrigerated Centrifuge and tube to a plastic plasma Transfer freeze. 145 Newborn Fetus Tumor-associated hypercalcemia Tumor-associated • • • Manage patients with solid tumors tumors with solid patients Manage hypercalcemia and Differential diagnosis of hyper- of diagnosis Differential calcemia 4.7 pmol/L 4.7 < Interpretive Information Interpretive Reference Range Reference Clinical Use PTH-related Protein (PTH-RP) Protein PTH-related • • Pyridinium Collagen Cross-links, Urine 36098X (PYD and DPYD) 36097X
Clinical Use Clinical Background • Assess rate of bone collagen Collagen fibers are linked together by degradation interchain molecules referred to as • Monitor response to therapy pyridinium cross-links. Collagen pyridinium cross-links have been Reference Range identified in all connective tissue PYD DPYD except skin. As collagen is broken nmol/mmol nmol/ down by collagenase, small breakdown creat mmol creat products are excreted in urine with Men the attached cross-links, including 24-h urine 20-61 3.7-19 pyridinoline (PYD) and deoxy- 2-h urine 23-65 6.2-26 pyridinoline (DPYD). Bone collagen is constantly turning over and is rich in Women* DPYD. Thus, urine PYD and, especially, 24-h urine 22-89 4.4-21 DPYD are useful markers for bone 2-h urine 25-83 5.7-23 matrix degradation and resorption. Children (24-h urine) 2-10 y 158-442 31-112 Method 11-14 y 107-398 17-101 • High-performance liquid chroma- 15-17 y 42-200 <59 tography (HPLC) Children (2-h urine) • Analytical sensitivity: 20 pmol/mL 2-10 y 153-401 39-120 (PYD); 13 pmol/mL (DPYD) 11-17 y 17-408 3.0-117 • Creatinine concentration also Tanner I 112-353 27-103 reported Tanner II 95-471 20-139 Specimen Requirements Tanner III 68-489 14-135 Tanner IV 17-448 10-136 24-Hour urine (36098X) 5 mL refrigerated aliquot of a 24-h urine; 1 mL minimum Creatinine Random 24-h urine Collect urine with 25 mL 6N HCl urine mg/dL g/24-h or 10 g of boric acid. Mix and aliquot. 0-6 mo 2-32 Record total volume on vial and 7-11 mo 2-36 request form. 1-2 y 2-128 2-Hour urine (36097X) 3-8 y 2-149 0.11-0.68 5 mL refrigerated aliquot of a 2-h 9-12 y 2-183 0.17-1.41 urine; 0.5 mL minimum 13-17 y 0.29-1.87 Discard first morning void. Collect Adults 0.63-2.50 urine for next 2 hours, in presence of >12 y 2 mL 6N HCl or 1 g of boric acid. Mix Male 20-370 well and aliquot. Female 20-320 Interpretive Information *Premenopausal. A value within the pre- menopausal range does not rule out osteo- • Osteoporosis porosis or the need for therapy. • Primary hyperparathyroidism • Paget’s disease • Relative to baseline indicates successful therapy
146 Alphabetical Test Section 16053X gene and gene THRB terpreted in light mutations. THRB ) gene that alter the the hormone- alter ) gene that Polymerase chain reaction (PCR) and chain reaction Polymerase sequencing DNA Analytical specificity: mutations in exons 3-10 of the regions intronic flanking their THRB Specimen Requirements whole blood 5 mL room temperature 3 mL minimum tube (EDTA) Collect in lavender-top of clinical and family data. Genetic of clinical and family data. Genetic counseling may be helpful for individuals and families undergoing for testing Method • • Resistance to thyroid hormone (RTH) to Resistance thyroid hormone (RTH) reducedis caused by target tissue to thyroid resulting hormone, response feedback of the normal in the loss secretion suppresses the that mechanism of thyrotropin (TSH). It is characterized serum levels of elevated by the presence thyroxine thyroid hormones of the free (T4) and triiodothyronine (T3) with In 90% or normal TSH. elevated slightly is caused by mutations in of cases, RTH the thyroid hormone receptor beta ( binding region of the protein. The disorder is usually inherited in an over and manner, autosomal dominant 100 different mutations have been described. mutations can give New rise of RTH. cases to isolated and other variation Since genetic accuracy of the problems can affect results test testing, direct mutation should always be in Clinical Background 147 Resistance to thyroid hormone to thyroid Resistance Screen individuals at risk for at risk for individuals Screen hormone to thyroid resistance Differential diagnosis of hyper- of diagnosis Differential thyroidism Negative (no mutations detected) (no mutations Negative Interpretive Information Interpretive present Mutation Reference Range Reference Clinical Use Resistance to Thyroid Hormone Thyroid to Resistance • • • Serotonin 818X 29851X
Clinical Use Clinical Background • Diagnose paraneoplastic carcinoid Carcinoid syndrome manifests with syndrome flushing, diarrhea, pain, and hepatomegaly. The ileum, stomach, Reference Range pancreas, duodenum, bronchus, ng/mL thymus, thyroid, testis, and ovary may Blood (818X) 22-180 be involved. Small bowel carcinoids most commonly metastasize to the Serum (29851X) liver. These tumors secrete increased Adults 26-165 amounts of serotonin, which metabo- Children (1-12 y) 81-349 lizes to 5-hydroxyindoleacetic acid Pediatric reference range from Brazilian J (5-HIAA). Measurement of serotonin Med Biol Res. 1993;26:309-317. concentration may be helpful in selected cases. Interpretive Information • Carcinoid syndrome Method • Medullary thyroid carcinoma • High performance liquid chroma- • Multiple endocrine neoplasia tography (HPLC) (MEN) types 1 and 2 • Fluorescent detection • Oat cell carcinoma of the lung Specimen Requirements • Islet cell tumors Whole blood (818X) • Depression 4 mL frozen whole blood 1 mL minimum Collect blood using the Serotonin Kit (provided by Quest Diagnostics) Serum (29851X) 2 mL frozen serum 1 mL minimum No additive red top preferred SST red top acceptable Patients should avoid avocados, bananas, tomatoes, plums, walnuts, pineapples, eggplant, tobacco, tea, and coffee for 3 d prior to specimen collection.
148 Alphabetical Test Section 30740X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 2 nmol/L sensitivity: Analytical Clinical Background The majority of circulating gonadal bound. are protein hormones steroid and dihydrotestosterone, Testosterone, circulating bound to are the estrogens (SHBG globulin binding sex hormone globulin, or testosterone-binding the liver. by produced which is TeBG), steroid total circulating Consequently, by altera- are affected levels hormone as well as concentration tions in SHBG them- the hormones of concentration of the gonadal Evaluation selves. system, therefore, hormone steroid serum of total measurements includes dihydrotestosterone, testosterone, to in addition species or estrogen the free hormones of measurements and SHBG. are increased concentrations SHBG and pregnancy and by estrogens Insulin by testosterone. decreased insulin in high SHBG levels lowers polycystic obesity, (eg, diabetes, states ovary syndrome). Method • • Specimen Requirements serum room temperature 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST form. sex on request age and Specify 149 Males Females Exogenous androgen therapy therapy androgen Exogenous Massive obesity Massive Virilization Hirsutism Testicular feminization Testicular Hyperthyroidism Male hypogonadism Male Alcoholism Pregnancy Estrogen administration Estrogen or abuse • • • • • • • • • • IIIIIIIVV 39-155 33-135 21-72 38-114 11-92 24-90 22-112 18-54 22-69 18-76 Assess abnormal serum gonadal serum gonadal abnormal Assess concentrations hormone 18-29 y18-29 y30-39 y40-49 y50-59 y60-69 7-49 y70-79 8-48 y80-91 9-45 6-112 18-47 14-102 3-9 y 17-54 11-100 y10-13 23-65 17-78 y14-17 20-63 17-95 stages Tanner 21-90 18-136 26-77 17-123 11-71 18-136 17-123 11-71 Children Adults nmol/L nmol/L Interpretive Information Interpretive Reference Range Reference Clinical Use Sex Hormone Binding Globulin Binding Hormone Sex • Somatostatin 34480X
Clinical Use Clinical Background • Diagnose somatostatin-producing Somatostatin, also referred to as tumors somatotropin release inhibiting factor (SRIF), is a neurohormone produced Reference Range as a 14-amino acid peptide in the brain Adults 10-22 pg/mL and hypothalamus and as a 28-amino acid N-extended product in the Interpretive Information gastrointestinal tract. In the pituitary • Somatostatinoma gland, SRIF inhibits secretion of growth hormone, TSH, and, under • Medullary thyroid carcinoma certain conditions, prolactin and • Pheochromocytoma ACTH. It exerts inhibitory effects on all endocrine and exocrine secretions • Following vagotomy of the pancreas and gut and a variety of endocrine-secreting tumors including insulinomas, glucagonomas, VIPomas, carcinoid tumors, and gastrinomas. Pancreatic somatostatinomas cause mild diabetes mellitus, cholelithiasis, steatorrhea, indigestion, and hypo- chlorhydria secondary to the inhibitory effects of secreted SRIF. SRIF may also be secreted by medullary thyroid carcinomas or pheochromocytomas.
Method • Extraction and radioimmunoassay (RIA) • Analytical sensitivity: 10 pg/mL
Specimen Requirements 1.8 mL frozen EDTA plasma 0.6 mL minimum Draw in pre-chilled lavender-top tube. Separate and freeze plasma immediately.
150 Alphabetical Test Section 36574X Radiobinding assay (RBA) assay Radiobinding Clinical Background thyroid with autoimmune Patients autoimmune including diseases, Graves and thyroiditis (Hashimoto) antibodies develop may rarely disease, T4 and/or (triiodothyronine) to T3 interfere(thyroxine). T3 antibodies by com- T3 assay measurement with anti-T3 immuno- with the peting the T3 assay. used in globulins con- T3 has a serum a patient When with is discordant that centrati\on serum eg, elevated test results, other of a detectable the presence T3 in it may then concentration, TSH serum if anti-T3 determine to be appropriate are present. antibodies Method • Specimen Requirements serum refrigerated 1 mL minimum 0.1 mL top preferred red No additive red top acceptable SST 151 Other autoimmune disease autoimmune Other Autoimmune thyroid disease thyroid Autoimmune • • Evaluate discordant serum T3 levels serum discordant Evaluate Negative Interpretive Information Interpretive Reference Range Reference Clinical Use T3 Antibody T3 • T3 Uptake 17732X
Clinical Use Clinical Background • Assess T4 (thyroxine) protein Thyroid hormones circulate bound to binding several binding proteins. The most • Indirect estimate of free T4 significant among these is thyroxine- concentration binding globulin (TBG). TBG levels are influenced by a variety of factors Reference Range including genetic abnormalities, Adults 0.79-1.16 pregnancy, oral contraceptives, glucocorticoids, androgens, and Interpretive Information renal disease. Fluctuations in TBG concentrations lead to changes in T4 • Hyperthyroidism and T3 levels that may generate a • Renal failure false diagnosis of thyroid dysfunction • Malnutrition in basically euthyroid patients. There are several methods of assessing • Hypothyroidism TBG-binding activity in serum. The T3 • Pregnancy uptake provides an indirect assessment • Acute hepatitis of TBG binding. Chemiluminescence- labeled T3 competitively binds to a solid phase-bound antibody or the patient TBG. The T3 bound to anti- body is measured relative to that in a standard serum. Increased antibody binding reflects decreased TBG binding (TBG-binding site saturation). Decreased antibody binding reflects increased TBG binding (binding site desaturation).
Method • Immunochemiluminometric assay (ICMA)
Specimen Requirements 1 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable
152 Alphabetical Test Section 34429X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 50 pg/dL sensitivity: Analytical Clinical Background (triiodothyronine) T3 circulating Most 0.3% only proteins; to plasma bound is is and state unbound free, the in exists intra- with for exchange available T3 receptors. cellular are used to diagnose T3 measurements of hyper- monitor treatment and an When T3, Total). (see thyroidism thyroxine- in circulating increase the as is suspected proteins binding the level, total T3 of an elevated cause this differentiate T3 assay can free true hyperthyroidism. from condition T3 immunoassay free Non-dialysis free T3 underestimate to tend methods of free absorption to nonspecific due matrix reagents. proteins or T3 to a free provide useful they do Although free T3 reliable the most T3 index, the equilibrium is measurement method. dialysis Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST 153 of pg/dL 1/3 Hypothyroidism (only Hypothyroidism Nonthyroidal illness Nonthyroidal Functional thyroid adenoma adenoma thyroid Functional Thyroid hormone resistance hormone Thyroid T3 thyrotoxicosis Graves disease Graves (T3-producing) cases) • • • • • • Clarify thyroid status in presence of status in presence thyroid Clarify binding abnormality protein possible Diagnose hyperthyroidism Diagnose <1 y1-9 y y10-13 y14-18 established Not 337-506 335-480 287-455 AdultsChildren 230-420 Interpretive Information Interpretive Reference Range Reference Clinical Use T3, Free, Non-dialysis Free, T3, • • T3, Free, Tracer Dialysis 36598X
Clinical Use Clinical Background • Diagnose hyperthyroidism Most circulating T3 is bound to plasma • Clarify thyroid status in presence of proteins. Only 0.3% exists in the free, possible protein binding abnormality unbound state and is available for ex- change with intracellular T3 receptors. Reference Range T3 measurements are used to diagnose Free T3 pg/dL and monitor treatment of hyper- Non-pregnant adults 210-440 thyroidism (see T3, Total). When an Pregnant women 200-380 increase in circulating thyroxine- binding protein is suspected as the Children cause of an elevated total T3 level, the <1 y Not established free T3 assay can differentiate this 1-9 y 282-518 condition from true hyperthyroidism. 10-13 y 286-556 14-17 y 242-501 Method Total T3 ng/dL • Free T3: equilibrium dialysis, Adults 60-181 radioimmunoassay (RIA) Pregnancy • Total T3: immunochemilumino- 1st trimester 81-190 metric assay (ICMA) 2nd and 3rd trimester 100-260 • Reported free T3 is the product of the 2: (total T3 x DFT3) where DFT3 Children is the dialyzable fraction of T3 <1 y Not established • Analytical sensitivity: 0.01% and 1-9 y 127-221 25 ng/dL for free T3 and total T3, 10-13 y 123-211 respectively 14-18 y 97-186 Specimen Requirements Interpretive Information 1 mL refrigerated serum • Graves disease 0.5 mL minimum • T3 thyrotoxicosis No additive red top preferred • Thyroid hormone resistance SST red top acceptable • Functional thyroid adenoma (T3-producing)
• Nonthyroidal illness • Hypothyroidism (only 1/3 of cases)
154 Alphabetical Test Section 967X Radioimmunoassay (RIA) Radioimmunoassay 0.05 ng/mL sensitivity: Analytical Clinical Background T3, (reverse 3,3´5´-Triiodothyronine 3,5,3´-triiodo- along with is, rT3) a deiodinated (T3), thyronine the (T4), of thyroxine metabolite secretorythe thyroid major product of T3 reverse however, Unlike T3, gland. inert. is metabolically process of 5´-monodeiodina- The rT3 and T4 to T3 that converts tion a in is inhibited to diodothyronine including variety of conditions wide poorly controlled malnutrition, fasting, trauma, surgery, mellitus, diabetes Consequently, systemic illnesses. and decreases, typically T3 level the serum often, the rT3 concentration and these in always, increases but not circumstances. Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.4 mL top preferred red No additive red top acceptable SST 155 Hypothyroidism Hyperthyroidism Low T3 syndrome Low Newborn Fetus Euthyroid sick syndrome sick Euthyroid • • • • • • Establish nonthyroidal illness as the illness nonthyroidal Establish function thyroid abnormal of cause tests 0.11-0.32 ng/mL 0.11-0.32 Interpretive Information Interpretive Reference Range Reference Clinical Use T3, Reverse T3, • T3, Total (Triiodothyronine) 859X
Clinical Use Clinical Background • Diagnose hyperthyroidism Triiodothyronine (T3) is primarily • Reflect tissue monodeiodination produced by extrathyroidal of T4 deiodination of T4, but 20% of it is secreted directly by the thyroid gland. Reference Range Circulating T3 is predominantly bound ng/dL to plasma proteins. Only 0.3% is Adults 60-181 unbound (free) and readily available for tissue exchange and interaction Pregnancy with its nuclear receptor. 1st trimester 81-190 Total T3 measurements are used to 2nd and 3rd trimester 100-260 diagnose and monitor treatment of Children hyperthyroidism. They are essential <1 y Not established for recognizing T3 toxicosis, more 1-9 y 127-221 fully defining the severity of hyper- 10-13 y 123-211 thyroidism, and for detecting 14-18 y 97-186 recurrent Graves disease. Changes in thyroxine-binding protein Interpretive Information concentrations alter the total T3 concentration without affecting the • Graves disease free T3 level; thus, free T3 assays can • T3 thyrotoxicosis differentiate most of these conditions • Hyperthyroidism, TSH from true hyperthyroidism (see T3, dependent Free, Tracer Dialysis). • Increased TBG A T3 measurement is usually not • Pregnancy helpful for diagnosing hypo- thyroidism, since the serum T3 • Nonthyroidal illness (low concentration typically remains T3 syndrome) normal in mild and moderate thyroid • Hypothyroidism (1/3 of cases) gland failure. Systemic illness and malnutrition reduce extrathyroidal • Decreased TBG T3 production, causing a low total T3 concentration that should not be confused with hypothyroidism.
Method • Immunochemiluminometric assay (ICMA) • Analytical sensitivity: 25 ng/dL
Specimen Requirements 1 mL refrigerated serum 0.5 mL minimum No additive red top preferred SST red top acceptable
156 Alphabetical Test Section 36576X Radiobinding assay (RBA) assay Radiobinding Clinical Background thyroid with autoimmune Patients autoimmune including diseases, Graves and thyroiditis (Hashimoto) antibodies develop may rarely disease, and/or T3 (thyroxine) to T4 T4 antibodies (triiodothyronine). interfere assay measurement with T4 the anti-T4 with by competing assay. T4 used in the immunoglobulins T4 has a serum a patient When with is discordant that concentration serum eg, elevated test results, other binding of thyroxine the absence T4 in of presence and excess (TBG) globulin serum TSH concentration, a detectable to it may be appropriate then are antibodies if anti-T4 determine present. Method • Specimen Requirements serum refrigerated 1 mL minimum 0.1 mL top preferred red No additive red top acceptable SST 157 Other autoimmune disease autoimmune Other Autoimmune thyroid disease thyroid Autoimmune • • Evaluate discordant serum T4 levels serum discordant Evaluate Negative Interpretive Information Interpretive Reference Range Reference Clinical Use T4 (Thyroxine) Antibody (Thyroxine) T4 • T4 Binding Proteins 36738X
Clinical Use Clinical Background • Evaluate patients with euthyroid Thyroid hormones normally circulate hyperthyroxinemia and hypo- bound to 1 of 3 classes of plasma thyroxinemic states proteins: thyroxine-binding globulin (TBG), transthyretin (prealbumin, Reference Range preALB), or albumin (see Test See next page Application and Interpretation section). Alterations in T4-binding Interpretive Information affinity and/or concentrations of these Mass of T4 bound to: proteins change the total and, in some TBG PreALB ALB cases, the free T4 concentrations. TBG deficiency ↓ NNThis thyroxine-binding protein panel TBG excess ↑ NNreveals the precise distribution of TBG variant ↓ NNprotein-bound T4 in plasma. TBPA excess N ↑ N Thyroxine-binding globulin excess, TBPA variants N ↑ N familial dysalbuminemic hyper- Dysalbuminemic NN↑ thyroxinemia (FDH), transthyretin- hyperthyroxinemia binding excess, and T4 antibodies can Euthyroid (sick) ↓↓N be differentiated. The panel can also hypothyroxinemia differentiate TBG deficiency and some molecular variants of TBG. Hypothyroidism ↓↓↓ Hyperthyroidism ↑↑↑Method Mass of T4 bound Quantify total T4 and binding proteins per unit mass of: • Immunochemiluminometric assay TBG PreALB ALB (total T4 and TBG) TBG deficiencyNNN• Fixed time nephelometry TBG excess NNN (prealbumin) TBG variant ↓ NN• Spectrophotometry (albumin) TBPA excessNNN TBPA variants N ↑ N Quantify T4 bound to each binding protein Dysalbuminemic NN↑ hyperthyroxinemia • Electrophoresis, radiobinding assay Euthyroid (sick) ↓↓N (TBG-, prealbumin-, albumin-bound hypothyroxinemia T4) Hypothyroidism ↓↓ ↓ Calculate T4 affinity of each binding Hyperthyroidism ↑↑↑protein • TBG-bound T4/TBG • Prealbumin-bound T4/prealbumin • Albumin-bound T4/albumin Specimen Requirements 4 mL refrigerated serum 2 mL minimum No additive red top preferred SST red top acceptable Continued
158 Alphabetical Test Section 36738X ) 159 continued ( μg/g μg/dL μg/mg μg/mL mg/dL 3700-5100 T4/prealbumin
3-8 y y9-13 y14-17 16.1-24.2 12.5-25.8 9.8-23.7 1st trimester trimester2nd trimester3rd All trimesters1-8 y y 6.4-15.2 9-13 7.4-15.2 y14-17 7.7-13.8 7.0-14.7 5.9-11.5 4.7-10.4 5.0-9.8 Adults y) (5-17 Children 0.5-1.0 0.5-1.2 Adults y) (5-17 Children 1.8-4.0 1.0-3.3 Adults y) (5-17 Children 2.6-5.8 2.1-6.2 MenWomenChildren MenWomen 13.5-30.9 12.7-25.1 17-34 21-43 Children AdultsPregnancy 4.8-10.4 Adults y) (5-17 Children 0.08-0.16 0.04-0.11 Adults y) (5-17 Children 2.0-3.0 Adults y) (5-17 Children 1.0-3.0 0.12-0.22 0.11-0.28 Reference Range Reference TBG Albumin-bound T4 Albumin-bound Prealbumin Prealbumin-bound T4 Prealbumin-bound Albumin TBG-bound T4 TBG-bound T4, Total T4, T4 Binding Proteins Proteins Binding T4 Albumin-bound T4/albumin T4/albumin Albumin-bound TBG-bound T4/TBG TBG-bound Prealbumin-bound T4, Free, Direct Dialysis 35167X
Clinical Use Clinical Background • Differentiate euthyroid hyper- Thyroid hormones are transported in thyroxinemia from hyperthyroidism blood bound to several binding • Differentiate euthyroid hypo- proteins. These include thyroxine- thyroxinemia from hypothyroidism binding globulin, prealbumin, and albumin. Only 0.03% of thyroxine Reference Range (T4) is unbound or free, but this free Adults ng/dL fraction provides the hormone 21-87 y 0.8-2.7 available to most tissues. Hyper- and hypothyroidism result from abnormal Pregnancy concentrations of serum free T4. Since 1st trimester 0.9-2.0 free T4 concentrations are never high 2nd trimester 0.8-1.5 enough to influence results of total T4 3rd trimester 0.8-1.7 immunoassays, total T4 assays measure Children (3-20 y) 1.0-2.4 only protein-bound T4. The most accurate method for Interpretive Information determining free T4 is the direct • Hyperthyroidism equilibrium dialysis method. Direct • Thyroid hormone resistance dialysis separates free T4 from protein- bound T4. The free T4 is then • Primary hypothyroidism measured directly from the protein- • Secondary hypothyroidism free dialysate. Results are independent of T4-binding protein concentrations and are unaffected by the presence of molecular variants of these proteins or by circulating thyroid autoantibodies.
Method • Direct equilibrium dialysis, radio- immunoassay (RIA) • Analytical sensitivity: 0.2 ng/dL • Analytical specificity: unaffected by T4 antibodies or variations in T4- binding proteins
Specimen Requirements 2 mL refrigerated serum 0.2 mL minimum No additive red top preferred SST red top acceptable
160 Alphabetical Test Section 866X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 0.1 ng/dL sensitivity: Analytical Clinical Background in are transported hormones Thyroid several binding to bound blood thyroxine- include These proteins. and prealbumin, globulin, binding of thyroxine Only 0.03% albumin. free this or free, but is unbound (T4) the hormone provides fraction and tissues. Hyper- to most available abnormal result from hypothyroidism of serum free T4. Since concentrations are free T4 concentrations serum to influence results high enough never T4 total T4 immunoassays, of total T4. protein-bound only measure assays T4 can distinguish free Determining hyperthyroxinemias the euthyroid hypothyroidism. from free T4 employed The most commonly 2-step 1-step and are the methods may methods These immunoassays. free T4 estimates falsely low provide of free absorption to nonspecific owing reagents. Such or matrix proteins T4 to a useful provide do, however, assays most accurate T4 index. The free T4 is the free for determining method dialysis method. equilibrium direct T4-binding of independent are Results are and concentrations protein of presence by the unaffected proteins or of these variants molecular autoantibodies. thyroid by circulating Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST 161 ng/dL Secondary hypothyroidism Primary hypothyroidism Thyroid hormone resistance hormone Thyroid Hyperthyroidism • • •• • Initial test of thyroid function in in function test of thyroid Initial thyroid with suspected patients also TSH) (see dysfunction Assess thyroid status in patients with in patients status thyroid Assess T4 concentrations total abnormal <1 y1-9 y y10-18 Not established 0.9-1.6 0.9-1.4 AdultsChildren 0.8-1.8 Interpretive Information Interpretive Reference Range Reference Clinical Use T4, Free, Non-dialysis Free, T4, • • T4, Total (Thyroxine) 17733X
Clinical Use Clinical Background • Diagnose hypo- and hyperthyroidism Thyroxine (T4, tetraiodothyronine) is when overt and/or due to pituitary the major secretory product of the or hypothalamic disease thyroid gland. In blood, T4 is bound to 1 of 3 classes of proteins: thyroxine- Reference Range binding globulin (TBG), transthyretin μg/dL (thyroxine-binding prealbumin), or Adult 4.8-10.4 albumin. Only the free T4 fraction Pregnancy (0.03%) is available for exchange with 1st trimester 6.4-15.2 tissues where it is deiodinated to triiodothyronine (T3). T3 then binds 2nd trimester 7.4-15.2 to nuclear receptors and brings about 3rd trimester 7.7-13.8 thyroid hormone actions. The serum All trimesters 7.0-14.7 total T4 concentration generally Pediatric reflects thyroid gland secretory activity. 1-8 y 5.9-11.5 It is elevated in most hyperthyroid 9-13 y 4.7-10.4 patients and low in most patients with 14-17 y 5.0-9.8 hypothyroidism. Although the total T4 assay is analyt- Interpretive Information ically precise, several factors limit • Hyperthyroidism the test's clinical accuracy. Changes in circulating T4-binding protein • Increased TBG concentrations alter the total T4 level • Familial dysalbuminemic without affecting the free T4 level or hyperthyroxinemia causing true thyroid dysfunction. • Increased transthyretin Serious systemic illness causes a low (preALB) total T4 in 25% of patients and a • Estrogen therapy modestly elevated T4 level in 2%; consequently, the serum T4 is not an • Pregnancy accurate measure of thyroid function • Primary hypothyroidism in severely ill patients. • Pituitary TSH deficiency The interpretation of T4 concen- trations and assessment of thyroid axis • Hypothalamic TRH deficiency function are greatly facilitated by • Nonthyroidal illness measurement of circulating TSH • Decreased TBG concentration. Method • Immunochemiluminometric assay (ICMA) • Analytical sensitivity: 0.8 μg/dL Specimen Requirements 1 mL refrigerated serum 0.5 mL minimum No additive red top tube preferred SST red top acceptable
162 Alphabetical Test Section 37351X ]) are more reliable. more ]) are ®’ D], and C-telopeptide C-telopeptide D], and ®’ Enzymatic 1.2 U/L sensitivity: Analytical level measures specificity: Analytical resistant that is phosphatase of acid inhibition to tartrate [CTx, CrossLaps [CTx, Clinical Background phosphatase acid resistant Tartrate by osteo- primarily is secreted (TRAP) erythrocytes also by but and clasts, be can levels Serum macrophages. resorption; of bone measure as a useful newer resorption the however, [NTx], (eg, N-telopeptide markers DYPD, [free deoxypyridinoline free Pyrilinks of bone marker is a potential TRAP with breast in patients metastasis in to increase levels appear cancer; bone number of to the proportion metastases. enzyme affected by is adversely TRAP has limited in serum, inhibitors and sensitivity, and analytical stability for bone. Erythrocytic specificity lacks in released during phosphatase acid non-bone other as as well clotting, vitro values. increase may falsely sources, Method • • • Specimen Requirements frozen serum 1.0 mL minimum 0.3 mL top preferred red No additive red top acceptable SST to serum separation; prior Refrigerate afterwards. immediately freeze 12-h fast required. 163 Hypoparathyroidism Fluoride excess excess Fluoride Anabolic steroid therapy steroid Anabolic Bisphosphonate therapy Bisphosphonate Advanced renal failure renal Advanced Primary hyperparathyroidism Paget's disease Paget's Metastatic cancer Metastatic Osteomalacia Osteoporosis • • • • • • • • • • Detect bone metastasis in patients patients in bone metastasis Detect breast cancer with Assess bone turnover bone Assess Assess osteoclast activity osteoclast Assess Adults U/L 3.0-6.0 Interpretive Information Interpretive Reference Range Reference Clinical Use Tartrate Resistant Acid Phosphatase Acid Resistant Tartrate • • • TBG (Thyroxine-Binding Globulin) 870X
Clinical Use Clinical Background • Differentiate quantitative TBG Thyroxine-binding globulin (TBG) is a derangements from thyroid glycoprotein produced in the liver. dysfunction TBG binds both thyroxine (T4) and triiodothyronine (T3) with high Reference Range affinity and accounts for 75% of μg/mL plasma protein thyroxine-binding Men 12.7-25.1 activity. Thus, an increase or decrease Women 13.5-30.9 in its circulating level alters total Children concentrations of T4 and T3 in blood, leading to potential confusion with 3-8 y 16.1-24.2 true thyroid gland function. A number 9-13 y 12.5-25.8 of diseases and medications, as well as 14-17 y 9.8-23.7 inherited alterations in TBG gene expression, can change the serum TBG Interpretive Information concentration (see Test Application • Pregnancy and Interpretation section). Common • Infancy causes of increased serum TBG include estrogen therapy (especially oral • Familial excess contraceptive agents), pregnancy, and • Hepatitis hepatitis. Conversely, serum TBG may • Estrogen treatment be decreased in cirrhosis, in the • Tamoxifen treatment nephrotic syndrome, and by androgens. • Androgenic steroid treatment The measurement of TBG can be used • Glucocorticoids (large dose) to establish the presence of TBG deficiency or excess suggested by • Nephrotic syndrome abnormal total serum T4 and T3 • Acromegaly concentrations in the presence of • Familial deficiency normal free levels of these hormones. Definitive documentation of a TBG derangement may avoid unnecessary diagnostic procedures and therapy in individuals with harmless congenital TBG anomalies, and in their relatives.
Method • Immunochemiluminometric assay (ICMA) • Analytical sensitivity: 3.5 μg/mL
Specimen Requirements 1 mL refrigerated serum (no additive red top tube); 0.5 mL minimum SST red top unacceptable
164 Alphabetical Test Section 5738X Radioreceptor assay Radioreceptor 2% inhibition sensitivity: Analytical Clinical Background are diseases thyroid Autoimmune autoantibodies, thyroid with associated against types directed several including receptor TSH receptor. the TSH be stimulatory, may autoantibodies in as of TSH, the actions mimicking they may disease. Alternatively, Graves the and block TSH binding inhibit Such TSH. of endogenous action produce autoantibodies blocking hypothyroidism neonatal transient thyroiditis. chronic atrophic and of the ability which measures TBII, its to binding TSH to inhibit antibodies of either the presence reflects receptor, the stimulatory inhibitoryor both and classes. immunoglobulin Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST 165 16% inhibition Transient neonatal Transient Neonatal Graves disease Graves Neonatal Postpartum autoimmune autoimmune Postpartum Atrophic thyroiditis Atrophic Graves disease Graves hypothyroidism thyroid disease thyroid • • • • • Diagnose and manage Graves Graves and manage Diagnose hypothyroidism, neonatal disease, dysfunction thyroid postpartum and Interpretive Information Interpretive Reference Range Reference Clinical Use TBII (Thyrotropin-binding TBII Immunoglobulin) Inhibitory Adults and children:Adults < Graves disease: 16% - 100% inhibition 16% - 100% disease: Graves • Testosterone, Calculated Free, 14966X Bioavailable, and Total
Clinical Use Clinical Background • Assess androgen status in male Most testosterone is transported in hypogonadism; female hirsutism, blood by sex hormone binding virilization, acne, and amenorrhea; or globulin (SHBG). Free testosterone in pediatric patients is the small amount of testosterone circulating unbound. Blood Reference Range testosterone levels are dependent on rates of production, interconversion, See next page. metabolic clearance, and SHBG concentration. SHBG levels are altered Interpretive Information by medications, disease, sex steroids, • Polycystic ovarian disease and insulin. SHBG increases with age • Cushing’s disease in men and decreases with androgen excess in hirsute women. Thus, • Congenital adrenal hyperplasia determination of free testosterone • Androgen resistance more accurately reflects the level of bioactive testosterone than measure- • ments of total testosterone and offers Hypogonadism greater sensitivity in diagnosis of hypo- • P-450c17 enzyme deficiency gonadism in aging men and evaluation of mildly hyperandrogenemic women. • Delayed puberty in boys In general, total testosterone by ICMA, • Gonadotropin deficiency a less technically demanding and • Testicular defects more economical procedure, is satisfactory for screening potential male hypogonadism. The Endocrine Society has stated that ICMA or other "direct" assays are unreliable for diagnosing patients with low levels (eg, <300 ng/dL) of testosterone. Method Testosterone, Total • Liquid chromatography tandem mass spectrometry (LC/MS/MS) Testosterone, Free • Calculation Testosterone, Bioavailable • Calculation SHBG • Immunochemiluminometric assay (ICMA) Albumin • Spectrophotometry Specimen Requirements 2.8 mL refrigerated serum (no additive red top tube); 1.3 mL minimum SST red top unacceptable Continued
166 Alphabetical Test Section 14966X ) continued ( 167 3.4 3.4 7.8 1.5 1.5 1.5 3.6 pg/mL Females for total total for 3.6-5.1 g/dL 5.4 < 1.31.3 < < 64.0 < 140.0 < Males ng/dL ng/dL pg/mL nmol/L nmol/L Testosterone, Total (Women, Children, (Women, Total Testosterone, IIIIIIIVV 39-155 33-135 38-114 21-72 24-90 11-92 22-112 18-54 22-69 18-76 18-29 y18-29 y30-39 y40-49 y50-59 y60-69 y70-79 7-44 y80-91 5-49 y80-84 8-112 8-46 15-49 9-107 3-9 y 23-38 8-96 10-89 y10-13 21-68 20-102 y14-17 20-63 stages 21-76 Tanner 18-136 17-123 26-77 18-136 17-123 11-71 11-71 12-13.9 y12-13.9 y14-17.9 < 8.0-210.0 < 18-69 y18-69 y70-89 y1-11.9 110.0-575.0 0.5-8.5 15.0-150.0 0.5-8.8 < 11-11.9 y11-11.9 y12-13.9 y14-17.9 < 4.0-100.0 < < 18-69 y18-69 y70-89 y1-10.9 46.0-224.0 0.2-5.0 6.0-73.0 0.3-5.0 < Albumin Children SHBG Adults Children Testosterone, Testosterone, Bioavailable Adults See Hypogonadal Males), LC/MS/MS testosterone referenceranges. Children Testosterone, Free Testosterone, Adults Reference Range Reference Bioavailable, and Total Total and Bioavailable, Testosterone, Calculated Free, Free, Calculated Testosterone, Testosterone, Free and Total, 36170X LC/MS/MS
Clinical Use Clinical Background • Assess androgen status in male hypo- Most testosterone is transported in gonadism; female hirsutism, viriliza- blood by sex hormone binding tion, acne, and amenorrhea; or in globulin (SHBG). Free testosterone pediatric patients is the small amount of testosterone circulating unbound. Blood Reference Range testosterone levels are dependent on Free Free rates of production, interconversion, Men % pg/mL metabolic clearance, and SHBG 18-69 y 1.5-2.2 35.0-155.0 concentration. SHBG levels are 70-89 y 1.5-2.2 30.0-135.0 altered by medications, disease, sex Women steroids, and insulin. SHBG increases 18-69 y 0.5-2.0 0.1-6.4 with age in men and decreases with 70-89 y 0.5-2.0 0.2-3.7 androgen excess in hirsute women. Pregnancy Thus, determination of free 1st trimester 0.15-0.66 0.5-6.0 testosterone more accurately reflects 2nd trimester 0.10-0.34 0.2-3.1 the level of bioactive testosterone than 3rd trimester 0.15-0.51 0.2-4.1 measurements of total testosterone Children, males and offers greater sensitivity in 5-9 y 0.44-1.78 <5.3 diagnosis of hypogonadism in 10-13 y 0.53-3.33 0.7-52.0 aging men and evaluation of mildly 14-17 y 1.05-2.91 18.0-111.0 hyperandrogenemic women. Children, females In general, total testosterone by ICMA, 5-9 y 0.28-1.81 0.2-5.0 a less technically demanding and 10-13 y 0.36-3.16 0.1-7.4 more economical procedure, is 14-17 y 0.41-2.34 0.5-3.9 satisfactory for screening potential See Testosterone, Total (Women, Children, male hypogonadism. The Endocrine Hypogonadal Males), LC/MS/MS for total Society has stated that ICMA or other testosterone reference ranges. "direct" assays are unreliable for diagnosing patients with low levels Interpretive Information (eg, <300 ng/dL) of testosterone. • Polycystic ovarian disease Method • Hyperthecosis Total Testosterone • Cushing’s disease • Liquid chromatography tandem • Congenital adrenal hyperplasia mass spectrometry (LC/MS/MS) • Precocious puberty • Analytical sensitivity: 1 ng/dL • Androgen resistance Free Testosterone • Hypogonadism • Tracer equilibrium dialysis, calculation • P-450c17 enzyme deficiency Specimen Requirements • Delayed puberty in boys 0.9 mL refrigerated serum (no additive • Gonadotropin deficiency red top tube); 0.38 mL minimum • Testicular defects SST red top unacceptable
168 Alphabetical Test Section 873X 200-300 ng/dL ng/dL 200-300 300 ng/dL, there there 300 ng/dL, Immunochemiluminometric assay Immunochemiluminometric (ICMA) 20 ng/dL sensitivity: Analytical Clinical Background testes the by secreted is Testosterone adrenal the by both and male in the ovarythe the It is and the female. in andro- circulating potent of the most the and perhaps hormones genic clinical assessment reliable for most Circulating effects. of androgenic sex to bound is largely testosterone (SHBG) globulin binding hormone free. only 2% is to albumin; and in decreased are levels Testosterone and secondaryprimary LH) (increased in the hypogonadism LH) (decreased in delayed puberty as well as in male, boys. screening utilized as a test is best This > if results are test: is very little chance of hypogonadism. is very of hypogonadism. chance little levels testosterone men with Aging to the or close just below frequently range may reference limit of the lower by as hypogonadal recognized not be Society has Endocrine The this assay. or other "direct" that ICMA stated for diagnosing are unreliable assays of testosterone. levels with low patients LC/MS/MS cases, the For such code be used (test should method 15983X). clinically significant men with Aging testoster- and symptoms hypogonadal < levels repeatedly one treat- testosterone benefit from may risk/benefit after adequate ment counseling. Method •• • Specimen Requirements serum refrigerated 2 mL minimum 0.5 mL top preferred red No additive red top acceptable SST request sex on test age and Specify form. 169 . Systemic diseases Systemic Testicular defects defects Testicular Gonadotropin deficiency Gonadotropin Delayed puberty (males) puberty Delayed Androgen resistance Androgen • • • • • Assess testicular function in males in function testicular Assess The preferred test for women, test for women, preferred The is men and hypogonadal children, Children, (Women, Total Testosterone, LC/MS/MS Males), Hypogonadal code 15983X) (test Males (20-60 y)Males ng/dL 241-827 Interpretive Information Interpretive Reference Range Reference Clinical Use Testosterone, Total, ICMA Total, Testosterone, • Testosterone, Total (Women, 15983X Children, Hypogonadal Males)
Clinical Use Clinical Background • Assess androgen status in male Testosterone is secreted by the testes in hypogonadism, in children, and in the male and by both the adrenal and female hirsutism, virilization, acne, the ovary in the female. It is the most and amenorrhea potent of the circulating androgenic Reference Range hormones and perhaps the most ng/dL ng/dL reliable for clinical assessment of Adults Men Women androgenic effects. Circulating 18-69 y 250-1100 2-45 testosterone is largely bound to sex 70-89 y 90-890 hormone binding globulin (SHBG) 70-94 y 2-40 and to albumin; only 2% is free. 1st trimester pregnancy 20-135 Testosterone levels are decreased in 2nd trimester pregnancy 11-153 primary (increased LH) and secondary 3rd trimester pregnancy 11-146 (decreased LH) hypogonadism in the Children Males Females male, as well as in delayed puberty in Cord blood* 17-61 16-44 boys. Testosterone concentrations may 1-10 d* <187 <24 be increased in hirsutism and 1-3 mo* 72-344 <17 virilization in the female. 3-5 mo* <201 <12 The Endocrine Society recommends 5-7 mo* <59 <13 use of a highly sensitive method 7-12 mo* <16 <11 (eg, LC/MS/MS, but not ICMA or 1-5.9 y <5<8 other "direct" assay) whenever low 6-7.9 y <25 <20 8-10.9 y <42 <35 testosterone levels are suspected 11-11.9 y <260 <40 (eg, women, children, and hypo- 12-13.9 y <420 <40 gonadal males). This includes aging 14-17.9 y <1000 <40 men with clinically significant hypo- Tanner stages gonadal symptoms and testosterone I<5<8 levels repeatedly <200-300 ng/dL; II <167 <24 these men may benefit from testoster- III 21-719 <28 one treatment after adequate risk/ IV 25-912 <31 benefit counseling. V 110-975 <33 *Data from J Clin Invest. 1974;53:819-828 Method and J Clin Endocrinol Metab. 1973;36:1132- 1142. • Liquid chromatography tandem mass spectrometry (LC/MS/MS) Interpretive Information • Analytical sensitivity: 1 ng/dL • Polycystic ovarian disease • Precocious puberty Specimen Requirements • Cushing's syndrome 0.5 mL refrigerated serum (no additive • Congenital adrenal hyperplasia red top tube); 0.18 mL minimum • Androgen resistance SST red top unacceptable • Hypogonadism • P450c17 enzyme deficiency • Delayed puberty in boys • Gonadotropin deficiency • Testicular defects
170 Alphabetical Test Section 874X Gas chromatography/mass Gas chromatography/mass spectrometry (GC/MS) 1 μg/L sensitivity: Analytical Clinical Background in testes the secreted by is Testosterone adrenal both the and by the male It is female. in the ovaries and gland of the circulating potent the most the and perhaps hormones androgenic of clinical assessment reliable for most Circulating effects. androgenic sex to bound is largely testosterone (SHBG) globulin binding hormone free. only 2% is to albumin; and inactive to is metabolized Testosterone tissues variety of body in a metabolites tissue. and adipose muscle including via hepatic is largely Inactivation enzymes family P450-3A cytochrome to by conjugation followed renal excretion. for glucuronides is a renal excretion hour Twenty-four for production surrogate reasonable rate. Method • • Specimen Requirements urine of a 24-h frozen aliquot 5 mL minimum 2.1 mL do not collection; during Refrigerate use preservatives. 24-h volume Record form. and request on vial 171 g/24-h μg/24-h μg/g creat enzyme deficiency enzyme c17 Delayed puberty in boys puberty Delayed P450 Testicular defects Testicular Gonadotropin deficiency Gonadotropin Hypogonadism Congenital adrenal hyperplasia adrenal Congenital Polycystic ovarian disease ovarian Polycystic Cushing's syndrome Cushing's Androgen resistance Androgen • • • • • • • • • MenWomen3-89-1213-17 20-170 Adults 5-38 0.11-0.68 0.17-1.41 0.29-1.87 0.63-2.50 MenWomen 13-110 3-47 Assess androgen status as cumulative cumulative status as androgen Assess period urinary over 24-h excretion Testosterone Creatinine Interpretive Information Interpretive Reference Range Reference Clinical Use Testosterone, Urine Testosterone, • Tetrahydroaldosterone, 39613X 24-Hour Urine
Clinical Use Clinical Background • Assess aldosterone production Tetrahydroaldosterone is a urinary • Diagnose hypo- and hyper- metabolite of aldosterone and serves aldosteronism as an index of aldosterone secretion. Measurement is useful in the diagnosis Reference Range of disorders of aldosterone produc- Tetrahydroaldosterone tion, since it provides an integrated Adults 6-79 μg/24-h measure of the 24-hour plasma aldos- terone level. 6-50 μg/g creat Creatinine g/24-h Method 3-8 0.11-.68 • Gas chromatography/mass 9-12 0.17-1.41 spectrometry (GC/MS) 13-17 0.29-1.87 • Analytical sensitivity: 1 μg/L >18 0.63-2.50 Specimen Requirements Interpretive Information 5.0 mL frozen aliquot of a 24-h urine • Primary hyperaldosteronism collection; 2.1 mL minimum • Secondary hyperaldosteronism Refrigerate during collection; do not use preservatives. • Hypoaldosteronism • Liddle's syndrome (pseudo- aldosteronism) • Apparent mineralocorticoid excess (AME) • Licorice root or tobacco chewing
172 Alphabetical Test Section 267X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 20 IU/mL sensitivity: Analytical Clinical Background directed immunoglobulins Circulating commonly are thyroglobulin against with autoimmune in patients present thyroiditis and, to a lesser (Hashimoto) Thyroglobulin Graves disease. extent, of precursor 660-kd protein is the Thyroglobulin hormones. thyroid of sera in the are present antibodies with of patients 70% approximately thyroiditis. autoimmune is to of this test principal use The goiter diffuse that a patient's confirm to is due hypothyroidism and/or may test The thyroiditis. autoimmune disease Graves help distinguish also Although goiter. toxic nodular from present are antibodies thyroglobulin microsomal than thyroid less often peroxidase (eg, thyroid antibodies with in patients anti-TPO) antibodies, diseases, thyroid autoimmune immunoglobulins for both screening sensitivity. diagnostic increases be may antibodies Thyroglobulin without in individuals detected disease. thyroid significant clinically the patient's do not define They status. functional thyroid Method • • Specimen Requirements serum room temperature 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST 173 Postpartum thyroiditis Postpartum Graves disease Graves Primary hypothyroidism Hashimoto thyroiditis (HT) thyroiditis Hashimoto (due to HT) (due • • • • Confirm presence of autoimmune of autoimmune presence Confirm disease thyroid <20 IU/mL <20 Interpretive Information Interpretive Reference Range Reference Clinical Use Thyroglobulin Antibody Thyroglobulin • Thyroglobulin Panel 30278X
Clinical Use Clinical Background • Screen patients with thyroid Thyroglobulin (Tg) is a 660-kd protein carcinoma for metastases after synthesized exclusively in the thyroid thyroid gland ablation gland. It serves as the precursor molecule for thyroid hormones, since • Indicate presence of thyroid gland it contains tyrosine moieties that are tissue in infants with congenital iodinated to form iodotyrosines and hypothyroidism coupled to form the thyroid hormones • Indicate deficient thyroglobulin T4 and T3. Most synthesized Tg is synthesis in infants with goitrous stored as colloid for later pinocytosis hypothyroidism and hydrolysis within thyroid follicular cells. Small amounts are secreted Reference Range directly from the endoplasmic reticulum during synthesis. Tg release Thyroglobulin ng/mL is increased in a variety of thyroid Adults 2.0-35.0 disease states. Thyroglobulin Antibody IU/mL Tg measurements are most useful in Adults and children <20* the postoperative monitoring of patients with papillary, follicular, and *Higher levels can interfere with thyro- Hürthle cell carcinomas. Tg globulin quantification. measurement also is helpful in the differential diagnosis of congenital Interpretive Information hypothyroidism and in detecting • Graves disease thyrotoxicosis caused by exogenous • Multinodular goiter medication (iatrogenic or factitious), in which case the serum Tg • Papillary and follicular thyroid concentration should be low. carcinomas Measurements are always preceded by • Thyroiditis assessment of Tg antibodies that interfere in the assay. In the presence • TSH-dependent hyper- of endogenous antibody, the Tg thyroidism measurement is not reliable.
• Thyroidectomy Method • Thyroid aplasia Thyroglobulin • Thyroglobulin synthetic defect • Immunochemiluminometric assay • Exogenous thyroid hormone use (ICMA) • Analytical sensitivity: 0.2 ng/mL • Analytical specificity: underestimation caused by thyroglobulin autoantibodies Thyroglobulin Antibody • Immunochemiluminometric assay (ICMA) • Analytical sensitivity: 20 IU/mL
Specimen Requirements 2 mL room temperature serum 0.8 mL minimum No additive red top preferred SST red top acceptable
174 Alphabetical Test Section 19584X HAMA precipitation HAMA Immunochemiluminometric (ICMA) assay 0.2 ng/mL sensitivity: Analytical under- specificity: Analytical by thyroglobulin caused estimation autoantibodies Immunochemiluminometric (ICMA) assay 20 IU/mL sensitivity: Analytical Clinical Background defined antibodies, Heterophilic immuno- animal against as antibodies inter- of a source can be globulins, immunoassays. in many ference characterized most frequently The interference from endogenous comes antibodies anti-mouse human the can react with which (HAMA), utilized antibodies monoclonal mouse in the presence HAMA assay. in the or lead to over- sample can patient of thyroglobulin. underestimation by is removed test, HAMA In this or to thyroglobulin prior precipitation determination. antibody thyroglobulin Method • Thyroglobulin • •••• • • Antibody Thyroglobulin • • • Specimen Requirements serum room temperature 2 mL minimum 1.5 mL top preferred red No additive red top acceptable SST 175 ng/mL IU/mL Exogenous thyroid hormone use hormone thyroid Exogenous Thyroglobulin synthetic synthetic Thyroglobulin defect Thyroid aplasia Thyroid Thyroidectomy TSH-dependent hyper- TSH-dependent Thyroiditis Papillary and follicular thyroid Papillary thyroid and follicular Multinodular goiter Multinodular Graves disease Graves thyroidism carcinomas • • • • • • • • • • Screen patients with thyroid with thyroid patients Screen or metastases for carcinoma recurrence *Higher levels can interfere levels *Higher thyro- with quantification. globulin Thyroglobulin Adults Antibody Thyroglobulin 2.0-35.0 Adults and childrenAdults <20* Interpretive Information Interpretive Reference Range Reference Clinical Use Thyroglobulin Panel with with Panel Thyroglobulin Treatment HAMA • Thyroid Peroxidase and 7260X Thyroglobulin Antibodies
Clinical Use Clinical Background • Confirm presence of autoimmune Thyroid peroxidase (TPO) antibodies thyroid disease are present in approximately 95% of patients with Hashimoto thyroiditis Reference Range and 85% of patients with Graves disease. Thyroglobulin (Tg) antibodies IU/mL are positive in about 60% and 30% of Thyroid Peroxidase Antibody <35 adult Hashimoto and Graves disease Thyroglobulin Antibody <20 patients, respectively. Tg antibodies are less frequently detected in children Interpretive Information with these diseases. For additional • Hashimoto thyroiditis information, see Clinical Background • Primary hypothyroidism for the individual tests (267X and 5081X). • Graves disease • Postpartum thyroiditis Method Thyroid Peroxidase Antibody • Immunochemiluminometric assay (ICMA) • Analytical sensitivity: 10 IU/mL Thyroglobulin Antibody • Immunochemiluminometric assay (ICMA) • Analytical sensitivity: 20 IU/mL
Specimen Requirements 1 mL refrigerated serum 0.6 mL minimum No additive red top preferred SST red top acceptable
176 Alphabetical Test Section 5081X Immunochemiluminometric assay Immunochemiluminometric (ICMA) 10 IU/mL sensitivity: Analytical Clinical Background is an (TPO) peroxidase Thyroid iodination the that catalyzes enzyme in thyroid hormone steps and coupling be to now known It is biosynthesis. antigen microsomal the principal directed are which antibodies against The thyroid diseases. in autoimmune test is to confirm use of this main and/or goiter a patient's diffuse that is due to autoimmune hypothyroidism help test may also The thyroiditis. toxic disease from Graves distinguish TPO antibody The goiter. nodular thyroid the replaced has measurement antibody (AMA) antimicrosomal measurement. may be detected antibodies TPO clinically without in individuals They disease. thyroid significant thyroid patient's define the do not status. functional Method • • Specimen Requirements serum refrigerated 1 mL minimum 0.5 mL top preferred red No additive red top acceptable SST 177 Postpartum thyroiditis Postpartum Graves disease Graves Primary hypothyroidism Hashimoto thyroiditis (HT) thyroiditis Hashimoto (due to HT) (due • • • • Confirm presence of autoimmune of autoimmune presence Confirm disease thyroid <35 IU/mL <35 Interpretive Information Interpretive Reference Range Reference Clinical Use Thyroid Peroxidase Antibody Peroxidase Thyroid (Anti-TPO) • Thyrotropin-releasing Hormone (TRH) 36588X
Clinical Use Clinical Background • Potential marker for TRH-secreting Thyrotropin-releasing hormone tumors (TRH) is a tripeptide produced in the hypothalamus, other central nervous Reference Range system sites, and the gastrointestinal <5 pg/mL tract, especially the pancreas. TRH stimulates TSH synthesis and release. Interpretive Information Most circulating TRH, which is rapidly • After TRH injection degraded, is probably derived from extra-hypothalamic tissues. Circulating • Fetus and newborn TRH levels are increased in fetal and • Ectopic (extrahypothalamic) neonatal serum because of increased production TRH production from fetal gut tissues • Primary hypothyroidism and placenta and low levels of TRH- (variable) degrading activity in fetal blood. Plasma TRH levels have not been shown to be useful in diagnosis of any known thyroid condition.
Method • Radioimmunoassay (RIA) • Analytical sensitivity: 5 pg/mL
Specimen Requirements 2 mL frozen plasma 0.6 mL minimum Collect blood in syringe or red-top tube and immediately transfer to a cold, special Nichols Institute PTH-RP collection tube; mix thoroughly. Centrifuge in refrigerated centrifuge. Transfer plasma to a plastic tube and freeze.
178 Alphabetical Test Section 36577X Hypopituitarism TSH deficiency Isolated disease Hypothalamic illness Nonthyroidal thyrotoxicosis Subclinical elderly Normal • • • • • • Radiobinding assay (RBA) assay Radiobinding Clinical Background antibodies develop may rarely Patients hormone stimulating to thyroid inter- antibodies These TSH (TSH). assay measurement with the TSH fere the anti-TSH with by competing assay. used in the immunoglobins TSH has a serum a patient When with is discordant that concentration results, it may be appropriate other test are antibodies if TSH to determine antibody a TSH For example, present. may be the cause of an inappropriately a patient TSH level in serum reduced free reduced simultaneously with T4 levels (free hormone thyroid of Other causes free T3). and/or also levels must TSH inappropriate be considered: Method • Specimen Requirements serum refrigerated 1 mL minimum 0.1 mL top preferred red No additive red top acceptable SST 179 Other autoimmune disease autoimmune Other Autoimmune thyroid disease thyroid Autoimmune • • Evaluate discordant serum TSH serum discordant Evaluate levels Negative Interpretive Information Interpretive Reference Range Reference Clinical Use TSH Antibody TSH • TSH, Ultrasensitive 8660X
Clinical Use Clinical Background • Diagnose hypo- and hyperthyroidism The serum TSH measurement is • Monitor T4-replacement or T4- 1 of the most important tools in the suppressive therapy diagnosis of thyroid disorders. TSH • Quantify TSH levels in the sub- secretion from the pituitary gland is normal range controlled by hypothalamic TRH and a negative feedback effect from Reference Range circulating, free thyroid hormones. Thus, in subjects with a normal Adults mU/L hypothalamic-pituitary system, there Men and non- 0.40-4.50 is an inverse correlation between pregnant women free thyroid hormone and TSH Pregnant women concentrations in serum. Increased 1st trimester 0.30-4.50 serum TSH is an early and sensitive 2nd trimester 0.50-4.60 indicator of decreased thyroid reserve and overt primary hypothyroidism. 3rd trimester 0.80-5.20 A third-generation TSH assay using Children chemiluminescent technology 1-4 d 3.20-35.00 produces functional sensitivities of 5-6 d Not established* 0.01 mU/L, permitting reliable 1-4 wk 1.70-9.10 quantification of serum TSH concen- 1-12 mo 0.80-8.20 trations in the subnormal range. This allows differentiation of euthyroid 1-19 y 0.50-4.30 from hyperthyroid patients. This *TSH levels decline rapidly during the assay also helps diagnose essentially first week of life in most children, but may remain transiently elevated in a few all patients with TSH-independent individuals despite normal free T4 levels. hyperthyroidism (Graves disease, For confirmatory testing following a etc.) with a single blood sample. In newborn thyroid screen, a free (or total) addition, the assay allows adjustment T4 level is usually required for proper of exogenous thyroxine dosage in interpretation of TSH levels in this age group. hypothyroid patients and in patients on suppressive thyroxine therapy for Interpretive Information thyroid neoplasia. • Primary hypothyroidism Method • Decreased thyroid reserve • Immunochemiluminometric assay (sub-clinical hypothyroidism) (ICMA) • TSH-dependent hyper- • Analytical sensitivity: 0.01 mU/L thyroidism • Thyroid hormone resistance Specimen Requirements 2 mL refrigerated serum (no additive • Graves disease red top tube); 0.5 mL minimum • Autonomous thyroid hormone secretion • TSH deficiency
180 Alphabetical Test Section 19537X HAMA precipitation HAMA Immunochemiluminometric (ICMA) assay 0.01 mU/L sensitivity: Analytical Clinical Background as defined antibodies, Heterophilic immuno- animal against antibodies of a source can be globulins, interference immunoassays. in many characterized most frequently The interference from endogenous comes antibodies anti-mouse human the can react with which (HAMA), utilized antibodies monoclonal mouse in the presence HAMA assay. in the or lead to over- sample can patient thyroid stimulating of underestimation is HAMA In this test, (TSH). hormone to TSH prior by precipitation removed determination. Method • • • Specimen Requirements serum refrigerated 2 mL minimum 1.5 mL top preferred red No additive red top acceptable SST 181 mU/L 0.40-4.50 TSH deficiency TSH Autonomous thyroid hormone hormone thyroid Autonomous Graves disease Graves Thyroid hormone resistance hormone Thyroid TSH-dependent hyper- TSH-dependent Decreased thyroid reserve thyroid Decreased Primary hypothyroidism secretion thyroidism (sub-clinical hypothyroidism) (sub-clinical • • • • • • • Monitor T4-replacement or T4- or T4-replacement Monitor therapy suppressive 1st trimester trimester2nd trimester3rd 1-4 dd5-6 0.30-4.50 0.50-4.60 1-4 wk mo1-12 0.80-5.20 y1-19 established* Not 3.20-35.00 1.70-9.10 0.80-8.20 0.50-4.30 *TSH levels decline rapidly during the but children, most in life of week first may remain transiently elevated in a few individuals despite normal free T4 levels. a following testing confirmatory For total) (or free a screen, thyroid newborn T4 levelis usually required forproper age this in TSH levels of interpretation group. Men and non- Men women pregnant Children TSH, Ultrasensitive TSH, women Pregnant Interpretive Information Interpretive Reference Range Reference Clinical Use TSH with HAMA Treatment HAMA with TSH • TSI (Thyroid Stimulating 30551X Immunoglobulin)
Clinical Use Clinical Background • Diagnose and manage Graves disease Autoimmune thyroid disease is associated with a variety of thyroid- Reference Range related autoantibodies. These <125% of basal activity include anti-thyroglobulin, anti-thyroid peroxidase, and TSH receptor anti- Interpretive Information bodies. The TSH receptor antibodies • Graves disease may be stimulatory, exerting a TSH-like effect, or inhibitory, blocking the effect • Hashitoxicosis of TSH. In the thyroid follicular cell, • Neonatal thyrotoxicosis TSH stimulates cyclic AMP (cAMP), which serves a pivotal role in thyroid cellular activation. Because TSI also stimulates cAMP production, the measurement of TSI bioactivity is accomplished by measuring its capacity to increase production of cAMP in a TSH receptor-expressing cell line. Studies have shown that TSI is present in 80% to 90% of patients with active Graves disease and absent in some patients with remission. In patients with Graves disease, monitoring TSI levels during pregnancy helps predict neonatal Graves disease. Patients with preexisting Hashimoto thyroiditis who develop hyperthyroidism will also often manifest measurable TSI bioactivity. TSH receptor antibodies can also be measured by their capacity to inhibit TSH binding to TSH receptors. In this case, they are referred to as TSH receptor binding inhibiting immunoglobulins (TBII, test code 5738X). Method • In vitro bioassay (luciferase) • Analytical sensitivity: 75% • Analytical specificity: under- estimation caused by thyrotropin blocking antibody presence Specimen Requirements 1 mL refrigerated serum 0.2 mL minimum No additive red top preferred SST red top acceptable
182 Alphabetical Test Section 920X Extraction, radioimmunoassay (RIA) radioimmunoassay Extraction, 12.5 pg/mL sensitivity: Analytical Clinical Background polypeptide intestinal Vasoactive neuropeptide acid a 28-amino is (VIP) in stimulation by neural produced It has broad of tissues. a variety effects on the cardiovascular, biological and respiratory gastrointestinal, in- include These effects systems. and of bicarbonate secretion creased decreased absorption and electrolytes water. and of electrolytes of the as part can occur VIPomas neoplasia 1 multiple endocrine type VIP is the syndrome. (MEN) of the watery mediator principal as (also known syndrome diarrhea Werner-Morrison cholera, pancreatic islet cell or diarrheogenic syndrome, syndrome, diarrhea Watery tumor). also been has elevated VIP, with tumors with neurogenic associated neuroblastoma, (ganglioneuroma, bronchogenic pheochromocytoma), cell hyperplasia. and islet tumors, Method • • Specimen Requirements plasma frozen EDTA 3 mL minimum 1.1 mL 183 MEN type 1 MEN VIPoma/watery diarrhea VIPoma/watery diarrhea syndrome • • Assess patients with suspected with suspected patients Assess (MEN) neoplasia endocrine multiple Diagnose watery diarrhea syndrome wateryDiagnose diarrhea (WDS) Adults and childrenAdults pg/mL <50 Interpretive Information Interpretive Reference Range Reference Clinical Use Vasoactive Intestinal Polypeptide Intestinal Vasoactive (VIP) • • Vitamin D, 1,25-Dihydroxy 4729X
Clinical Use Clinical Background • Measurement of bioactive vitamin D Vitamin D is a fat-soluble vitamin that • Differential diagnosis of hypo- is synthesized from cholesterol when calcemia the skin is exposed to ultraviolet light; • Monitor patients with renal osteo- it can also be derived from the diet. dystrophy or chronic renal failure The liver hydroxylates vitamin D to 25-hydroxyvitamin D [25(OH)D], the Reference Range major circulating species of vitamin D. The kidney, along with placenta pg/mL and granuloma tissues, hydroxylates Adults 15-60 25(OH)D to form 1, 25-dihydroxy- Children vitamin D [1,25(OH)2D], the 3-17 y 27-71 bioactive form of the vitamin. Kidney 1-hydroxylation of 25(OH)D Interpretive Information is stimulated by parathyroid hormone • Chronic granulomatous (PTH) and hypophosphatemia. Levels disorders of 1,25(OH)2D are approximately • Hyperparathyroidism 1,000- fold less than those of 25(OH)D.
• Hypercalcemia associated with 1,25(OH)2D increases intestinal lymphoma absorption of calcium and is required • Type II vitamin D-dependent to maintain adequate bone calcifica- rickets tion. Vitamin D deficiency is assessed with measurements of 25(OH)D. • Severe vitamin D deficiency Measurements of 1,25(OH)2D assess • Type I vitamin D-resistant rickets bioactive vitamin and may be useful in • Hypoparathyroidism renal disease, parathyroid disease, and vitamin D resistance or dependence. • Pseudohypoparathyroidism • Renal osteodystrophy, renal Method failure • Extraction, chromatography, • Tumor-induced osteomalacia radioreceptor assay • Tumor-induced hypercalcemia • Analytical sensitivity: 5 pg/mL (except with lymphoma) Specimen Requirements 3 mL frozen serum 1.1 mL minimum No additive red top preferred SST red top acceptable
184 Alphabetical Test Section
2 , ; 3 3 D 2 is is 17306X and and 2 3 3 ; or D 3 2 D 3 2 ,25(OH) α ; 1 2 D 2 , doxercalciferol; and , doxercalciferol; , alfacalcidiol 2 3 is used in a high potency potency a high is used in and 25OHD 2 2 ). Thus, analytical methods methods ). Thus, analytical 2 ,25(OH) (OH)D (OH)D α α α (cholecalciferol) and vitamin D and vitamin (cholecalciferol) 1 Liquid chromatography tandem tandem chromatography Liquid spectrometrymass (LC/MS/MS) for 4 ng/mL sensitivity: Analytical 25OHD Analytical specificity: no cross- specificity: Analytical D with vitamin reaction 1 1 calcitriol; 25,26(OH) calcitriol; 3 (ergocalciferol). Vitamin D (ergocalciferol). both quantitate can accurately that and for diagnosis are essential forms D with vitamin patients monitoring as differentiating as well deficiency and other intoxication between disorders. hypercalcemic Method • • • obtained from foods of plant origin. origin. of plant from foods obtained D Vitamin 25OHD Specimen Requirements serum room temperature 0.3 mL 0.15 mL minimum top preferred red No additive red top acceptable SST preferred. h) fasting is (8-12 Overnight Clinical Background is the D (25OHD) 25-Hydroxyvitamin D vitamin form of circulating major form active of the precursor the and Because D). (1,25-dihydroxyvitamin measure- 25OHD long half-life, of its vitamin for assessing are useful ments in patients. D status vitamin in 2 forms: D occurs Vitamin D origin of animal from foods obtained light-stimulated from ultraviolet and in of 7-dehydrocholesterol conversion vitamin D whereas the skin, (50,000 IU) formulation for treating for IU) formulation (50,000 Both deficiency. vitamin D severe over-the-counter are used in forms and fortified foods and supplements to their respective are metabolized 25OHD forms (ie, 25OHD 185 levels levels 2 Not established Not established Not 2 3 Type II vitamin D-dependent D-dependent II vitamin Type Primary hyperparathyroidism Tuberculosis Hyperphosphatemic tumoral tumoral Hyperphosphatemic Sarcoidosis Obesity Intestinal malabsorption Intestinal Nephrotic syndrome with syndrome Nephrotic Vitamin D-resistant metabolic metabolic D-resistant Vitamin Severe cholestatic or cholestatic Severe Osteomalacia Secondary hyperparathyroidism Nutritional rickets Nutritional Vitamin D intoxication Vitamin rickets calcinosis marked proteinuria marked bone disease due to multiple to multiple disease due bone control for seizure drugs parenchymal liver disease liver parenchymal • • • • • Decreased, but usually not below not below but usually Decreased, range reference • • • • • • • • • Diagnose vitamin D deficiency or D deficiency vitamin Diagnose intoxication 25OHD reflect <20 ng/mL levels 25OHD The optimal D deficiency. vitamin 25OHD ng/mL. is 30-50 level >4 ng/mL suggest compliance with with compliance suggest >4 ng/mL supplements. Total 25OHDTotal 25OHD ng/mL 20-100 Levels vary with exposure to sunlight, varyto sunlight, Levels exposure with summer months. in the peaking Interpretive Information Interpretive Reference Range Reference Clinical Use Vitamin D, 25-Hydroxy, LC/MS/MS 25-Hydroxy, D, Vitamin • VMA (Vanillylmandelic Acid), 39517X Urine 1710X
Clinical Use Clinical Background • Diagnose catecholamine-producing Urinary vanillylmandelic acid (VMA) tumors is the end product of catecholamine metabolism and reflects catecholamine Reference Range production by chromaffin cells of the adrenal medulla and the sympathetic 24-Hour urine (39517X) nervous system. VMA mg/24-h Pheochromocytomas are rare tumors Adults <6.0 of the chromaffin cells located in or Children near the adrenal glands. These tumors 3-8 y <2.3 are diagnosed on the basis of elevated levels of urinary metanephrines, 9-12 y <3.4 urinary VMA, and plasma and/or urine 13-17 y <3.9 catecholamines. Measurement of Creatinine g/24 -h homovanillic acid (HVA) has little value in identifying patients with Adults 0.63-2.50 pheochromocytoma but differentiates Children those with neuroblastoma. 3-8 y 0.11-0.68 Neuroblastomas are malignant tumors 9-12 y 0.17-1.41 that usually occur before 2 years of age; both VMA and HVA levels help 13-17 y 0.29-1.87 in diagnosing these tumors. Random urine (1710X) Gangliomas are rare, benign, well- VMA mg/g creat differentiated tumors in young adults Adults 1.1-4.1 and are associated with excess production of catecholamines and Children their metabolites. Birth-6 mo 5.5-26 7-11 mo 6.1-20 Method 1-2 y 2.5-21 • High-performance liquid chromato- 3-8 y 1.7-6.5 graphy (HPLC), electrochemical 9-12 y 1.4-5.1 detection 13-17 y 1.5-3.6 • Analytical sensitivity: 0.5 mg/L • Creatinine concentration also Creatinine mg/dL reported 0-6 mo 2-32 Specimen Requirements 7-11 mo 2-36 1-2 y 2-128 10 mL room temperature aliquot of urine; 5 mL minimum 3-8 y 2-149 9-12 y 2-183 Collect 24-h urine in 25 mL 6N HCl. Record 24-h volume on vial and request >12 y form. Adjust pH of random urine to Males 20-370 <3.0 using 6N HCl. Females 20-320 It is preferable for the patient to be Interpretive Information off medications for 3 days prior to collection; however, common • Pheochromocytoma antihypertensives cause minimal or no • Neuroblastoma interference. Patients should avoid alcohol, coffee, tea, tobacco, nicotine • Ganglioblastoma patch, bananas, citrus fruit, and • Stress strenuous exercise prior to collection
186 Test Application
and Interpretation
Test Application and Interpretation
PREFACE
Advances in our understanding of endocrine physiology and pathophysiology and the availability of highly sensitive and specific hormone assays permit increasingly accurate endocrine diagnosis and differential diagnosis. As a result, dynamic tests measuring endocrine gland reserve or autonomy are less often necessary; however, they are still useful in evaluation of hormonal systems that change rapidly, such as growth hormone. Stimulation and suppression tests are still applied to the assessment of selected patients with pituitary and adrenal disorders. In this test manual, we have attempted to summarize the state of the art with regard to endocrine assay applications, dynamic tests, and their interpretation circa 2007. We hope that this guide provides helpful information for physicians
entrusted with the care of patients with known and suspected endocrine Test & Application
disorders. Interpretation
189 Test Application and Interpretation
DISORDERS OF ADRENAL FUNCTION
Background Physiology The hypothalamic-pituitary-adrenal axis (Figure 1) functions as a "closed loop" control system. Adrenocorticotropic hormone (ACTH) stimulates adrenal cortisol production, which, by negative feedback at the hypothalamic and pituitary levels, inhibits corticotropin releasing hormone (CRH) and ACTH release, respectively. CRH is a 41-amino acid, hypothalamic, neurosecretory hormone that stimulates ACTH secretion. Arginine vasopressin (AVP), usually known for its antidiuretic properties, potentiates CRH stimulation of ACTH. ACTH is secreted in a pulsatile and diurnal pattern. The lowest concentrations of both ACTH and cortisol are seen at 6 to 11 PM and the highest concentrations at 6 to 10 AM. The striking diurnal variation in cortisol/ACTH secretion is also associated with a diurnal variation in sensitivity to ACTH suppression by exogenous glucocorticoids, such as dexamethasone. The most sensitive time for suppression is 10 to 11 PM, when as little as 0.125 to 0.25 mg of dexamethasone will suppress the next morning's ACTH surge and increased cortisol production. Figure 2 shows the mean concentrations of cortisol plotted against time of day.
Figure 1. Features of the hypothalamic-pituitary-adrenal axis. CRH, corticotropin releasing hormone; AVP, arginine vasopressin; CBG, cortisol-binding globulin; DHEA-S, dehydroandrostenedione sulfate, an adrenal androgen precursor.
190 Test Application and Interpretation Test & Application Interpretation
Figure 2. Temporal pattern of plasma cortisol concentrations in normal subjects with regular sleep hours.
The differential diagnosis of adrenal gland dysfunction has been greatly facilitated by availability of sensitive and specific assays for ACTH and adrenal hormones, their precursors, and metabolites. These methods now often allow accurate diagnosis based on single blood samples. Table 1 summarizes the application of commonly available tests for patient diagnosis. In some instances, diagnosis requires further assessment of pituitary-adrenal reserve or adrenal steroid biosynthetic pathway integrity. In such patients, stimulation of the adrenal glands with ACTH, or suppression of the pituitary- adrenal axis with dexamethasone, provides important supplementary information. Pituitary reserve can be tested with CRH or metyrapone (see section on Dynamic Tests). It is important to recognize that most steroid hormone immunoassays are subject to cross-reaction with closely related steroids. Thus, assays that do not employ extraction or purification of the sample prior to immunoassay may be misleading in diagnosing disorders of enzyme function, such as congenital adrenal hyperplasia. Steroid hormones and their urinary metabolites can be definitively characterized by gas chromatography/mass spectrometry (GC/MS). Steroid excretion patterns assessed in this way can provide discriminating diagnostic information as a supplement to serum assays. Liquid chromatography, tandem mass spectrometry (LC/MS/MS) technology now is available for highly sensitive and specific measurement of steroid hormones and their metabolites in serum or urine. Quest Diagnostics Nichols Institute has moved from the extraction, chromatography, and radioimmunoassay technology to LC/MS/MS as the next generation platform for steroid hormone assay. There is a high correlation between steroid hormone concentrations measured by extraction, chromatography, and immunoassay and by LC/MS/MS.
191 Test Application and Interpretation
Table 1. Hormone Changes in Adrenocortical Disorders Direct Discriminating Disorder ACTH Cortisol Aldosterone Renin Precursor Adrenal insufficiency ↑↑N or ↓ N or ↓ΝΑ Cushing’s syndrome* Adrenal ↓ ↓ ↑ † N or ↓ NA Pituitary N or ↑ ↓ ↑ N or ↓ NA Ectopic ACTH ↑ ↓ ↑ ↓ NA ACTH deficiency N or ↓ N or ↑↓ NNA Glucocorticoid ↑↓ ↑ ↓↑ mineralocorticoids resistance Congenital adrenal N or ↑↑ or ↓ N or ↓ N or ↓↑ discriminating hyperplasia precursor/product ratio Primary hyper- N ↓ N ↑↑ aldosterone to 18-OH aldosteronism corticosterone ratio Dexamethasone Ν↓ Ν ↑↑ 18-OH cortisol suppressible hyperaldosteronism Apparent mineralo- ↓↓ Ν ↓↓ Cortisone to corticoid excess cortisol ratio Hypoaldosteronism N ↑Ν ↓↑ 18-OH corticosterone to aldosterone ratio Pseudohypo- N ↑Ν ↑Non-responsive to aldosteronism aldosterone Hyporeninemic NN or ↓ N ↓↑ prorenin hypoaldosteronism N, normal; NA, not applicable. *24-hour urine cortisol is increased. †ACTH and cortisol are secreted with striking diurnal changes. Thus, a ”normal” 8:00 AM cortisol/ ACTH that does not change diurnally can cause Cushing’s syndrome.
Abbreviations ACTH Adrenocorticotropic hormone Aldo Aldosterone 3β-HSD 3β-Hydroxysteroid dehydrogenase CAH Congenital adrenal hyperplasia Compound S 11-Deoxycortisol CRH Corticotropin releasing hormone DHEA Dehydroepiandrosterone L-Dopa Levodopa 17-KGS 17-Ketogenic steroids 17-OHCS 17-Hydroxycorticosteroids P-450scc(CYP11A1) Adrenal side-chain cleavage enzyme P-450c21(CYP21A2) Adrenal 21-hydroxylase P-450c17(CYP17) Adrenal and gonadal 17-hydroxylase P-450c11(CYP11B1) Adrenal 11β-hydroxylase P-450c11ase(CYP11B2) Adrenal aldosterone synthetase 192 Test Application and Interpretation
Adrenal Insufficiency Adrenal insufficiency has 2 major causes: ACTH deficiency (secondary adrenal insufficiency) and decreased responsiveness to ACTH (primary adrenal insufficiency). These causes can be further classified as shown in Table 2. With severe primary or secondary adrenal insufficiency, the serum cortisol concentration is very low. However, with partial adrenal insufficiency, basal serum cortisol levels are normal or low, and diurnal rhythmicity may overlap the normal range. ACTH levels, however, are increased. ACTH deficiency is associated with low levels of both cortisol and ACTH. In some instances, particularly in patients with mild deficiency, ACTH stimulation testing may be useful. For primary or secondary disease, the standard 30- or 60-minute ACTH stimulation test is useful. Normal rapid ACTH stimulation test results for infants and children are shown in Tables 3 and 4; test results for adults are shown in Figures 3 and 4 and Table 5. For secondary disease (ACTH deficiency), the CRH test, metyrapone test, or prolonged ACTH test may be helpful in selected patients (see “Dynamic Test Test & Application Application and Interpretation” section). Interpretation
Adrenal Biosynthetic Enzyme Deficiencies Classical Congenital Adrenal Hyperplasia (CAH) Classical CAH is uniquely manifest in infancy by inefficiency of cortisol synthesis; such inefficiency is caused by genetic mutations that result in deficient activity of 1 of the adrenal biosynthetic enzymes (Figure 1). The most common deficiency is P-450c21 (CYP21A2) activity, accounting for 95% of CAH patients. P-450c11 (CYP11B1) deficiency accounts for another 4%; 3β-hydroxysteroid dehydro- genase (3β-HSD) and P-450scc (CYP11A1) deficiencies constitute the remainder of cases in infancy. CAH is virilizing in all of these disorders, except P-450scc deficiency, because of the increased production of DHEA or androstenedione induced by ACTH-mediated adrenal hyperactivity in the presence of blocked cortisol synthesis (Figure 5). Newborn screening programs now commonly screen for CYP21A2 deficiency by measuring 17-hydroxyprogesterone levels in filter paper blood samples.
Table 2. Differential Diagnosis of Adrenal Insufficiency Pituitary dependent Primary adrenal insufficiency CRH dependent Congenital adrenal hyperplasia Hypothalamic tumors Congenital adrenal hypoplasia Idiopathic, acquired Congenital ACTH unresponsiveness Congenital Autoimmune disease ACTH deficiency, congenital Infectious diseases Hypopituitarism Tuberculosis Congenital causes Fungi Neoplastic Cytomegalovirus Craniopharyngioma Hemorrhage Infection Metastatic tumors Sarcoidosis Sarcoidosis Head trauma Amyloidosis Adrenoleukodystrophy
193 Test Application and Interpretation
Table 3. Serum Adrenal Steroid Concentrations in Infants. Observed Range Before (B), After (A), and Response (U, range) to Rapid ACTH Test* Premature† Term Steroid 26-28 wk 34-36 wk 1-6 mo Pregnenolone B 260-2100 203-1024 10-150 A 962-3179 637-1888 110-359 U 70-2673 162-1685 20-282 Progesterone B 18-640 − 5-53 A 52-1348 − 74-200 U 29-796 − 35-165 17-OH pregnenolone B 375-3559 559-2906 52-828 A 2331-11440 831-9760 633-3286 U 1219-9799 346-8911 229-3104 17-OH progesterone B 124-841 186-472 13-173 A 285-1310 334-1725 85-250 U 50-596 18-1253 52-193 DHEA B 236-3640 223-3640 26-505 A 1320-8952 727-7821 67-1453 U 408-8610 32-7219 28-1343 Androstenedione B 92-892 90-837 6-78 A 145-1248 183-1367 21-114 U 40-718 13-1084 9-76 11-Deoxycortisol B 110-1376 70-455 10-200 A 206-2504 81-645 101-392 U 15-1128 40-190 5-366 Cortisol B 1-11 3-34 3-22 A 6-52 16-76 27-50 U 4-41 6-44 19-41 Deoxycorticosterone B 20-105 28-78 7-48 A 44-320 28-95 40-158 U 17-215 1-67 13-144 Corticosterone B 235-1108 201-5030 78-2500 A 1667-8251 2240-11900 2225-4974 U 1338-8016 2039-10141 1149-4789 18-OH corticosterone B 10-670 38-779 5-300 A 35-1500 152-2183 130-465 U 16-830 114-2183 21-394 Aldosterone B 5-635 12-736 2-71 A 13-1046 42-1365 5-166 U 8-517 28-629 3-123 *Results in ng/dL except cortisol (μg/dL); data from extraction, chromatography, RIA (Ref. 23). †Samples obtained on postnatal days 2-4.
194 Test Application and Interpretation
Table 4. Observed Ranges for Serum Adrenal Steroids in Infants and Children. Values Before (B), After (A), and Response (U) to Rapid ACTH Test Pubertal Pubertal Steroid <1 y 1-5 y 6-12 y Male Female Pregnenolone B 10-137 10-48 15-45 15-84 24-50 A 49-359 34-135 38-104 33-218 37-149 U 19-282 4-114 16-73 6-193 9-101 Progesterone B 5-80 8-64 5-93 6-1286 17-145 A 74-200 51-233 38-204 32-1069 35-223 U 35-192 19-192 22-170 0-104 0-192 17-OH pregnenolone B 13-788 9-98 10-177 19-346 50-516 A 373-3125 43-702 67-624 84-817 239-1525 U 200-3000 15-680 60-500 65-750 108-1280 Test & Application
17-OH progesterone B 11-173 4-114 7-69 12-190 18-220 Interpretation A 85-466 50-350 75-218 69-313 80-422 U 50-275 30-300 50-250 7-281 9-287 DHEA B 26-500 9-42 11-153 25-400 69-686 A 18-1100 21-98 34-322 62-509 95-1557 U 5-600 5-70 20-220 22-386 26-1233 Androstenedione B 6-78 5-51 7-68 17-151 43-221 A 21-139 12-68 12-98 2-215 58-319 U 10-75 5-60 5-60 8-121 9-118 11-Deoxycortisol B 10-200 7-210 14-136 11-151 15-130 A 80-390 98-360 95-322 87-283 78-250 U 5-350 50-280 30-180 35-241 34-233 Cortisol B 3-23 5-25 5-23 4-15 4-16 A 32-60 22-40 17-40 15-45 16-35 U 17-40 5-25 5-20 5-32 7-26 Deoxycorticosterone B 7-57 4-49 5-34 2-12 4-30 A 20-157 26-143 19-138 13-63 12-74 U 26-110 23-135 16-130 10-53 7-43 Corticosterone B 78-1750 120-2030 155-1365 111-598 115-1219 A 2225-6505 2150-7540 1775-7500 1723-5100 1472-5060 U 1140-5120 960-7300 1490-7300 1380-4700 1003-4740 18-OH corticosterone B 5-310 7-155 10-74 11-82 5-73 A 67-470 49-370 79-360 69-322 73-1472 U 22-395 33-333 69-310 58-254 22-1467 Aldosterone B 2-130 2-37 3-21 2-32 1-14 A 5-167 13-85 14-50 10-34 10-33 U 4-122 7-54 4-40 0-22 7-25 Results in ng/dL except cortisol (μg/dL); ACTH 1-24 (250 μg) given as intravenous bolus; age groups ranged from 15-22 children (M and F) except pubertal (n=43, 23M, 20F, Tanner II-V); data from extraction, chromatography, RIA (Ref. 21, 22).
195 Test Application and Interpretation
50
40
30
20 Cortisol (ng/dL)
10
0 0 15306090 Minutes Figure 3. Rapid ACTH stimulation results in normal adults (glucocorticoid pathway); steroid analyses by extraction, chromatography, and radio- immunoassay.
35
30
25
20
15
10 Aldosterone (ng/dL) Aldosterone
5
0
0 15306090 Minutes Figure 4. Rapid ACTH stimulation results in normal adults (mineralocorticoid pathway); steroid analyses by extraction, chromatography, and radio- immunoassay. 196 Test Application and Interpretation
Table 5. Rapid ACTH Stimulation Results in Normal Adults Range of Blood Levels in ng/dL (Cortisol, μg/dL) Steroid 0 15 min 30 min 60 min 90 min Glucocorticoid pathway 17-Hydroxypregnenolone 29-189 267-856 305-847 293-913 252-846 17-Hydroxyprogesterone 27-122 63-183 74-211 72-187 58-180 Dehydroepiandrosterone 230-955 472-1681 545-1785 545-1846 545-1855 Androstenedione 56-134 69-206 88-271 72-288 98-292 11-Deoxycortisol 21-133 82-232 82-292 82-261 82-246 Cortisol (μg/dL) 5.8-19 13-30 14-36 14-41 18-43
Mineralocorticoid pathway Test & Application Progesterone 5-33 -- 11-44 21-44 21-69 Interpretation Deoxycorticosterone 3-10 -- 11-35 14-33 13-30 18-Hydroxycorticosterone 11-46 43-131 45-151 54-161 61-233 Aldosterone 2-9 6-33 5-27 5-20 4-15 Steroid analysis by extraction, chromatography, and radioimmunoassay. Data from Quest Diagnostics Nichols Institute Clinical Correlations Department; from healthy young adults (10 females and 10 males).
Figure 5. Adrenal gland biosynthetic pathways. The enzymes involved are 1) P-450scc (CYP11A1), 2) 3β-HSD, 3) P-450c21 (CYP21A2), 4) P-450c11 (CYP11B1), 5) P-450c11ase (CYP11B2), and 6) P-450c17 (CYP17).
197 Test Application and Interpretation
The differential diagnosis of congenital virilizing adrenal hyperplasia in infancy includes P-450c21, P-450c11, and the rare 3β-HSD deficiencies. Diagnosis of these disorders in infancy, and especially in premature infants, is complicated by the normal, transient deficiency of 3β-HSD activity in the fetal adrenal cortex (which resolves during the early weeks and months of life) and by the apparent deficiency of P-450c11 activity in premature infants <30 weeks of gestational age. Blockage of the adrenal biosynthetic pathway is manifest by increased enzyme precursor levels or an increased steroid precursor to product ratio imposed by the deficient enzyme activity. Normal baseline and ACTH-stimulated precursor/ product ratios for childhood and adolescent ages are shown in Tables 6-8. Tables
Table 6. Normal Ranges in Children for Adrenal Enzyme Precursor/Product Ratios. Values Before (B) and After (A) Rapid ACTH Test* Precursor 6 mo - 1 y 1-5 y 6-12 y Pubertal Ratios Product B A B A B A B A 3β-HSD 17-OH pregnenolone 2-22 2-20 0.3-3.0 0.5-3.3 0.5-6.0 0.3-5.3 0.4-3.4 0.5-6.3 17-OH progesterone DHEA 0.8-6.5 1.5-13 0.6-9.0 0.7-7.5 2.0-4.5 1.1-5.8 1.5-4.0 1.8-4.9 Androstenedione
P450c17 Pregnenolone 0.1-2.9 0.1-0.5 0.3-3.6 0.2-1.5 0.2-2.8 0.2-0.9 0.1-1.7 0.1-1.0 17-OH pregnenolone Progesterone 0.2-5.2 0.4-1.1 0.2-3.5 0.5-1.5 0.2-2.6 0.2-0.9 0.2-2.2 0.2-1.5 17-OH progesterone
P450c21 Progesterone 0.3-7.0 0.9-4.0 0.5-10 0.6-6.0 0.9-8.4 0.9-3.7 1.3-14 1.2-6.6 Deoxycorticosterone 17-OH progesterone 0.4-3.1 0.5-2.0 0.3-2.1 0.5-1.6 0.2-2.1 0.5-1.6 0.2-3.7 0.4-2.7 11-Deoxycortisol
P450c11 11-Deoxycortisol 0.8-10 2.4-10 1.0-6.8 3.8-11 1.2-9.0 2.8-9.0 1.8-12 3.4-11 Cortisol
P450c11ase 18-OH corticosterone 1.3-5.0 2-13 1.2-6.0 1.9-15 2.6-7.1 5-12 2.0-5.7 3.4-13 Aldosterone *Values in ng/dL except 11-deoxycortisol = ng/dL ng/dL cortisol μg/dL. Measurements by extraction, chromatography, and radioimmunoassay; data from references 21, 22, and Quest Diagnostics Nichols Institute Clinical Correlations.
198 Test Application and Interpretation
Table 7. Normal Ranges in Infants for Adrenal Enzyme Precursor/Product Ratios. Values Before (B) and After (A) Rapid ACTH Test* Premature Premature Term Infants Precursor Product Ratios 26-28 wk 34-36 wk 1-6 mo BA B A BA 3β-HSD 17-OH pregnenolone 1.1-5.2 3.6-11 1.8-6.5 3.6-12 2-22 3-20 17-OH progesterone
DHEA 1.0-8.4 3.4-15 1.6-5.0 2.2-7.8 2.2-6.5 2.8-13 Androstenedione
P450c17 Test & Application
Pregnenolone 0.3-0.7 0.3-5.0 0.2-0.7 0.2-1.3 0.1-0.7 0.03-0.3 Interpretation 17-OH pregnenolone
Progesterone 0.2-1.8 0.1-1.5 −−0.2-2.1 0.4-1.1 17-OH progesterone
P450c21 Progesterone 1.6-9.4 1.1-9.8 −−0.3-7.0 0.9-4.0 Deoxycorticosterone
17-OH progesterone 0.4-2.4 0.3-2.1 0.9-4.8 0.8-4.2 0.4-3.1 0.5-2.0 11-Deoxycortisol
P450c11 11-Deoxycortisol 25-300 10-189 3-115 3-26 0.8-10 2.4-10 Cortisol
P450c11ase 18-OH corticosterone 1.0-4.5 0.8-2.6 1.1-10 1.2-11 1.3-5.0 2-13 Aldosterone *Values in ng/dL except 11-deoxycortisol = ng/dL ng/dL cortisol μg/dL. Premature data derived during first week; measurements by extraction, chromatography, and radioimmunoassay; data from references 23, 24.
9 and 10 summarize neonatal urine GC/MS steroid reference ranges. Figures 6-9 illustrate the urine GC/MS steroid precursor-product ratios in normal vs CAH infants with proven 21-hydroxylase or 3β-HSD deficiency. Diagnostic ratios for infants with 21-hydroxylase, 17-hydroxylase, 11β-hydroxylase, and 3β−HSD deficiencies are listed in the “Alphabetical Test Section” (CAH Profile, Neonatal Random Urine). As in childhood and adolescence, normal ratios in adults are
199 Test Application and Interpretation
Table 8. Adrenal Enzyme Precursor/Product Ratios in Infants and Children with Documented Deficiencies of Adrenal P-450 Enzymes
Precursor P-450c21 Product Ratios Type 1 Type II P-450c11 P-450c17 P-450c11ase 3β-HSD 17-OH pregnenolone 0.2 0.2 3 2 * 17-OH progesterone * (0.3) (2) (1) *
DHEA 0.7 3 3 2 * Androstenedione * (5) (0.6) (7) *
P450c17 Pregnenolone 0.2 0.7 0.4 15 * 17-OH pregnenolone * (0.2) (0.4) (35) *
Progesterone 0.1 0.1 0.8 22 * 17-OH progesterone * (0.1) (0.6) (16) *
P450c21 Progesterone 200 41.22 * Deoxycorticosterone * (10) (0.6) (2) *
17-OH progesterone 250 20 0.1 0.5 4 11-Deoxycortisol * (60) (0.1) (0.7) (1.5)
P450c11 11-Deoxycortisol 20 4 850 28 7 Cortisol * (4) (350) (17) (10)
P450c11ase 18-OH corticosterone 1.0 6 2 110 150 Aldosterone (8) (4) (120) (180) Values in ng/dL except 11-deoxycortisol = ng/dL ng/dL cortisol μg/dL. Measurements derived from extraction, chromatography, and radioimmunoassay. Ratio values in parentheses are post ACTH stimulation. Ratios were calculated from data published in reference 24, with permission. Bold print indicates discriminating ratios. *No results available.
similar before and after ACTH and do not exceed 10 for the 3β-HSD, P-450c17, and P-450c21 enzymes. Normal adult precursor/product ratios for P-450c11 and P-450c11ase do not exceed 15. Table 8 summarizes the adrenal enzyme precursor/ product ratios in infants and children with documented deficiencies of adrenal P-450 enzymes.
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Table 9. Pediatric Urine Steroid Reference Ranges (μg/g creat, GC/MS Analysis) Steroid LOQ 1 Day Old 2-4 Days Old >16 Days Old 17α−Hydroxy- 10 30-420 15-200 40-90 pregnanolone 15β,17α-Dihydroxy- 20 85-490 25-270 45-80 pregnanolone
16α-Hydroxy-DHEA 100 5000-220,000 2000-180,000 200-16,650
Pregnanetriol 2 45-620 20-280 5-70
Tetrahydro-11- 10 70-670 30-470 15-60
deoxycortisol Test & Application Interpretation 15β,17α-Dihydroxy- 100 4900-43,900 1800-59,000 630-3300 pregnenolone
Pregnanetriolone 2 20-280 <260 <10
16α-Hydroxy- 50 4400-98,900 3460-150,840 75-10,910 pregnenolone
Pregnenetriol 10 <360 <390 20-180
Tetrahydrocortisone 100 620-13,160 530-7430 1600-5570
6α-Hydroxytetrahydro- 40 <120 <170 <40 11-deoxycortisol
Tetrahydro-11-dehydro- 150 * * 320-1490 corticosterone
α-Cortolone 10 25-520 20-380 40-600
β-Cortolone 50 120-1360 150-1175 170-1350 6α-Hydroxytetrahydro- 10 30-540 20-250 10-100 11-dehydrocorticosterone
LOQ,, lowest limit of quantitation. *Prior to 16 days of age, measurement of this steroid is compromised by interference from other fetal steroids. From Quest Diagnostics Nichols Institute Clinical Correlations Department.
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Table 10. Diagnostic Urine Ratios in Normal Neonates and Patients with CAH (GC/MS Analysis) Age Normals 21-OH 3β-HSD 11β-OH 17-OH Steroid Ratio (Days) n = 59 n = 32 n = 2a n = 2b n = 1c 21-OH Deficiency 15β-,17α-(OH)2 - pregnanolone x 100 1 3.8-17.4 47.4-400 Denominator 2-4 1.0-14.4 116-498 >16 1.0-2.6 170-1627 67.1; 150 33.6; 21.4 4.4 17α-OH-pregnanolone x 100 1 2.0-22.0 29.9-423 Denominator 2-4 1.0-11.0 103-974 >16 1.0-4.0 248-1470 74.8; 224 36.4; 22.0 8.0 Pregnanetriol x 100 1 2.0-30.0 36.6-648 Denominator 2-4 1.0-13.0 112-286 >16 <3.0 76.6-1344 22.6; 103 16.4; 5.8 4.1 Pregnanetriolone x 100 1 0.7-15.9 40.6-461 Denominator 2-4 <14.5 45.1-331 >16 <0.2 83.3-961 8.3; 4.6 6.4; 3.4 12.9 17-OH Deficiency 5α-THA x 100 1<8.0 <55.4 Denominator 2-4 <11.0 <7.8 >16 <34.3 <75.4 ND; 57.0 ND; ND 50.4 6α-OH-THA x 100 1 1.0-28.0 ND Denominator 2-4 1.0-11.0 ND >16 1.0-2.0 ND ND; ND NMC; 29,983 NMC 16α-OH-pregnenolone 1 0.3-1.3 1.2-4.4 16α-Hydroxy-DHEA 2-4 0.1-2.2 1.9-11.4 >16 0.2-0.7 1.0-11.7 0.9; 0.4 9.5; 8.1 156 11β-OH Deficiency THS x 100 1 2.9-25.0 13.6-34.0 Denominator 2-4 1.1-26.0 10.4-49.1 >16 0.5-1.4 1.5-136 15.6; 27.0 366; 97.2 14.2 6α-OH-THS x 100 1<13.0 ND Denominator 2-4 <11.0 ND >16 ND ND ND; ND 190,482; ND 117,723 3β-HSD Deficiency Pregnenetriol x 100 1<24.4 <27.4 Denominator 2-4 <17.1 <36.6 >16 0.5-2.4 <287.9 356; 149 36.4; 1.2 ND Pregnenetriol 1 0.1-10.8 <0.4 Pregnanetriolone 2-4 0.2-95.2 <0.4 >16 5.6-28.6 <1.5 42.8; 32.5 2.6; 1.7 ND Denominator = sum of tetrahydrocortisone, alpha, and beta cortolone; THA, tetrahydro-compound A, tetrahydro-11-dehydrocorticosterone; ND, not detected, numerator level below detectable limit of assay; NMC, not measured due to contamination of numerator component; THS, tetrahydro-sub- stance S, tetrahydro-11-deoxycortisol. From Quest Diagnostics Nichols Institute Clinical Correlations Department (Ref. 18). aOne patient; samples collected when 7 and 15 days of age. bTwo patients, 14 and 49 days of age. cOne 7 day old patient.
202 Test Application and Interpretation
10000.0
1000.0
100.0 Ratio Value Ratio 10.0
1.0 Test & Application Interpretation
0.1 ABC
Figure 9. 1 5β,17α-(OH)2pregnanolone x 100/Denominator. FigureA, 21-hydroxylase; 6. 15β,17α-(OH) B,2pregnanolone 3β-HSD; C, x normal 100/Denominator. samples.
10000
1000
100 Ratio Value Ratio 10
1
0 AB C Figure 6. 17α-Hydroxypregnanolone x 100/Denominator. Figure 7. 17α-Hydroxypregnanolone x 100/Denominator. A, 21-hydroxylase; B, 3β-HSD; C, normal samples.
203 Test Application and Interpretation
10000.0
1000.0
100.0
10.0 Ratio Value Ratio
1.0
0.1
0.0 ABC
Figure 7.8. Pregnanetriol xx 10100/Denominator. 0/Denominator. A, 21 -hydroxylase; B, 3β-HSD; C, normal samples.
10000.0
1000.0
100.0
10.0 Ratio Value Ratio
1.0
0.1
0.0 AB C FigureFigure 9. 8. Pregnanetriolone Pregnanetriolone x 100/Denominator. x 100/Denominator. A, 21-hydroxylase; B, 3β-HSD; C, normal samples. Figures 6-9. Diagnostic ratios for 21-hydroxylase and 3β-HSD deficiencies using GC/MS analysis. Shown are 4 urinary metabolites relative to the major cortisol metabolites (denominator, sum of tetrahydrocortisone, alpha, and beta cortolone). The patient and normal data points are grouped according to patient/subject age at time of urine collection (day 1, days 2-4, >5 days). In patients with 3βHSD deficiency, only the pregnanetriolone ratio is normal. Statistical analysis of data from the 4 ratios (ie, 21-OH patients vs. normal subjects) for the differ- ent collection times were P <0.001, <0.001, and 0.001 for 0-1, 2-4, and >5d, respectively. A, 21-hydroxylase; B, 3β-HSD; C, normal samples. See Ref. 18.
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Nonclassical Disease (Partial Deficiency in Steroid Biosynthesis)
Nonclassical, late-onset, partial, or attenuated forms of P-450c21, 3β-HSD, and P-450c11 adrenal biosynthetic enzyme deficiencies are more common than severe deficiencies producing CAH. The overall prevalence of CAH approximates 1 in 8,000 births but is higher in the Eskimo and Jewish populations. The prevalence of nonclassical P-450c21 deficiency varies from 1 in 27 Ashkenazi Jews to 1 in 1,000 in other Caucasians. Nonclassical 3β-HSD and P-450c11 deficiencies are much less common, but precise prevalence data are not available. Among Caucasian hyperandrogenic women, nonclassical P-450c21 deficiency affects 1% to 2% while nonclassical 3β-HSD deficiency and P-450c11 deficiency probably each account for less than 1%. P-450c21 gene mutations have been characterized in patients with nonclassical P-450c21 enzyme deficiency, but the genetic defects for the β 3 -HSD and P-450c11 varieties have not been demonstrated. It has been proposed Test & Application that most hirsute women suspected to have nonclassical 3β-HSD enzyme Interpretation deficiency have, instead, a nonspecific dysregulation of P-450c17 involving the adrenal glands and ovaries. The common clinical manifestations of nonclassical disease include hirsutism and oligomenorrhea, which result from the excess androgen secretion. Androgenic symptoms usually are mild; clitoromegaly, male habitus, and temporal baldness are infrequent manifestations. Diagnosis is based on elevated precursor and/or precursor-to-product ratios, and detection usually requires ACTH stimulation. Nonclassical disease is confirmed if the discriminating precursor/product ratio is clearly elevated. Stimulated ratios usually exceed 40 in nonclassical P-450c21 deficiency patients. In postpubertal females, a morning follicular phase serum 17-hydroxyprogesterone level <200 ng/dL excludes a diagnosis of nonclassical P-450c21 enzyme deficiency. ACTH stimulation testing is recommended only when the 8 AM fasting serum 17-hydroxyprogesterone level exceeds this level. Nonclassical P-450c21 deficiency is diagnosed by a post-ACTH 17-hydroxyprogesterone level >1,000 ng/dL, a value 4 times the upper normal limit (Figure 3). For the diagnosis of nonclassical 3β-HSD in postpubertal subjects, it is recommended that the post-ACTH 17-hydroxypregnenolone level exceed 1,500 ng/dL, a value 3 SD above the mean of control subjects (Figure 3), and the 17-hydroxypregnenolone/17-hydroxyprogesterone or DHEA/ androstenedione ratio should exceed 10, a value exceeding the approximate 3 SD value of control pubertal subjects (Table 6). For the diagnosis of nonclassical P-450c11 deficiency in postpubertal patients, the 11-deoxycortisol response to ACTH should exceed 350 ng/dL and the 11-deoxycortisol/cortisol ratio should exceed 15, values approximating 3 SD above the mean for normal subjects (Tables 5 and 6).
205 Test Application and Interpretation
Cushing’s Syndrome Cushing’s syndrome, the clinical manifestations of adrenal cortical hormone excess, can be the result of excessive pituitary or ectopic ACTH production, autonomous adrenal cortical steroid secretion, or exogenous glucocorticoid medication (Table 11). Endogenous Cushing’s syndrome is associated with increased 24-hour excretion of urinary cortisol, while administration of synthetic glucocorticoids (eg, prednisone, dexamethasone) is typically accompanied by decreased cortisol excretion, since the adrenal axis is suppressed (Table 12). If endogenous cortisol hypersecretion is suspected, the simplest approach to diagnosis is collection of a 24-hour urine sample for measurement of free cortisol (with creatinine to assess the completeness of collection). If excessive urine free cortisol is confirmed on two 24-hour urine collections, Cushing's syndrome is usually present, particularly if the value is >200 μg/24-h. Depression, alcohol abuse, and other stresses can also produce lesser degrees of excessive urine free cortisol excretion (pseudo-Cushing's). Alternative approaches to confirm hypercortisolism are to 1) determine the suppressibility of serum cortisol using the overnight dexamethasone suppression test (see "Dynamic Test Application and Interpretation" section), or 2) measure the serum or salivary cortisol level at 11 PM to detect a disrupted circadian rhythm.
Once endogenous hypercortisolism is established, differentiating the cause as pituitary tumor, ectopic neoplasm, or adrenal tumor can be accomplished by plasma ACTH measurement, dynamic testing, and imaging procedures (Table 11). In patients with ACTH-secreting pituitary adenomas (Cushing’s disease), plasma ACTH is typically 30 to 150 pg/mL. An exception is Nelson syndrome, in which previous adrenalectomy is associated with increased production of ACTH and growth of a pituitary corticotropinoma. The ectopic ACTH syndrome is often associated with very high plasma ACTH levels, although these may overlap the range of ACTH-secreting pituitary adenomas; ectopic ACTH production is also characterized by secretion of "big" molecular species of ACTH and, rarely, by co-secretion of corticotropin releasing hormone (CRH). Autonomous adrenal cortisol production by a benign or malignant neoplasm is likely if plasma ACTH
Table 11. Differential Diagnosis of Cushing’s Syndrome Pituitary dependent ACTH adenoma Adrenotroph hyperplasia Intermittent Cushing’s Adrenal disease Adenoma Carcinoma Macronodular disease Micronodular dysplasia Other (McCune Albright syndrome, Carney syndrome) Ectopic (cancer-associated) Cushing’s Ectopic ACTH Ectopic CRH Pseudo-Cushing’s (alcohol-related) Exogenous glucocorticoid administration (eg, prednisone)
206 Test Application and Interpretation
Table 12. Hormone Changes in Cushing’s Syndrome Plasma High Dose or Overnight Cause Plasma ACTH Cortisol Dexamethasone (PM) Suppression Pituitary-dependent N or slightly ↑ ↑ Yes Adrenal disease ↓ or undetectable ↑ No Ectopic Cushing’s* ↑↑↑ ↑↑ Usually no Pseudo-Cushing’s N or slightly ↑ N or ↑ Usually yes *Plasma CRH measurements may also be indicated. ACTH levels in some patients overlap those of pituitary dependent disease. levels are low or undetectable, although mishandling of plasma samples may lead
to misleadingly low ACTH concentrations due to hormone degradation. Test & Application Interpretation Imaging of the pituitary and/or adrenal glands is usually best deferred until a biochemical diagnosis has been established, because false-negative and false- positive findings are common. Only 50% of patients with surgically proven ACTH-secreting pituitary adenomas have a lesion that is visualized by MRI. Conversely, up to 20% of normal individuals may have radiological findings consistent with a pituitary adenoma, which may be an entirely incidental lesion. Similarly, as many as 3% of normal individuals may have adrenal cortical masses seen on CT or MR imaging. The most definitive contemporary test to distinguish pituitary from ectopic ACTH hypersecretion, and to localize pituitary tumors, is sampling of the pituitary venous effluent by bilateral inferior petrosal sinus (IPS) catheterization. Measurement of ACTH in simultaneously obtained IPS and peripheral venous samples can identify the true source of endogenous ACTH production, particularly when additional samples are obtained after CRH stimulation (see “Dynamic Test Application and Interpretation” section).
Renin-Angiotensin ⎯ Aldosterone System
Background Physiology The renin-angiotensin aldosterone system (RAAS) modulates, in association with vasopressin, body salt and water metabolism (Figure 1). Renal sodium excretion is regulated by the RAAS to maintain sodium homeostasis relative to salt intake. Renal renin secretion is stimulated by decreased blood volume and decreased renal perfusion pressure. Renin, in turn, stimulates cleavage of circulating angiotensinogen substrate to produce angiotensin I, an inactive decapeptide. Angiotensin converting enzyme (ACE) converts angiotensin I to angiotensin II, an octapeptide, and angiotensin III, a heptapeptide. Both angiotensin II and III stimulate aldosterone secretion via the type I angiotensin receptor. Angiotensin II also acts as a vasoconstrictor, and the net effect of the increase in renin and angiotensin II levels is expansion of extracellular and blood volume and increased blood pressure. Increased blood volume inhibits renin, reduces angiotension II and III levels, and decreases aldosterone secretion, resulting in increased sodium excretion.
207 Test Application and Interpretation
Vasopressin (antidiuretic hormone or ADH) modulates plasma osmolality, within narrow limits, by regulating renal free water excretion (urine osmolality). Increased serum sodium concentration (increased osmolality) stimulates pituitary ADH secretion which increases renal tubular water reabsorption to expand volume and reduce extracellular sodium concentration. Decreased sodium concentration inhibits ADH secretion decreasing urine osmolality, increasing free water excretion, and increasing serum sodium concentration. Thus, the kidney plays an essential role in sodium and water balance and blood pressure. Several genes are involved in renal tubular sodium reabsorption from the glomerular filtrate, and mutations in these genes have been associated with the rare Mendelian disorders of body sodium homeostasis (Ref. 8-9). These disorders and the prominent phenotypic manifestations are summarized in Table 13. Although rare, these disorders highlight the important relationship between salt intake, the RAAS, and blood pressure. Attempts to define the more subtle genetic alterations in sodium homeostasis predisposing to hypertension have provided mixed results, but positive correlations of hypertension with expression of the AGT gene for angiotensinogen and the ENaC gene have been demonstrated in humans and in animal models. Assessment of Disorders of the RAAS The key elements of the RAAS (renin and aldosterone) can be measured in blood. The tests are useful in evaluating secondary causes of hypertension, such as renovascular hypertension and primary aldosteronism (Figure 1). In some circumstances, a knowledge of the renin status in essential hypertension can be useful in determining therapy, since patients with high-renin hypertension respond better to renin-lowering agents, while low-renin cases may be better treated with diuretics.
Table 13. Heritable Disorders of Renal Sodium Handling Salt + Genes Inheritance Disorder Hypertension Wasting Aldo Renin K Involved Pattern Liddle syndrome Yes No ↓↓↓ENaC GOF dominant mutations Gordon syndrome Yes No ↓↓↑WNK LOF mutations increase NCCT activity Pseudohypo- No Yes ↑↑↑ENaC LOF dominant aldosteronism or recessive mutations Bartter syndrome No Yes ↑↑↓NKCC2 LOF recessive ROMK2 mutations* CLCNKB Barttin Gitelman syndrome No Yes ↑↑↓NCCT LOF recessive mutations Aldo, aldosterone; GOF, gain of function; LOF, loss of function; ENaC, epithelial sodium channel; WNK, with no lysine potassium kinase; NKCC2, sodium-potassium-chloride cotransporter; ROMK2, inward rectifier outer medullary potassium channel; CLCNKB, basolateral chloride channel; Barttin, cofactor for chloride channel kidney B (CLC-KB); NCCT, sodium chloride cotransporter. *ROMK2 or CLCNKB LOF mutations lead to decreased NKCC2 activity.
208 Test Application and Interpretation
Renin can be measured in 2 types of assays. In the classical method, the level of renin is determined by generating angiotensin I during incubation with angiotensinogen and measuring the angiotensin by RIA. This assay is called plasma renin activity (PRA). The renin direct assay is a newly developed procedure that utilizes an antibody directed to the renin molecule itself. The concentration of renin, rather than renin activity, is measured. Renin measurements should be made in plasma under controlled or known conditions of dietary sodium intake and body position. A diurnal variation is normally present and samples are typically obtained at 7 to 9 AM. A low-salt diet (<40 mEq/d) and diuretics stimulate renin and aldosterone, while a high-salt diet (>150 mEq/d) suppresses the system. If there is doubt about the general sodium status in the patient, a 24-hour urine measurement of sodium excretion is useful for interpretation. Aldosterone is regulated by the renin-angiotensin system but may be autonomously secreted by an adrenal tumor. In the former situation, renin and aldosterone parallel each other. In the case of primary aldosteronism, Test & Application Interpretation renin is suppressed and aldosterone inappropriately increased. The measurement of renin and aldosterone, in association with serum potassium concentration, provides important differential diagnostic information for the assessment of disorders of the renin-aldosterone axis (Table 14). The effect of mineralocorticoids (ie, increase renal potassium excretion in exchange for reabsorbed sodium) tends to reduce serum potassium levels in thypermineralocorticoid states. Serum potassium concentrations are increased in he
Table 14. Assessment of Disorders of the Renin-Aldosterone System Disorder Renin Aldosterone K+ (Serum) Primary aldosteronism ↓↑ ↓ Renin secreting tumor ↑↑N or ↓ Pseudohyperaldosteronism (apparent ↓↓ ↓ mineralocorticoid excess, AME) Dexamethasone-suppressible ↓↑ ↓ hyperaldosteronism Hypoaldosteronism (adrenal) ↑ N or ↓ ↑ Addison’s disease ↑↓ ↑ Hyporeninemic hypoaldosteronism ↓↓ or N ↑ Mineralocorticoid resistance ↑↑ ↑ (pseudohypoaldosteronism) Bartter syndrome ↑↑ ↓ Renovascular hypertension N or ↑ N or ↑ N or ↓ Diuretic medication ↑↑N or ↓ Congestive heart failure ↑↑N or ↓ Liver disease ↑↑N or ↓ Nephrosis ↑↑N or ↓
209 Test Application and Interpretation states of deficient mineralocorticoid production or action. Patients with secondary hyperaldosteronism caused by congestive heart failure, liver disease, or nephrosis may manifest hypokalemia, but these states are clinically evident. In Bartter syndrome, hyperaldosteronism is caused by juxtaglomerular hyperplasia and is associated with hypokalemic alkalosis in the absence of hypertension or edema.
Primary Aldosteronism Primary aldosteronism is an uncommon, but usually curable, cause of hypertension. Since aldosterone is the major regulator of potassium balance, hyperaldosteronism is associated with potassium wasting. One marker of the disorder is hypertension with unexplained hypokalemia (and alkalosis). Screening hypertensive patients for hypokalemia should be accomplished prior to the use of antihypertensive therapy, especially diuretics. Hypokalemia per se is normally associated with low aldosterone levels, so the combination of an inappropriate serum aldosterone (>15 ng/dL) and a low renin (<0.2 ng/dL) is diagnostic in the presence of hypokalemia. An elevated (>20) aldosterone/PRA ratio, or an aldosterone/direct renin ratio >5, also is useful in the diagnosis of primary aldosteronism (see Ref. 20). Two maneuvers are sometimes useful to confirm the diagnosis. An infusion of saline or administration of mineralocorticoid (Florinef®’ 0.4 mg/d x 2 days) will in each case reduce plasma aldosterone to <5 ng/dL. Two etiologies are recognized. Conn syndrome is caused by an aldosterone- secreting adrenal adenoma and is characterized by elevated levels of the precursor 18-hydroxycorticosterone with values >50 ng/dL. Less often (20%), the idiopathic form is caused by unexplained bilateral hypersecretion of aldosterone, in which case precursor levels are normal. The diagnosis of suspected primary hyperaldosteronism in patients with hypertension and hypokalemia requires measurements of serum and urine aldosterone and plasma renin (or renin activity) concentrations. 18-Hydroxycorticosterone and 18-hydroxycortisol levels in serum and urine are increased in patients with adrenal adenoma, and levels may be useful to differentiate adenoma from idiopathic hyperplasia. Serum 18-hydroxycortisol levels in >300 ng/dL or 18-hydroxycorticosterone levels >100 ng/dL are suggestive of adrenal adenoma. Diagnosis in some cases may be enhanced by aldosterone suppression testing using saline or fluorohydrocortisone (see “Dynamic Test Application and Interpretation” section). CT or MRI scans identify most adrenal tumors. Rarely, bilateral adrenal vein catheterization is utilized to determine whether the disorder is unilateral or bilateral. Recent reports emphasize the importance of diagnosing primary aldosteronism, since in addition to the hypertension, aldosterone excess leads to a cardiomyopathy and increased incidence of heart failure. Renovascular Hypertension Renovascular hypertension is a relatively common cause of primary hypertension and is more common in the elderly (atherosclerosis) and in a cohort of young women (fibronodular). Hypokalemia is observed only in the more severe cases. Suspicion and either a family history (fibronodular) or sudden development of hypertension in an older patient may be the only clues. Peripheral renin is 210 Test Application and Interpretation usually, but not invariably, increased. Aldosterone levels may be either normal or increased. Vascular studies are the key to diagnosis. The best indicator of a positive response to vascular surgery is demonstration of increased renin or renin activity levels (x2) from the ischemic kidney. Dexamethasone-suppressible Hyperaldosteronism Dexamethasone-suppressible hyperaldosteronism is a rare form of hypertension characterized by high levels of aldosterone, suppressed renin (or PRA), and a dominant mode of inheritance. Chronic ACTH administration stimulates a continued rise in aldosterone concentration; dexamethasone administration produces a rapid and complete suppression of serum aldosterone. Urinary excretion of 18-hydroxycortisol is increased. The pathogenesis appears to be abnormal regulation of aldosterone secretion by ACTH, perhaps due to intergenic gene promotor sequence recombination such that ACTH simulates transcription of the P-450c11 isozyme normally active in the zona glomerulosa. Test & Application Interpretation Pseudohyperaldosteronism Pseudohyperaldosteronism, a syndrome of apparent mineralocorticoid excess (AME), was first described in children with hypertension and hypokalemia. In adults, the syndrome has been associated with licorice ingestion and Cushing’s syndrome. The mechanism has recently been discovered. Adjacent to the mineralocorticoid receptor is the enzyme 11β-hydroxysteroid dehydrogenase. This enzyme normally protects the aldosterone receptor from cortisol, converting cortisol to cortisone. In AME or after heavy licorice ingestion, the enzyme is inactive and cortisol is free to act as a mineralocorticoid. In these patients, both renin and aldosterone are suppressed. Diagnosis can be confirmed by measuring urine cortisol and cortisone metabolites (THF/THE).
Adrenal Diagnosis via Urine GC/MS Metabolite Analysis Gas chromatography/mass spectrometry (GC/MS) of urinary adrenal steroids offers a useful supplement to serum precursor/product analysis of adrenal steroid enzymatic abnormalities. This approach allows comprehensive precursor/product and pattern analysis of adrenal steroid excretion using small- volume (5-mL) random urine samples. Production data can be estimated from analysis of 24-hour or other timed-interval urine collections. In newborn infants, urine can be collected in a clean diaper and squeezed out into a test tube. Urinary steroids are measured with the methods described by Shackleton or Ulick, et al (Ref. 27, 28). The free and conjugated steroids are first extracted from urine, whereupon the glucuronides and sulfates are cleaved by enzyme hydrolysis. The freed steroids are re-extracted, chemically derivatized to render them heat stable, and separated by fused-silica gas chromatography prior to mass spectrometric quantitation. Table 15 summarizes the measured urinary steroids and their abbreviations. Table 10 summarizes normal and abnormal urine steroid metabolite precursor/product ratios in neonates, children, and adolescents. Normative data for urinary steroid metabolite excretion in adults are shown in Table 16. Table 17 summarizes normal and abnormal steroid precursor/product ratios for the assessment of adrenal biosynthetic enzyme deficiencies in adolescents and adults.
211 Test Application and Interpretation
Table 15. Urinary Steroid Analyte Abbreviations Abbreviations Trivial Name AN Andosterone ET Etiocholanolone DHEA Dehydroepiandrosterone 5-AD-17β 5-Androstene-3β,17β-diol 11-OXO-ET 11-Oxo-etiocholanolone 17α-HP 17α-Hydroxypregnanolone 11β-OH-AN 11β-Hydroxy-androsterone 11β-OH-ET 11β-Hydroxy-etiocholanolone 16α-OH-DHEA 16α-HydroxyDHEA PD Pregnanediol PT Pregnanetriol 5-AT 5-Androstene-3β, 16α, 17β-triol THS Tetrahydrosubstance S Estriol Estriol PT’ONE 11-Oxo-pregnanetriol 5-PT 5-Pregnene-3β, 17α, 20α-triol THE Tetrahydrocortisone THA Tetrahydro-11-dehydrocorticosterone THB Tetrahydrocorticosterone 5α-THB 5α-Tetrahydrocorticosterone THF Tetrahydrocortisol 5α-THF 5α-Tetrahydrocortisol α-Cortolone α-Cortolone THALDO Tetrahydroaldosterone β-Cortolone β-Cortolone α-Cortol α-Cortol Cortisol Cortisol 11-OXO-AN 11-Oxoandrosterone 18-OXO-THF 18-Oxotetrahydrocortisol β-Cortol β-Cortol Cortisone Cortisone The steroids are listed approximately in order of their elution from the gas chromatography column.
212 Test Application and Interpretation
Table 16. Urinary Steroid Excretion Normal Values (n = 98) Metabolites Men Women μg/24-h Range μg/24-h Range AN 320-5400 240-2300 ET 430-3300 245-2300 DHEA 21-2710 21-2710 5-AD-17β 17-485 7-175 11-OXO-ET 78-1165 78-1165 17α-HP 59-470 16-460 11β-OH-AN 195-1500 130-900 11β-OH-ET 14-680 14-680 16α-OH-DHEA 81-2600 42-1040
PD 49-385 57-4540 Test & Application
PT 71-1000 47-790 Interpretation 5-AT 95-1320 45-680 THS 1.8-63.5 1.8-63.5 Estriol 0.72-8.17 0.35-16.77 PT’ONE 0.2-26.4 0.9-13.4 5-PT 10-480 47-210 THE 445-5960 330-3430 THA 36-270 36-270 THB 36-245 36-245 5α-THB 37-440 37-440 THF 280-2820 210-1680 5α-THF 100-2660 100-1250 α-Cortolone 47-1400 90-1100 THALDO 6-79 6-79 β-Cortolone 8-560 10-220 α-Cortol 30-490 25-215 Cortisol 7-185 13-115 11-OXO-AN 15-111 8-87 18-OXO-THF <42 <10 β-Cortol 8-340 17-215 Cortisone 19-245 24-160 Data developed by Quest Diagnostics Nichols Institute Clinical Correlations Department using a sample of 48 females and 50 males. All were apparently healthy, non-medicated employees aged 18 to 50 years.
213 Test Application and Interpretation
Table 17. Normal Ranges for Urinary Adrenal Enzyme Precursor/Product Ratios in Adolescents and Adults (GC/MS Analysis) Precursor Normal Ratios Product Male Female Affected 3β-HSD DHEA 0.05 0.10 THE + THR + 5α-THF 0.01-0.27 0.01-0.60 >5 5PT 0.05 0.05 THE + THF + 5α-THF 0.01-0.1 0.02-0.08 >5 AN + ET 0.66 0.60 THE + THF + 5α-THF 0.4-1.2 0.4-1.0 >5
P450c17 THA + THB + 5α-THB 0.10 0.10 THE + THF + 5α-THF 0.06-0.2 0.05-0.2 >20 AN + ET 0.17 0.15 THA + THB + 5α-THB 0.04-0.30 0.08-0.25 >20
P450c21 Classic Late Onset 17HP 0.04 0.03 THE + THF + 5α-THF 0.01-0.12 0.01-0.10 >5 0.17-1.0 PT 0.10 0.10 THE + THF + 5α-THF 0.06-0.12 0.02-0.15 >5 0.25-2.0 P’TONE 0.003 0.004 THE + THF + 5α-THF 0.002-0.008 0.001-0.005 >5 0.08-0.5
P450c11 AN + ET 3.0 4.1 11β-OH-AN + 11β-OH-ET 1.0-4.3 2.3-4.5 >10 THS 0.01 0.01 THE + THF + 5α-THF 0.006-0.036 0.005-0.022 >7
P450c11ase 18-OH-THA 3.2 2.6 THALDO 2.6-5.6 1.7-5.0 >10 THA + THB + 5α-THB (or 6α-OH-THA) 0.10 0.10 THE + THF + 5α-THF 0.06-0.2 0.05-0.2 >1.0 THA + THB + 5α-THB (or 6α-OH-THA) 20 15 THALDO 3.6-22 10-22 >50 From Ref. 8, 29.
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References 1. Stewart PM. The adrenal cortex. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Philadelphia, PA: WB Saunders; 2003:491-551. 2. Clark AJL, Grossman AB. Adrenal insufficiency. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:2343- 2351. 3. Cavagnini F, Giraldi FP. Adrenal causes of hypercortisolism. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:2353-2386. 4. Ilias I, Torpy DJ, Pacak K, et al. Cushings syndrome due to ectopic cortico- tropin secretion: twenty years experience at the National Institutes of Health. J Clin Endocrinol Metab. 2005;90:4955-4962. Test & Application 5. Malchoff DM, Malchoff CD. Generalized glucocorticoid resistance. In: Interpretation DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier- Saunders; 2006:2387-2392. 6. Conton P, Maccino B, Montero F. Adrenal cancer. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:2455- 2460. 7. Dluhy RG, Lawrence JE, Williams GH. Endocrine hypertension. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Philadelphia, PA: WB Saunders; 2003:552-586. 8. O’Shaughnessy KM, Karet FE. Salt handling and hypertension. J Clin Invest. 2004;113:1075-1081. 9. Jeunemaitre X. Renin-angiotensin-aldosterone system polymorphisms and essential hypertension: where are we? J Hypertens. 2003;21:2219-2222. 10. Kasperlik-Zaluska AA, Czarnocka B, Czech W. Autoimmunity as the most frequent cause of idiopathic, secondary adrenal insufficiency: report of 111 cases. Autoimmunity. 2003;36:155-159. 11. Root AW, Shulman DI. Clinical adrenal disorders. In: Pescovitz OH, Eugster EA, eds. Pediatric Endocrinology, Philadelphia, PA: Lippincott Williams and Wilkins, 2004:568-600. 12. Wajnrajch MP, New MI. Defects in adrenal steroidogenesis. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:2393-2416. 13. Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J Med. 2003;349:776-788. 14. Clayton PE, Miller WL, Oberfield SE et al. Consensus statement on 21- hydroxylase deficiency from the European Society for Paediatric Endocrinology and the Lawson Wilkins Pediatric Endocrine Society. Horm Res. 2002;58:188-195. 15. Olgemoller B, Roscher AA, Liebl B, et al. Screening for congenital adrenal hyperplasia: adjustment of 17-hydroxyprogesterone cut-off values to both age and birth weight markedly improves the predictive value. J Clin Endocrinol Metab. 2003;88:5790-5794.
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16. Homma K, Hasegawa T, Nogai T, et al. Urine steroid profile analysis in cytochrome P450 oxidoreductase deficiency: implication for the backdoor pathway to dihydrotestosterone. J Clin Endocrinol Metab. 2006;91:2643-2649. 17. New MI, Carlson A, Obeid J, et al. Update: prenatal diagnosis of congenital adrenal hyperplasia in 595 pregnancies. Endocrinologist. 2003;13:233-239. 18. Caulfield MP, Lynn T, Gottschalk ME, et al. The diagnosis of congenital adrenal hyperplasia in the newborn by gas chromatography/mass spectrometry analysis of random urine specimens. J Clin Endocrinol Metab. 2002;87:3682-3690. 19. New MI. Extensive clinical experience: nonclassical 21-hydroxylase deficiency. J Clin Endocrinol Metab. 2006;91:4205-4214. 20. Mulatero P, Stowasser M, Loh KC, et al. Increased diagnosis of primary aldosteronism, including surgically correctable forms, in centers from five continents. J Clin Endocrinol Metab. 2004;89:1045-1050. 21. Lashansky G, Saenger P, Fishman K, et al. Normative data for adrenal steroidogenesis in a healthy pediatric population: age- and sex-related changes after adrenocorticotropin stimulation. J Clin Endocrinol Metab. 1991;73:674-686. 22. Lashansky G, Saenger P, Dimartino-Nardi J, et al. Normative data for the steroidogenic response of mineralocorticoids and their precursors to adrenocorticotropin in a healthy pediatric population. J Clin Endocrinol Metab. 1992;75:1491-1496. 23. Hingre RV, Gross SJ, Hingre KS, et al. Adrenal steroidogenesis in very low birth weight preterm infants. J Clin Endocrinol Metab. 1994;78:266-270. 24. Adrenal steroid response to ACTH. Pediatrics, Endocrine Sciences, Calabasses Hills, CA. May 1991. 25. Viardot A, Huber P, Puder JJ, et al. Reproducibility of nighttime salivary cortisol and its use in the diagnosis of hypercortisolism compared with urinary free cortisol and overnight dexamethasone suppression test. J Clin Endocrinol Metab. 2005;90:5730-5736. 26. Agha A, Tomlinson JW, Clark PM, et al. The long-term predictive accuracy of the short synacthen (corticotropin) stimulation test for assessment of the hypothalamic-pituitary-adrenal axis. J Clin Endocrinol Metab. 2006;91:43-47. 27. Shackleton CH. Mass spectrometry in the diagnosis of steroid-related disorders and in hypertension research. J Steroid Biochem Mol Biol. 1993;45:127- 140. 28. Ulick S, Chan CK, Wang JZ. Measurement of 4 urinary C-18 oxygenated corticosteroids by stable isotope dilution mass fragmentography. J Steroid Biochem Mol Biol. 1991;38:59-66. 29. Corvol P, Persu A, Gimenez-Roqueplo AP, et al. Seven lessons from two candidate genes in human essential hypertension: angiotensinogen and epithelial sodium channel. Hypertension. 1999;33:1324-1331.
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DISORDERS OF ANTERIOR PITUITARY FUNCTION
Background Physiology The hypothalamic-pituitary complex (Figure 1) is the central control system for most of the important endocrine glands. It functions as a neuroendocrine transducer in order to translate neural system output into endocrine hormone secretion. The hypothalamus is comprised of multiple cell clusters, or nuclei, that regulate synthesis and secretion of a variety of neurotransmitters and hormone releasing factors, including corticotropin releasing hormone (CRH), growth hormone releasing hormone (GHRH), gonadotropin releasing hormone (GNRH), thyrotropin releasing hormone (TRH), dopamine \(DA), and somatostatin (SS), which acts as a release inhibiting factor. The pituitary portal vascular system transports these factors to the anterior pituitary where
production/secretion of adrenocorticotropic hormone (ACTH), growth Test & Application
hormone (GH), luteinizing/follicle stimulating hormones (LH/FSH), thyroid Interpretation stimulating hormone (TSH), and prolactin (PRL) are regulated by the corticotropic, somatotropic, gonadotropic, thyrotropic, and lactotropic cells comprising the anterior pituitary tissue mass. The releasing hormones bind to and activate their respective receptors (CRHR, GHRHR, GNRHR, TRHR, SSR, DAR) in the tropic cells, thereby regulating synthesis and secretion of the anterior pituitary hormones. Arginine vasopressin (AVP) and oxytocin (OT) are produced by the hypothalamic magnocellular neurons whose axons terminate in the posterior pituitary wherein the hormones are stored and secreted. Regulation of PRL secretion is more complex and involves stimulation by TRH, vasoactive
Figure 1. The hypothalamic-pituitary neuroendocrine transducer system functions to translate neuronal output into endocrine hormonal activity. The pituitary portal vascular system transports the releasing hormones dopamine (DA) and somatostatin (SS) to the anterior pituitary where they bind to their cognate receptors to modulate their respective peripheral endocrine organs.
217 Test Application and Interpretation intestinal peptide (VIP), OT (via a short portal system), pituitary adenylate cyclase-activating peptide (PACAP), and galanin and inhibition by DA. GH secretory control also is complex and involves stimulation by GHRH and inhibition by SS. The regulation of these factors within the hypothalamus involves a variety of stimuli (see "Disorders of the Growth Hormone Insulin-like Growth Factor Axis"). A cascade of homeodomain proteins and transcription factors is involved in hypothalamic-pituitary embryogenesis and function (Figure 2). Mutations of the homeobox genes sonic hedgehog (SHH), ZIC1, and SIX3 have been described in patients with holoprosencephaly. HESXI mutations have been identified in siblings with septo-optic dysplasia in association with midline brain defects and pituitary hypoplasia. GLI3 mutations have been associated with the Pallister-Hall syndrome involving hypothalamic dysgenesis and hypopituitarism. Transcription factors participating in early pituitary development include the Rathke Pouch gene (RPX), PITX, and LHX genes. Genes participating in later stages of pituitary development, PROP1 and PITI, program development and function of the pituitary cells producing GH, TSH, and PRL.
Hypopituitarism Hypopituitarism refers to deficiency of 1 or more of the pituitary hormones. ACTH, TSH, gonadotropin, and GH deficiencies are reviewed in separate sections. Multiple pituitary hormone deficiencies are associated with combined manifestations of GH deficiency, hypothyroidism, adrenal insufficiency, and/or hypogonadism. The causes of hypopituitarism are summarized in Table 1. Mutations of the homeobox genes sonic hedgehog (SHH), ZIC1, and SIX3 have been identified in patients with holoprosencephaly and HESX-1 mutations in association with septo-optic dysplasia. GLI3 defects have been described in the Pallister-Hall syndrome associated with hypothalamic disorganization and hypopituitarism (Figure 2).
Figure 2. A cartoon displaying the cascades of homeodomain proteins and transcription factors mediating hypothalamic-pituitary embryonic development. See text for details. 218 Test Application and Interpretation
Mutation of the TRHR gene, the GHRHR gene, and the GNRHR gene has been reported in association with isolated TSH, GH, and gonadotropin hormone deficiencies, respectively (Figure 1). TRH and GHRH deficiencies are usually diagnosed during infancy and childhood because of their profound effect on growth. GNRH deficiency (Kallmann's syndrome) usually is not recognized prior to puberty. This deficiency can be due to mutations in the X-linked Kal-1 gene or the autosomal Fgf1 receptor gene. Other forms of idiopathic hypogonadotropic hypogonadism are due to mutations in the GP54 receptor gene which may play a role in the timing of puberty. This is discussed in more detail in the gonadal section. A variety of mutations in pituitary transcription factor genes and pituitary hormone genes have been characterized in hypopituitary patients. The phenotypic features of patients with pituitary transcription factor gene mutations are summarized in Table 2 and are discussed further in related sections. Test & Application
The pituitary hormones are easily measured in peripheral blood. Hypothalamic Interpretation hormones are not secreted into peripheral blood, but can be detected in blood where they are derived from ectopic tissue sources. Quantification of selected hypothalamic hormones in peripheral blood can be useful in the diagnosis of patients with excessive pituitary hormone secretion caused by ectopic hypothalamic hormone production from extrapituitary tumors.
Table 1. Causes of Hypopituitarism Developmental anomalies Vascular events Basal encephalocele Postpartum (Sheehan necrosis) Holoprosencephaly Internal carotid aneurysm Median facial anomalies Inflammatory diseases Septo-optic dysplasia Encephalitis Pituitary hypoplasia Meningitis, bacterial Congenital hormone deficiencies Granulomatous disease Hypothalamic Histiocytosis X TRH Autoimmune hypophysitis GHRH Destructive lesions LHRH (Kallmann’s syndrome) Intraventricular hemorrhage Isolated pituitary hormone deficiencies Surgery ACTH Radiation TSH Trauma GH Neoplasia LH, FSH Hypothalamic tumors Multiple pituitary hormone deficiencies Pituitary adenomas PIT1 mutation Metastatic carcinoma PROP1 mutation Pinealoma HESX1 mutation Craniopharyngioma LHX3 and LHX4 mutation
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Table 2. Phenotypic Features of Patients with Pituitary Trancription Factor Gene Mutations Gene Mutations PROP1 PIT1 LHX3 LHX4 HESX1 TPIT GH D A Α DDN Prolactin D A A V ΝΝ TSH D D Α VVN LH/FSH D N Α NNN ACTH V ΝΝ V Ν D Post pituitary N N N E E N D, decreased; A, absent; N, normal; V, variable; E, ectopic. Modified from Parks JS, Brown MR. Ref. 7.
Pituitary Adenomas
General Features Adenomas of the anterior pituitary gland are relatively common and are variable with regard to their cell of origin and function. Incidental pituitary adenomas have been documented in up to 20% of adult humans at autopsy. Most of these do not produce signs or symptoms. The relative prevalence of the clinically significant pituitary adenomas is summarized in Table 3. The pituitary adenomas are classified by their hormonal secretion(s), and most are characterized by the clinical effects of the excessive hormone production (Table 4). The most common secreting pituitary adenoma is the prolactin-secreting prolactinoma. The least common of the functional varieties are the gonadotropinomas and thyrotropinomas. Most gonadotroph-derived adenomas, also referred to as a chromophobe adenoma, are nonsecreting and present with compressive symptoms associated with multiple hormone deficiencies, including hypogonadism or mild elevations in prolactin. Most of these nonsecreting tumors show immunostaining for 1 or more hormones, including the alpha subunit, LH beta, FSH beta, and, less frequently, TSH beta subunits, and ACTH. Some of these tumors secrete alpha, LH beta, and FSH beta subunits which are detectable in serum and can be used as tumor markers. LH, FSH, and alpha subunit respond paradoxically to TRH stimulation. Thyrotrope secreting tumors lead to hyperthyroidism with an elevated or inappropriately normal TSH. The application of hormone assays to pituitary tumor characterization is summarized in Table 4.
Table 3. Relative Prevalence (%) of Pituitary Adenomas* Prevalence (%) Prolactinomas 29 GH-producing 15 GH + prolactin-producing 8 Corticotroph adenomas 16 Gonadotropinomas 7 Thyrotropinomas 1 Null cell adenomas 27 *From Melmed S, Kleinberg D (Ref 2). 220 Test Application and Interpretation
Table 4. Hormone Products Associated with Pituitary Adenomas Hormone or Pro- Acro- Cushing’s Thyro- Gonado- Non- secretory Precursor lactinoma megaly Disease tropinoma tropinoma Adenoma ACTH √ GH √ PRL √ TSH √ LH √ FSH √ Alpha √√√
Subunit Test & Application
Lipoprotein √ Interpretation Endorphin √ Chromogranin √√√ √ √ √ IGF-I √ IGFBP-3 √
Prolactinoma Serum prolactin levels normally are <15 ng/mL in males and <25 ng/mL in females. Causes of hyperprolactinemia are shown in Table 5.
Table 5. Causes of Hyperprolactinemia Physiologic Newborn Pregnancy Lactation Intracranial disease Hypothalamic disorders Pituitary stalk disorders Pituitary adenoma Idiopathic hyperprolactinemia Chest wall stimulation Herpes zoster Breast infection or tumor Surgical or accidental traumas Breast stimulation Drugs (eg, phenothiazines, metoclopramide) Miscellaneous Hypothyroidism Chronic renal failure Big/macroprolactinemia
221 Test Application and Interpretation
A prolactinoma usually is associated with clinical evidence of gonadal dysfunction and/or symptoms related to a pituitary mass effect. Galactorrhea is present in 30% to 80% of women with prolactinomas but is uncommon in men. Basal blood samples are adequate to diagnose hyperprolactinemia. Stimulation testing usually is not required and does not aid in differential diagnosis of prolactinoma. Borderline hyperprolactinemia is best confirmed by measurements in single samples collected on 3 separate days or in 3 separate samples collected at 30-minute intervals via indwelling catheter. Prolactin (PRL) is a stress hormone, and stress should be avoided when sampling blood. Postprandial and post-breast examination sampling should also be avoided. Most prolactinomas are associated with serum PRL levels >100 ng/mL, while levels in drug-induced hyper- prolactinemia tend to be lower. Mild elevation of prolactin of <100 ng/mL is also seen in stalk compression due to pituitary or other parasellar masses. The stalk compression relieves the lactotrophs from the normal inhibitory dopaminergic tone. The diagnosis of prolactinoma is confirmed in patients without other obvious causes of hyperprolactinemia by pituitary imaging techniques (Table 5). PRL exists in a variety of forms: the 26 kd monomer, the 40-60 kd dimeric or "big" prolactin, and the >100 kd macroprolactin, which is the monomer complexed with IgG. Macroprolactin is biologically inactive but cross-reacts in several PRL immunoassays. Since the presence of macroprolactin may lead to an overestimation of the biologically active form of PRL, macroprolactin should be specifically measured in patients with apparent hyperprolactinemia that is not consistent with the clinical presentation.
Acromegaly Acromegaly with or without increased prolactin secretion accounts for some 15% of clinically significant pituitary adenomas. The prevalence ranges from 38 to 69 cases per million population. Clinical features are related to sustained over secretion of GH and IGF-I. The primary pituitary somatotroph adenomas generally are slow growing, and clinical manifestations develop insidiously over many years. These include coarsening of the facial features, enlargement of the hands and feet, excessive sweating, amenorrhea, impotence, diabetes mellitus, hypertension, skin tags, and a variety of seemingly unrelated complaints, such as jaw malocclusion, carpal tunnel syndrome, colonic polyps, arthritis, and heart failure. Rarely, hyperthyroidism due to increased TSH secretion may coexist. The diagnosis is confirmed by demonstrating excessive GH secretion. Measurement of GH in random serum samples may be inadequate, since normal values vary during the day. In patients with suggestive clinical features and normal GH levels, the glucose suppression test may be useful. Patients with acromegaly fail to suppress serum GH to <1 ng/mL following ingestion of 75 g of glucose (see Dynamic Test section). Additional confirmation is obtained by measurement of serum IGF-I and IGFBP-3 levels. Both are GH-dependent proteins, and levels usually are increased in patients with acromegaly. In patients with confirmed GH hypersecretion, pituitary imaging is conducted to identify a pituitary mass. In the absence of a pituitary tumor, an ectopic source of growth hormone releasing hormone (GHRH) must be considered. Ectopic acromegaly usually can be diagnosed by measuring serum GHRH levels in peripheral blood and by CT or MR imaging of the chest, abdomen, and/or pelvis. See the “Growth Hormone
222 Test Application and Interpretation
Insulin-like Growth Factor Axis” section for further information. Treated patients should have lifelong monitoring of IGF-1 and GH suppression testing since recurrences are frequent and the recent broadening of therapeutic options with somatostatin analogues and GH antagonists gives additional opportunities for intervention. Failure to suppress GH in response to glucose and persistently high IGF-1 levels are predictors of increased morbidity and mortality and thus should be normalized if possible.
Cushing’s Disease See section on “Disorders of Adrenal Function.”
Thyrotropinoma
TSH-secreting adenomas account for <1% of all pituitary tumors. They are Test & Application characterized by hyperthyroidism and measurable (nonsuppressed) levels of Interpretation circulating TSH. Such TSH-dependent hyperthyroidism must be distinguished from other causes of inappropriate TSH secretion (ie, thyroid hormone resistance syndromes). Co-secretion of TSH and prolactin, or GH, may occur, and alpha subunit levels may be increased. In most (88%) patients with thyrotropinoma, there is disproportionate secretion of alpha subunit and an alpha subunit/TSH ratio >1.0; in non-tumorous hyperplasia the ratio does not exceed 1.0. The diagnosis is confirmed by pituitary imaging. Although thyroid hormone resistance and TSH secreting adenomas are 2 distinct clinical entities, an association of these tumors with thyroid hormone resistance or a thyroid hormone receptor defect has been described, suggesting a possible causal relationship in some of these patients.
Gonadotropinoma Previously, many macroadenomas now recognized by immunostaining to be gonadotropin-producing were classified as nonsecreting or chromophobe adenomas, because they usually do not produce a recognizable clinical syndrome. Thus, the classification of gonadotropinomas versus chromophobe adenomas was variable. Gonadotroph adenomas constitute 80% to 90% of all nonfunctioning pituitary adenomas using molecular, cell culture, and immunostaining techniques. Because of the minimal symptoms, gonadotropinomas are not detected early and present more commonly as macroadenomas than microadenomas, often with visual or neurological symptoms related to the mass effect. The tumors frequently produce increased serum FSH levels but only uncommonly increased serum LH concentrations. The increased biologically active levels of FSH may lead to an ovarian hyperstimulation syndrome, and elevations of LH, FSH, or both may lead to testicular enlargement or precocious puberty. About 15% of patients evidence increased alpha subunit concentrations, and both intact gonadotropin and subunit levels may increase (>30%) in response to TRH stimulation. The alpha subunit test can help differentiate primary hypogonadism from hypogonadism due to a tumor secreting nonfunctional LH and FSH.
223 Test Application and Interpretation
A pituitary adenoma is likely to be of gonadotroph or thyrotroph origin if the serum prolactin level is <100 ng/mL, the patient has no acromegalic features, the serum IGF-I concentration is not increased, and there is no clinical or laboratory evidence of Cushing's syndrome. In men, elevated basal levels of FSH, alpha subunit, and FSH and LH beta subunits, alone or in combination with responses of these to TRH, are strong evidence for a gonadotroph adenoma. In women, measurements of FSH are not usually helpful unless the FSH concentration is markedly increased in the presence of a normal LH value. The response of intact FSH or LH levels and of the alpha and LH beta subunits to TRH may be helpful.
References 1. Cone RD, Low MJ, Elmquist JK, Cameron JL. Neuroendocrinology. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Philadelphia, PA: WB Saunders; 2003:81-176. 2. Melmed S, Kleinberg D. Anterior pituitary. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Phila- delphia, PA: WB Saunders; 2003:177-277. 3. Jostel A, Lissett CA, Shalet SM. Hypopituitarism. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:397-409. 4. Bronstein MD. Disorders of prolactin secretion and prolactinomas. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier- Saunders; 2006:485-510. 5. Melmed S. Acromegaly. In: DeGroot LJ, Jameson JL, et al, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:411-428. 6. Snyder PJ. Gonadotroph adenomas. In: DeGroot LJ, Jameson JL, et al, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:465-473. 7. Parks JS, Brown MR. Consequences of mutations in pituitary transcription factor genes. In: Pescovitz OH, Eugster EA, eds. Pediatric Endocrinology. Phila- delphia, PA: Lippincott Williams and Wilkins; 2004:80-89. 8. Wu W, Rosenfeld MG. Development of the pituitary. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:291- 308. 9. Braunstein GD. Hypothalamic syndromes. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:373-386. 10. Melmed S. Evaluation of pituitary masses. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:387-395. 11. Raynaud R, Gueydan M, Saveanu A, et al. Genetic screening of combined pituitatry hormone deficiency: Experience in 195 patients. J Clin Endocrinol Metab. 2006;91:3329-3336. 12. Daly AF, Jaffrain-Rea ML, Ciccarelli A, et al. Clinical characterization of famil- ial isolated pituitaty adenomas. J Clin Endocrinol Metab. 2006;91:3316-3323.
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DISORDERS OF CALCIUM AND BONE METABOLISM
Background Physiology The parathyroid glands, usually 4 in number, are small derivatives of the pharyngeal pouches located on the posterior surfaces of the bilobed thyroid gland (Figure 1). These glands are the only source of parathyroid hormone (PTH). PTH secretion is modulated by changes in concentration of free calcium in blood plasma; decreased concentrations of calcium stimulate and increased concentrations inhibit PTH secretion. PTH is an 84-amino acid protein hormone that acts 1) on the kidney tubules to increase phosphorus (P) excretion and enhance calcium reabsorption, 2) mobilize calcium from bone tissue, and 3) stimulate the 1α-hydroxylation of 25(OH) vitamin D [25(OH)D] to 1,25(OH)2D. This hydroxylation greatly augments the biological activity of vitamin D, and 1,25(OH)2D acts on the gut to increase absorption of dietary Test & Application calcium. All of these actions tend to increase the blood calcium concentration Interpretation and increase the circulating blood calcium/phosphorus ratio. 1,25(OH)2D also acts to potentiate bone mineralization.
Phosphorus
Figure 1. The control of blood calcium by parathyroid hormone (PTH) and vitamin D. See text for details.
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PTH is secreted predominantly as an intact molecule, but it is rapidly cleaved in peripheral tissues to amino terminus and carboxy terminus fragments. The amino terminal fragment is biologically active and has a relatively short plasma half-life. The carboxyterminal species include a 7-84 peptide and a variety of shorter fragments. They have a longer half-life but are biologically inactive. The most reliable assays for PTH measure the intact molecule. Vitamin D is a fat-soluble vitamin stored in precursor form in skin and other tissues. Hydroxylation to 25(OH)D occurs in liver, and this form is the major circulating form of the vitamin. It serves as a precursor for 1α-hydroxylation to the active moiety [1,25(OH)2D], and circulating levels of 25(OH)D serve as a marker for the adequacy of vitamin D nutrition. Vitamin D is ingested as vitamin D2 (ergocalciferol), as D3 (cholecalciferol), or as precursor ergosterol, which is activated to ergocalciferol in skin by ultraviolet light. Disorders of the system occur because of congenital absence or acquired damage to the parathyroid glands (hypoparathyroidism); adenoma or hyperplasia of the glands (primary hyperparathyroidism); vitamin D deficiency; molecular defects in the PTH receptor (pseudohypoparathyroidism), the calcium sensing receptor (familial hypocalcemic hypercalciuria, neonatal severe primary hyperparathyroidism, and autosomal dominant hypocalcemia), the vitamin D receptor (vitamin D resistance), or 1α-hydroxylase (vitamin D dependence); and renal disease that limits phosphorus excretion and 1,25(OH)2D production with resulting hypocalcemia and (secondary) hyperparathyroidism. Parathyroid hormone-related protein (PTH-RP) is a protein with a 14-amino acid amino terminus analogous to the amino terminus of PTH. It binds to and activates the PTH receptor and can produce hypercalcemia. It is secreted by fetal tissues and plays a role in fetal calcium metabolism and fetal bone development. In adults, the hormone is produced in large amounts by the lactating breast and is secreted in breast milk. The physiological significance of PTH-RP in children and adults is not clear. It is, however, produced by a variety of tumor tissues and is the most common cause of the hypercalcemia of malignancy. Calcitonin (CT) is derived from the thyroid gland C-cells, which are widely dispersed in the interfollicular tissue of the gland. CT secretion is stimulated by increased plasma calcium levels and has the capacity to inhibit the effect of PTH on bone tissue. CT levels are relatively increased in the fetus and may have a role in fetal bone anabolism. However, there is a limited role for CT in calcium or bone metabolism in children and adults; CT deficiency has no significant effect on calcium homeostasis. CT serves as a marker for medullary thyroid carcinoma, a malignancy involving the thyroid C-cells.
Laboratory Assessment of Disorders of Calcium Metabolism The availability of sensitive and specific tests for several hormones modulating serum calcium levels has greatly improved our ability to assess disorders of calcium metabolism (Tables 1 and 2). Medullary thyroid carcinoma is discussed in the “Pheochromocytoma, Medullary Thyroid Carcinoma, and Multiple Endocrine Neoplasia” section.
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Table 1. Disorders of Calcium Metabolism Disorder Phenotype Etiology Vitamin D deficiency Hypocalcemia, defective bone Dietary deficiency mineralization, rickets, osteo- malacia Vitamin D dependence, Hereditary rickets, growth Vitamin D, 21α-hydroxy- type I retardation, hypocalcemia lase mutation Vitamin D dependence, Hereditary rickets, alopecia, Vitamin D receptor type II (vitamin D growth retardation, hypocalcemia mutations resistance) Hypoparathyroidism Hypocalcemia, neuromuscular Autoimmune embryonic irritability, cataracts, increased defect, surgery, radiation, bone density PTH deficiency, other Test & Application
Pseudohypoparathy- Albright's hereditary osteo- Gsa protein defect, GNAS Interpretation roidism (PTH resis- dystrophy (variable expression), mutations tance) generalized hormone resistance Autosomal dominant Normal; resistance limited to Imprinting defect in hypocalcemia PTH target tissues GNAS gene Neonatal hypocalcemia Transient neonatal hypocalcemia Maternal hypercalcemia Hyperparathyroidism Increased PTH secretion, Parathyroid adenomas, hypercalcemia, nephrolithiasis, MEN, HRPT2 mutation decreased bone mineralization Tumor hypercalcemia Hypercalcemia, PTH suppression Tumor secretion of PTH-RP Familial benign hyper- Osteomalacia Calcium sensing receptor calcemia (hypocalci- (CaSR) mutation, uric hypercalcemia) heterozygous Neonatal severe Polyuria, hypotonia, bone CaSR mutation heterozy- hyperparathyroidism demineralization, fractures gous or homozygous
Secondary hyperpara- Hypocalcemia, osteodystrophy 1,25(OH)2 vitamin D thyroidism (CRF) deficiency, parathyroid hyperplasia
Tertiary hyperpara- Hypocalcemia, osteodystrophy 1,25(OH)2 vitamin D thyroidism (ESRD) deficiency, parathyroid autonomy Medullary thyroid Increased calcitonin secretion, MEN or sporadic C-cell carcinoma C-cell malignancy carcinoma PTH-RP, parathyroid hormone related protein; CRF, chronic renal failure; ESRD, end stage renal disease.
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Table 2. Assessment of Disorders of Calcium-Related Hormones Blood Measurements Urine Disorder PTH CT Ca PO 25D 1,25D PTH-RP Calcium Vitamin D deficiency ↑ NN or ↓↓ ↓ ↑ N or ↓ N ↓ Vitamin D ↑ N ↓ N or ↓ N or ↑↓ N ↓ dependence, type I Vitamin D dependence, N or ↑ N ↓ N or ↓ N ↑ N ↓ type II (vitamin D resistance) Hypoparathyroidism N or ↓ N ↓↑ NN or ↓ N ↓ Pseudohypoparathyroid- ↑ NN or ↓ N or ↑ NN or ↓ N ↓ ism (PTH resistance) Autosomal dominant N or ↓ N ↓↑ N or ↓ NN N N or ↑ hypocalcemia Neonatal hypocalcemia N or ↓↑ ↓ ↑ NN N or ↓↓ Hyperparathyroidism ↑ N ↑ N or ↓ NN or ↓ N ↑ Tumor hypercalcemia ↓ N ↑ N or ↓ NN or ↓ N or ↑ N or ↑ Familial benign N or ↑ N ↑↓ or N N N N ↓ hypercalcemia (hypo- calciuric hypercalcemia) Neonatal severe ↑ N ↑↑ or N N N N ↓ primary hyperpara- thyroidism Secondary hyper- ↑↑N ↑↓ or N ↓ or N N ↓ parathyroidism (CRF) Tertiary hyperpara- ↑↑ ↑ ↑ ↑ ↓ or N ↓ NN/A thyroidsm (ESRD) Medullary thyroid- N ↑ NN N N N N carcinoma PTH, parathyroid hormone; CT, calcitonin; Ca, calcium; PO, phosphorus; 25D, 25-hydroxyvitamin D; 1,25 D, 1,25-dihydroxyvitamin D; PTH-RP, parathyroid hormone related protein; CRF, chronic renal failure; ESRD, end stage renal disease; N/A, not applicable. Evaluation of Hypercalcemia Laboratory tests are essential to evaluate patients with hypercalcemia (Table 1). The parathyroid hormone (PTH) assay plays a central role in differentiating the 2 most common causes of hypercalcemia (hyperparathyroidism and tumor- associated hypercalcemia), particularly when the PTH concentration is interpreted in relationship to the ambient serum or plasma calcium level (Figure 2). It is extremely useful in defining the most fruitful pathway for subsequent diagnostic evaluation of the hypercalcemic patient. Parathyroid hormone-related peptide (PTH-RP), the mediator of hypercalcemia in some patients with malignancy, can be assayed directly. Vitamin D measurements are also helpful in identifying patients with exogenous vitamin D intoxication and excessive endogenous vitamin D activation (eg, sarcoidosis and lymphoma). Other causes of hypercalcemia include hyperthyroidism, immobilization, thiazide diuretics, vitamin D intoxication, vitamin A intoxication, the milk-alkali syndrome, and lithium therapy. Other than lithium therapy, which causes increased PTH secretion through a shift in the dose response curve relating serum calcium to PTH secretion, these causes of hypercalcemia are associated with suppression of serum PTH levels.
228 Test Application and Interpretation Test & Application Interpretation
Figure 2. Relationship of serum calcium and intact PTH (IRMA or ICMA) in various disease states. The hatched square indicates normal pediatric range. S, hypoparathyroidism; U, primary hyperparathyroidism; O, hypercalcemia of malignancy.
Familial hypocalciuric hypercalcemia (FHH), a familial benign hypercalcemia, is an autosomal dominant trait. The hypercalcemia is asymptomatic in most subjects and most prominent in the first decade of life. The hypocalciuria is due to increased renal tubular calcium reabsorption. Serum PTH levels are in the normal range but inappropriate for the serum calcium level. The defect is due to a loss of function mutation of the gene on chromosome 3 for the calcium-sensing receptor that is expressed in kidney, parathyroid cell, and other tissues. The heterozygous state causes FHH (as a dominant negative allele), while the homozygous state produces severe neonatal hypercalcemia. Diagnosis requires demonstrating hypocalciuria and reduced renal calcium clearance in the context of a family history of these disorders. Mutational analysis of the calcium sensing receptor confirms the diagnosis in those with a family history and is necessary for the diagnosis in patients without a family history.
Evaluation of Hypocalcemia The causes of hypocalcemia are listed in Table 1. Hypoparathyroidism can be due to damage to the parathyroid glands (surgery, infiltrative disease, irradiation, chemotherapeutic drugs, sepsis), to autoimmune disorders, or to molecular genetic defects. The latter involve autosomal defects linked to the PTH gene on chromosome 11 as well as the yet to be defined X-linked variety. Activating mutations in the calcium sensing receptor causing autosomal dominant hypocalcemia (ADH) are also causative.
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Pseudohypoparathyroidism, or PTH resistance, is due to a defect in the renal PTH receptor-adenylate cyclase complex (type I) or a more distal defect blocking the renal phosphaturic response to cAMP (type II). Vitamin D deficiency, dependence, and resistance often are associated with rickets or osteomalacia. Type I vitamin D dependence is due to a functional defect in the 25(OH) vitamin D, 1α-hydroxylase enzyme in the kidney, impairing activation of 25(OH) vitamin D to 1,25(OH)2 vitamin D. Type II dependence is due to a defective vitamin D receptor. Transient neonatal hypocalcemia is due to chronic fetal hypercalcemia associated with maternal hyperparathyroidism. Recovery of parathyroid gland function, which is suppressed in utero, is delayed, and there is a period of relative hypoparathyroidism lasting several days to 2 to 4 weeks in the early neonatal period.
Vitamin D Deficiency Vitamin D is essential for optimal gastrointestinal absorption of calcium and maintenance of bone mineral density (BMD). Deficiency is relatively common, especially in the elderly. Daily requirements vary with age, calcium intake, and sunlight exposure. An intake of 400 IU/d is recommended for infants and children and for adults with minimal sunlight exposure. In the elderly, efficacy of UV light-stimulated synthesis of vitamin D in the skin is reduced; thus, a minimal daily intake of 600 IU is recommended. To prevent osteoporotic fractures in elderly individuals, 800 IU/d of vitamin D and 1200 mg/d of calcium are recommended. The recommended upper limit of intake is 2,000 IU/d.
Endogenous vitamin D is D3 (cholecalciferol), whereas supplemental vitamin D in the United States is primarily D2 (ergocalciferol). The adequacy of vitamin D levels is assessed by measuring serum levels of 25(OH)D; however, some immunoassays underestimate D2 and thus total circulating 25(OH)D. The tandem mass spectrometry method, on the other hand, accurately detects and quantifies both D2 and D3 and thus is the method of choice when monitoring patients taking vitamin D supplements. Figure 3 shows the relationship of serum 25(OH)D levels vs PTH concentrations in apparently healthy 18 to 65 year-old individuals. PTH concentrations increase perceptibly when 25(OH)D levels fall below 30 ng/mL and are clearly increased at 25(OH)D concentrations of 10 ng/mL in agreement with earlier studies. BMD of the femoral neck also has been shown to be relatively decreased at serum 25(OH)D levels <30 ng/mL, and secondary hypoparathyroidism with increased bone turnover is associated with serum 25(OH)D concentrations <15 ng/mL. Optimal serum 25(OH)D concentrations appear to fall in the 30 to 80 ng/mL range, particularly in elderly people.
Vitamin D Metabolism in Renal Failure Bone and mineral metabolism is gradually altered as renal failure progresses in chronic kidney disease (CKD). By the time of progression to end stage renal disease (ESRD), all subjects with renal failure are affected by metabolic bone disease and are at increased fracture risk. A host of factors contribute to this 230 Test Application and Interpretation
70
60
50
40
30 Intact PTH (pg/mL)
Intact PTH (ng/mL) 20
10 Test & Application Interpretation 0 10-19 20-29 30-39 40-49 50-59 60-69 70-79
25(OH)D25(OH)D (ng/mL) (ng/mL)
Figure 3. 25-Hydroxyvitamin D vs. PTH levels in 282 apparently healthy individuals with normal serum calcium (8.8-10.1 mg/dL) and serum creatinine (0.5-1.4 mg/dL) levels. disease and are at increased fracture risk. A host of factors contribute to this including 1) the loss of 1α-hydroxylation of 25(OH) vitamin D in the kidneys, 2) the inability to excrete phosphorus, 3) hypocalcemia, and 4) increased resistance to the action of PTH. These factors lead to a gradual increase in PTH levels. The above usually occurs in a population already prone to increased risk for 25- hydroxyvitamin D deficiency. A host of factors in subjects with advanced renal failure or on dialysis contribute to this increased risk such as 1) relatively decreased exposure to sunlight, 2) decreased efficiency in skin synthesis of vitamin D3, 3) diabetes, 4) renal wasting of vitamin D and vitamin D binding protein in subjects with the nephrotic syndrome, and 5) loss of vitamin D in dialysate of subjects on peritoneal dialysis. The net effect is a decreased substrate for 1α-hydroxylation and a further decrease in 1,25(OH)2 vitamin D levels. The initial rise in PTH levels leads to a state of secondary hyperparathyroidism in which a normal calcium level is maintained at the expense of a high serum PTH level (Table 1). This is seen as early as stage 3 CKD (Table 3). If this state of secondary hyperparathyroidism is left untreated, the constant stimuli for increased PTH synthesis leads to parathyroid hyperplasia with lack of sensing of ambient ionized calcium and eventual autonomy of the parathyroid glands. On occasion, neoplasia with adenomatous transformation in one or more of the glands is observed. This leads to a state of tertiary hyperparathyroidism characterized by high PTH levels and hypercalcemia. In both secondary and tertiary hyperparathyroidism, calcium is mobilized from the skeleton leading to renal osteodystrophy.
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Table 3. Frequency of Measurement of PTH and Calcium/Phosphorus by Stage of CKD GFR Range Measurement of CKD Stage Measurement of PTH mL/Min/1.73 m2 Calcium/Phosphorus 3 30-59 Every 12 months Every 12 months 4 15-29 Every 3 months Every 3 months 5 <15 or dialysis Every 3 months Every month
To prevent this sequence of events, it is recommended that all the causes of renal bone disease be treated as early as the disease is identified. The National Kidney Foundation for Bone Metabolism and Disease in Chronic Kidney Disease (K/DOQI Clinical Practice Guidelines) recommends the monitoring of serum calcium, phosphorus, and PTH levels, as indicated in Table 4, and maintaining PTH levels within the parameters provided in Table 3. This can be achieved through a variety of measures including control of phosphorus levels through phosphate binders, correction of 25-hydroxyvitamin D deficiency (<20 ng/dL) and insufficiency (<32 ng/dL) through pharmacologic doses of D2 or D3. After vitamin D sufficiency is achieved, it is appropriate to treat with active vitamin D metabolites such as calcitriol (Table 5).
Metabolic Bone Disease Bone is an unusual tissue because it is largely mineral and undergoes an active process of remodeling. Bone remodeling occurs in 2 stages, resorption followed by replacement or formation. These processes are mediated by different bone cell types, osteoclasts and osteoblasts, respectively; however, the processes are coupled so that, usually, no net bone is lost. During resorption, the matrix or scaffolding of bone, which is composed of mineralized deposits and proteins, is solubilized and digested by acid and enzymes produced by the osteoclasts. Tartrate resistant acid phosphatase (TRAP) is one such enzyme and is a marker of osteoclastic activity. The matrix protein degradation products (cross-linked N- telopeptide [NTx], C-telopeptide (CTx), deoxypyridinoline [DPYD], pyridinoline [PYD], and hydroxyproline from bone collagen) are released into the extracellular environment and are excreted in the urine. Measurement of these breakdown products allows assessment of the rate of bone resorption.
Osteoblasts, unlike osteoclasts, synthesize collagen and other matrix proteins and are responsible for deposition of the matrix, which is then mineralized. Collagen is synthesized as a large precursor procollagen protein, which is processed Table 4. Target Range of Intact Plasma PTH by Stage of CKD
CKD Stage GFR Range Target “intact” PTH mL/min/1.73m2 pg/mL (pmol/L) 3 30-59 35-70 (3.85-7.7)* 4 15-29 70-110 (7.7-12.1)* 5 <15 or dialysis 150-300 (16.5-33.0)† *Opinion-based. †Evidence-based.
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Table 5. Recommended Supplementation for Vitamin D Deficiency/Insufficiency in Patients with CKD Stages 3 and 4 Serum 25(OH)D Ergocalciferol Duration Definition (Vitamin D2) Comment ng/mL (nmol/L) Dose Months <5 (12) Severe vitamin 50,000 IU/wk 6 Measure 25(OH)D D deficiency orally x 12 wks, levels after 6 months then monthly 5-15 (12-37) Mild vitamin D 50,000 IU as 6 Assure patient deficiency single I.M. adherence; dose x 4 weeks, measure 25(OH)D then monthly at 6 months 16-30 (40-75) Vitamin D 50,000 IU/month 6 insufficiency orally processed prior to its incorporation into bone matrix. Osteoblasts also synthesize Test & Application noncollagenous matrix proteins such as osteocalcin (OC) and enzymes such as Interpretation bone specific alkaline phosphatase (BSAP). Circulating levels of OC and BSAP reflect osteoblast activity and bone formation. Table 6 summarizes the biochemical markers of bone turnover. Tables 7 and 8 list the methodologies and normal values for the bone resorption and formation markers. Reference range curves for normal values in children from 2 months to 18 years have been recently published (Ref 16).
Clinical Application of Bone Markers Biochemical markers of bone metabolism are useful diagnostic tools because they reflect acute changes in bone-remodeling activity. They complement measurement of bone density, which measures the cumulative balance of bone resorption/formation over a longer time interval.
Diagnosis of Bone Disorders Table 9 outlines the use of bone metabolism markers in the diagnosis of a number of bone-related disorders. Levels of all markers are clearly elevated in Paget's disease and in primary hyperparathyroidism. In addition to their usefulness in diagnosing these disorders, DPYD and NTx are also useful for monitoring the activity of these disease processes and their response to therapy.
Table 6. Biochemical Markers of Bone Turnover Biochemical Marker Abbreviation Resorption Deoxypyridinoline DPYD Pyridinoline PYD Cross-linked N-telopeptide NTx Collagen type I C-telopeptide CTx Tartrate resistant acid phosphatase TRAP Formation Osteocalcin OC Bone specific alkaline phosphatase BSAP
233 Test Application and Interpretation 3.0-6.0 3.0-6.0 Serum Assess bone turnover y; U, urine; BCE, bone collagen equivalent;bone collagen BCE, mg/24-h U/L Urine (2nd AM Urine void or 24-h) Hydroxyproline TRAP Assess bone turnover quid chromatograph C Colorimetric Enzymatic nmol/mmol creat Urine (2nd AM Urine void, 2-h, 24- h) Assess bone turnover resistant acid phosphatase; HPLC, high pressure li See individual assays in Alphabetical Test Section Seeindividual assays in AlphabeticalTest nmol/mmol nmol/mmol creat Urine (2nd AM void, 2-h, 24-h) PYD Collagen Cross-links PYD Collagen DPYD PYD Assess bone turnover assay; immunoassay; assay; immunoassay; creat AM void) Free DPYD Free Assess bone turnover PYD, pyridinoline; TRAP, tartrate PYD, pyridinoline; TRAP, pg/mL nmol/mmol Serum Urine (2nd CTx y; ECL, electrochemiluminescence ptide; DPYD, deoxypyridinoline; deoxypyridinoline; ptide; DPYD, creat AM void) Paget’s disease) Paget’s NTx 18-29 y30-39 y40-49 y 64-640 60-650 40-465 18-29 y30-39 y40-49 y50-68 y 87-1200 70-780 60-700 87-345 18-29 y30-59 y 12-99 9-60 U, 2nd AM void, 2-h 2nd AM void, U, U, 24-h 4-64S 5-79 3.3-13.5 5.7-23.3 25.4-82.8 4.4-21.1 22.1-88.9 7-49 U, 2nd AM void, 2-hU, 2nd AM void, U, 24-hS 5-87 2.1-8.1 6.2-25.6 22.7-65.2 3.7-18.7 20.0-60.8 9-73 Children Women, premenopausal Women, Men MethodAnalytical sensitivity rangesReference nmol 10BCE/L pg/mL 30 BCE/mmol nmol ICMA 20 nmol/L 13 pmol/mLpmol/mL 20 μg/mL 2 ECL 1.2 U/L ICMA HPLC HPL Sample Type Urine (2nd Clinical Use Monitor therapy (osteoporosis & Table 7. Bone 7. Resorption Markers Table CTx, C-telope NTx, N-telopeptide; ICMA, immunochemiluminometric assa S, serum.
234 Test Application and Interpretation
Table 8. Bone Formation Markers BSAP* Osteocalcin† Sample type Serum Serum Method IRMA IRMA Analytical Sensitivity 2.0 μg/L 2.0 ng/mL Reference ranges μg/L μg/L ng/mL Adults Men Women Men 11.3-35.4 18-29 y 8.4-29.3 4.7-17.8 Women 7.2-27.9 30-39 y 7.7-21.3 5.3-19.5 40-49 y 7.0-18.3 5.0-18.8 50-68 y 7.6-14.9
50-76 y 5.6-29.0 Test & Application Interpretation Children* Male Female Children† 2-24 mo 25.4-124.0 25.4-124.0 6-9 y 40-2-108.0 6-9 y 41.0-134.6 41.0-134.6 10-13.9 y 35.8-165.5 10-13 y 43.8-177.4 24.2-154.2 14-17.9 y 14-17 y 13.7-128.0 10.5-75.2 Males 27.8-194.1 Females 16.3-68.7 Clinical use Monitor therapy in osteo- Assess bone turnover porosis and Paget’s disease BSAP, bone specific alkaline phosphatase, BAP, skeletal alkaline phosphatase; osteocalcin, bone gla protein, BGP. *Pediatric data from Int J Biol Markers. 1996;11:159-164. †Pediatric data from Quest Diagnostics Nichols Institute Clinical Correlations Department.
Because of the coupling of bone resorption and formation, utilizing results from a single biochemical marker of bone turnover, either formation or resorption, is not as informative as the use of markers for both processes. It is likely that the relative values or ratios of formation and resorption markers will prove to be more informative in evaluating bone disorders and their treatment than the use of single markers or absolute levels.
Table 9. Effect of Disease State on Biochemical Markers of Bone Turnover Resorption Markers Formation Markers Disease DPYD PYD NTx OC BSAP Osteoporosis ↑↑ ↑ ↑ Normal Normal Paget’s Disease ↑↑↑ ↑↑↑ ↑↑↑ ↑↑ ↑↑↑ Hyperparathyroidism ↑↑ ↑↑ ↑↑ ↑↑ ↑↑ ↑, marginally elevated relative to normal; ↑↑, elevated relative to normal; ↑↑↑, highly elevated relative to normal.
235 Test Application and Interpretation
Diagnosis of Osteoporosis The current practice for diagnosis of osteoporosis is bone densitometry. Since bone density can decline at a rate of 1% to 3% per year, bone densitometry is a good method of detecting established osteoporosis. It has limited usefulness, however, for immediate follow-up of treatment of osteoporotic patients because subtle changes in bone density cannot be detected. Measurements of biochemical markers of bone formation and resorption (Table 6), unlike bone densitometry, are particularly well suited for monitoring the efficacy of treatment. These markers will change rapidly if treatment, such as estrogen therapy, is effective.
In osteoporosis, the rate of bone resorption may be only modestly increased after menopause, as indicated by the overlap in the reference ranges for these markers in premenopausal and postmenopausal women. Thus, the usefulness of these markers in diagnosis of primary osteoporosis is limited. The markers are most useful in cases of high bone turnover. Additionally, in the majority of cases, the biochemical markers will be most useful in evaluation of the efficacy of treatment. Patients with high levels of bone turnover markers may be the most suitable for treatment with antiresorptive agents. In this case, following the bone turnover markers at a later time will be especially useful in determining efficacy of treatment.
Efficacy of Therapy Measurement of the levels of biochemical markers can be used to determine whether a drug therapy is effective within 2 to 6 months. A decrease in the levels of the markers of >25% indicates effective intervention.
Follow-up to Therapy A similar approach can be used to determine the appropriate dose of drug. By following the changes in biochemical bone markers, the level of drug administered can be fine-tuned to achieve the desired level of inhibition of bone resorption to maintain an acceptable rate of bone remodeling.
References 1. Bringhurst FR, Demay MB, Kronenberg HM. Hormones and disorders of mineral metabolism. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Philadelphia, PA: WB Saunders; 2003:1303-1371. 2. Bouillon R. Vitamin D: from photosynthesis, metabolism, and action to clinical applications. In: DeGroot LJ, Jameson JL, et al, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:1377-1418. 3. Levine MA. Hypoparathyroidism and pseudohypoparathyroidism. In: DeGroot LJ, Jameson JL, et al, eds. Endocrinology. 5th ed. Philadelphia, PA: WB Saunders; 2006:1611-1636.
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4. Root AW, Diamond FJ Jr. Calcium metabolism, normal homeostasis. In: Sperling MA, ed. Pediatric Endocrinology. 2nd ed. Philadelphia, PA: WB Saunders; 2002:65-95. 5. Diamond FB Jr., Root AW. Disorders of calcium metabolism in the newborn and infant. In: Sperling MA, ed. Pediatric Endocrinology. 2nd ed. Philadelphia, PA: WB Saunders; 2002:97-110. 6. Diaz R, Brown EM. Familial hypocalciuric hypercalcemia and other disorders due to calcium-sensing receptor mutations. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:1595-1609. 7. St. Arnaud R, Glorieux FH. Genetic defects in vitamin D metabolism and action. In: DeGroot LJ, Jameson JL, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:1637-1652. 8. Delmas PD, Chapurlat RD. Osteoporosis. In: DeGroot LJ, Jameson JL, et al, eds. Endocrinology. 5th ed. Philadelphia, PA: Elsevier-Saunders; 2006:1751- Test & Application 1769. Interpretation 9. Goodman WG, Juppner H, Salusky B, Sherrard DJ. Parathyroid hormone (PTH), PTH-derived peptides, and new PTH assays in renal osteodystrophy. Kidney Internat. 2003;63:1-11. 10. Strewler GJ. The physiology of parathyroid hormone related protein. N Engl J Med. 2000;342:177-185. 11. Raisz L, Kream BE, Lorenzo JA. Metabolic bone disease. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Philadelphia, PA: WB Saunders; 2003:1373-1410. 12. Dulipsingh L, DeSouza MJ, Ikram Z, Fall P, Willard A, Fang Z, Prestwood K, Raisz L. Clinical utility of urinary markers of bone turnover in response to antiresorptive therapies. The Endocrinologist. 2003;13:293-299. 13. Steelman J, Zeitler P. Treatment of symptomatic pediatric osteoporosis with cyclic, single day intravenous pamidronate infusions. J Pediatr. 2003;142:417- 423. 14. National Kidney Foundation. K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease. Am J Kidney Dis. 2003;42(Suppl 3):S1-S201. 15. Tomita T, Millard DM. C-cell hyperplasia in secondary hyperparathyroidism. Histopathol. 1992;21:469-474. 16. Rauchenzauner M, Schmid A, Heinz-Erian P, et al. Sex- and age-specific reference curves for serum markers of bone turnover in healthy children from 2 months to 18 years. J Clin Endocrinol Metab. 2007;92:443-449.
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238 Test Application and Interpretation
DISORDERS OF CARBOHYDRATE METABOLISM
Background Physiology In the fasting or post absorptive state, blood glucose concentration is maintained in a narrow range (about 70 to 100 mg/dL) by a balance of hormonal actions (Figure 1). Principal among these is the level of circulating bioactive insulin. In response to rising glucose levels, pancreatic insulin secretion is stimulated, glucose uptake by liver and peripheral tissues is increased, glycogen synthesis is augmented, and hepatic glucose output is suppressed. The net result is a reduction in blood glucose concentration with an increase in carbohydrate stores (Figure 1). Hypoglycemia results in reduced insulin secretion and stimulation of glucagon, growth hormone, cortisol, and, when severe, catecholamine secretion.
These counter-regulatory hormones initiate glycogenolysis, gluconeogenesis, and Test & Application
increased hepatic glucose output to raise blood glucose at the expense of Interpretation carbohydrate and substrate stores.
Hyperglycemic Disorders The commonest disorders of carbohydrate metabolism are the several types of diabetes mellitus involving decreased insulin secretion or action.Type 1 diabetes mellitus, also referred to as insulin dependent diabetes mellitus (IDDM), is characterized by persistent hyperglycemia, usually with ketosis and low levels of
Figure 1. Features of carbohydrate metabolism. See text for details.
239 Test Application and Interpretation circulating insulin, C-peptide, and proinsulin. Type 1 diabetes is due to chronic autoimmune pancreatic islitis with progressive destruction of the insulin- secreting beta cells of the pancreatic islets. Insulin and islet cell autoantibodies are present in most patients with type I diabetes at diagnosis and the disease usually is manifest during the first 2 decades of life. Other autoantibodies and autoimmune diseases, especially autoimmune thyroid disease, are present in 10% to 30% of the patients with type I diabetes. Type 2 diabetes mellitus, sometimes referred to as adult onset disease or non- insulin dependent diabetes mellitus (NIDDM), represents a more heterogeneous group of disorders associated with insulin resistance and variable degrees of hyperinsulinemia and hyperglycemia without ketosis. It is the predominant form of the disease worldwide. Autoimmunity may contribute to the pathogenesis in type 2 disease since 7% to 10% of adult diabetes mellitus is associated with positive autoimmune markers.
Type 1 Diabetes Research during the past two decades has identified circulating autoantibodies directed against a variety of pancreatic islet cell antigens (ICA) in patients with IDDM and their first-degree relatives. These antigens are summarized in Table 1. The pancreatic beta cell autoimmune destructive process occurs over an extended period of time during which the patient is asymptomatic and euglycemic. Clinical glucose intolerance develops as the residual islet beta cell mass is progressively reduced to 10% or less. Continuing destruction leads to overt diabetes mellitus. The prolonged prediabetic stage of the disease makes it possible to utilize immunologic markers to identify individuals at risk and estimate the severity of the autoimmune process. The presence of autoantibodies directed to 2 or more characteristic autoantigens indicates a more severe autoimmune process and is associated with a higher risk for progression to overt disease.
Table 1. Antigens for Which Autoantibodies Have Been Detected in Patients with IDDM Insulin Glutamic acid decarboxylase (GAD) IA-2 (ICA 512, 40 kd)* Phogrin (IA-2b, 37 kd)* GM2-1 ganglioside 52 kd ICA α† 69 kd ICA α† Carboxypeptidase H Pancreative sulfatide Glucose transporter *A member of the protein tyrosine phosphatase (PTP) family. †Protein with unknown function.
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Cytoplasmic islet cell autoantibody measured by immunofluorescence using histologic sections of human or monkey pancreas is semi-quantitative and difficult to standardize. Direct methods using radioassay or ELISA formats have been developed utilizing purified or recombinant protein antigens. These include insulin, GAD, and ICA-512 (IA-2) antigens. These assays, used in combination, provide high sensitivity and reliability for the diagnosis of pancreatic islet beta cell autoimmune disease. The assays allow identification of at-risk individuals in families with type 1 diabetes, the identification of late onset type 1 disease in adult patients, and, with studies of first phase insulin release in prediabetic individuals, allow monitoring of progress of the autoimmune state. Diagnosis of the prediabetic phase allows patient monitoring and early institution of insulin therapy after documentation of hyperglycemia prior to metabolic decompensation (eg, ketoacidosis). Diagnosis of the prediabetic state will be essential to early institution of immunomodulatory therapies as these become
available. Test & Application Interpretation Verge et al (Diabetes. 1996;45:926, Ref. 2) studied 882 first degree relatives (median age 13 years) of patients with type 1 diabetes. Fifty of the relatives developed diabetes during a median follow-up of 2 years (0.1-11.3 years). Among the 50, 98% had autoantibodies to 1 or more antigens (GAD, ICA 512, insulin), and 80% had 2 or more autoantibodies. None of 200 control subjects expressed more than a single autoantibody. Table 2 summarizes the risk of IDDM in the 882 relatives according to the number of antibodies detected.
Kulmala et al (J Clin Invest. 1998;101:327, Ref. 3) assessed autoantibody prevalence (ICAA, GAD, IA-2, insulin) in an unselected population of 755 siblings of children with type 1 disease. The risks for disease in siblings with 4, 3, 2, 1, or no antibodies were 40%, 70%, 25%, 2%, and 0.8%, respectively. Sensitivity of combined GAD and IA-2 was 81% and the positive predictive value was 41%. Bilbao et al (Hormone Res. 2000;54:181, Ref. 5) reported that of 448 recent onset type 1 patients, 90% were positive for at least 1 antibody and 68% presented with 2 or more autoantibodies. Caveats of autoantibody testing include lack of high risk of diabetes for most individuals expressing only a single autoantibody unless there is loss of first phase insulin secretion (<1st percentile of normals) on intravenous glucose tolerance testing Table 2. Risk of Development of Overt IDDM in First Degree Relatives of IDDM Patients Relative to the Presence of Insulin, GAD and/or ICA512 Autoantibodies* Number of Risk (%) of IDDM within Autoantibodies 3 Years 5 Years 10 Years 0<1<1<1 1 8 15 23 2304372 3 49 >95 *Verge et al. Diabetes. 1996;45:926. (Ref. 2)
241 Test Application and Interpretation testing. This is particularly true for individuals with the protective HLA alleles DQA1*0102, DQB1*0602, relatives, and autoimmune polyglandular syndrome II (APS-II) patients with high titer islet cell immunofluorescence but only GAD autoantibodies. Insulin autoantibodies are predominantly, but not exclusively, found in children developing type 1 diabetes. Subcutaneous insulin therapy, even with human insulin, induces insulin antibodies and thus positive results are not reliable if an individual has had more than approximately 1 week of insulin therapy. Of note, GAD and ICA 512 often remain positive for up to 2 years after the development of symptomatic disease. Finally, a small subset of individuals developing type 1 diabetes lack GAD, insulin, and ICA 512 autoantibodies. Risk factors for autoimmune type 1 disease are summarized in Table 3.
Table 3. Diagnosis of Increased Diabetes Risk Transient hyperglycemia ≥1 Ab: high risk insulin dependence (<10% with ≥1 Ab) Gestational diabetes (<10% with ≥1 Ab) ≥1 Ab: high risk insulin dependence First degree relatives of patient with type 1 diabetes: Offspring of father with type 1 ≥2 Ab: high risk; 0 Ab: risk <1/300 Offspring of mother with type 1 ≥2 Ab: high risk; 0 Ab: risk <1/300 Sibling ≥2 Ab: high risk; 0 Ab: risk <1/300 Parent ≥2 Ab: high risk; 0 Ab: risk <1/300 Monozygotic twin ≥1 Ab: high risk; 0 Ab: risk > general population Potential renal transplant donor Increased risk with antibody as indicated for relatives (see above); intravenous GTT and OGTT useful DQA1*0102/DQB1*0602 “protected” 1 Ab: low risk; ≥2 Ab: unknown risk; relative may be low APS II patient High titer single antibody: only Graves disease modestly increased risk Addison’s disease Myasthenia gravis Celiac disease APS I patient High titer single antibody: only modestly increased risk General population (<1/300 express >2 Ab) High/moderate risk HLA alleles ≥2 Ab: similar to sibling risk DQA1*0102/DQB1*0602 0 Ab: risk <1/300; ≥2 Ab: risk currently unknown Ab, antibody; APS, autoimmune polyglandular syndrome.
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Maturity Onset Diabetes of the Young (MODY) Maturity onset diabetes of the young (MODY) is a subclass of type 2 disease recognized in children, adolescents, and young adults and usually transmitted as an autosomal dominant trait. The patients may or may not be obese. Six mutated genes have been described in MODY patients (Table 4). All of the nuclear factors and IPF-1 function as transcription factors regulating insulin biosynthesis. Heterozygous mutations cause diabetes related to beta cell dysfunction. Homozygous mutation of IPF-1 has been shown to produce islet cell aplasia. Other defects limiting insulin secretion may be involved in MODY presenting with obesity and with or without ketoacidosis.
Type 2 Diabetes
The pathogenesis of type 2 diabetes involves interaction of genetic and Test & Application
environmental factors. Cardinal features include insulin resistance, defective Interpretation insulin secretion in response to glucose stimulation, and increased hepatic glucose production. Factors predisposing to insulin resistance include a familial history of diabetes, increased body fat content (body mass index, BMI), particularly central (intra-abdominal) adiposity, and relative inactivity. Insulin resistance leads progressively to relative insulin deficiency and increasing fasting plasma glucose. Other associated features have been described as the metabolic syndrome, which the WHO has defined as a waist circumference/ hip circumference ratio >0.90, BMI >30, increased fasting plasma glucose (>110 mg/dL), dyslipidemia (serum triglycerides >150 mg/dL and HDL cholesterol <35 mg/dL), and blood pressure >140/90 mm Hg. The syndrome in middle-aged men is associated with a 2- to 3-fold increase in cardiovascular disease mortality and a 2-fold increase in overall mortality (Ref 8).
Type 2 diabetes has become a significant health problem in childhood and adolescence now accounting for 8% to 45% of new pediatric diabetic patients. The increasing prevalence has paralleled the increase in BMI in children during the past 20-30 years, and the pathogenesis appears to mirror that in adult patients.
Table 4. Mutated Genes in MODY Patients MODY Type Mutated Gene Prevalence MODY 1 HNF 4α Rare MODY 2 Glucokinase Common MODY 3 HNF 1α Common MODY 4 IPF-1 Rare MODY 5 HNF 1β Rare MODY 6 Neuro D1/BETA 2 Rare HNF, hepatocyte nuclear factor; IPF, islet-promoting factor also known as IDX-1, a homeobox gene; Neuro D1/BETA 2, neurogenic differentiation 1/beta cell E-box transactivator.
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Risk factors include a positive family history, obesity, polycystic ovarian syndrome (PCOS), acanthosis nigricans, ethnicity (higher prevalence in minority populations), and intrauterine programming with increasing insulin resistance leading to relative insulin deficiency and overt diabetes. Although the absence of diabetes autoimmune markers is presently a prerequisite for the diagnosis of type 2 diabetes in pediatric patients, the prevalence of such markers appears to be similar to the 7% to 10% prevalence in adult type 2 patients. Female patients with variable degrees of insulin resistance associated with insulin autoantibody, variable carbohydrate intolerance, acanthosis nigricans, and PCOS have been referred to as type B insulin resistant. Males also manifest the disease but without gonadal dysfunction. Diabetes also may be due to a variety of insulin receptor mutations. Females with insulin resistance type A associated with inactivating mutations in the insulin receptor gene also display acanthosis nigricans and PCOD; these features are believed to be secondary to the hyperinsulinemia, but the mechanism is not yet understood. The Rabson- Mendenhall syndrome is manifest by leprechaun-like features, acanthosis nigricans, and severe insulin resistance due to insulin receptor mutation. Heterozygous mutations in the transcription factor peroxisome proliferator- activated receptor γ (PPARγ) are associated with severe insulin resistance and early onset type 2 diabetes. Additionally, a common polymorphism in PPARγ (Pro12Ala) predisposes to insulin resistance with an increased risk of type 2 diabetes. Impaired islet beta cell function also has been associated with a maternally transmitted mutation in mitochondrial transfer RNA with variable phenotypic expression resembling type 1 or type 2 diabetes. These disorders are summarized in Table 5, which details the blood tests available for assessing patients with hyperglycemia.
Diagnosis of Diabetes Mellitus The diagnostic criteria for impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and diabetes mellitus are shown in Table 6. Venous blood from an antecubital vein is preferable to capillary blood samples for glucose tolerance testing. Plasma or serum can be used for glucose measurements. Standard tests are done in the morning after 12 to 14 hours of fasting. No formal dietary preparation is necessary unless the patient has been on a restricted diet. Criteria for testing for diabetes mellitus in pediatric and asymptomatic adult subjects are summarized in Tables 7 and 8.
Preventing and Managing Complications The major complications of diabetes include dyslipidemia, cardiovascular disease, ophthalmopathy, nephropathy, and neuropathy. Laboratory tests are useful tools for prevention and management of these complications. The American Diabetes Association recommends the following tests be considered for initial laboratory evaluation of a patient diagnosed with diabetes (Ref. 15):
• Hemoglobin A1c
244 Test Application and Interpretation ation, early death subunit Kir6.2 phenotype, severe phenotype, severe Other tissue autoantibodies present; present; Other tissue autoantibodies usually childhood onset Insulin receptor autoantibodies; acanthosis nigricans; other autoimmune disease Often obese diabetic state; risk of Transient disease permanent mutation of ATP-sensitive Activating potassium-channel inheritance Usual dominant in females; PCOD nigricans; Acanthosis insulin receptor mutation Insulin receptor mutation, leprechaun- precocious like features, puberty Insulin receptor mutation, abnormal facies, growth retard trait; insulin dominant Autosomal responsive Several genetic forms transmission Maternal 2 Early onset type insulin resistance Other Test & Application Interpretation Ketosis No No No No No Yes Insulin Receptor Receptor Antibody †
↑ ± Insulin N or Antibody* 2 2 ovarian syndrome; V, variable: type 1 or type 2 phenotype. 2 type 1 or type variable: V, ovarian syndrome; No No No No No No Yes No NoNo No NoNo Yes No NoNo No No No No No No No No No No Yes No NoNo No No No No No 7% 7% Islet Cell Islet Antibodies ↑ ↑
↑ ↑ Blood Measurements N or N or N or ↑ ↓↓ NN or or NN N or N or ↑ ↑↑ ↑↑ ↑ ↑↑ ↓ ↓ ↑↑ ↑ ↑↑ ↑ ↑↑ ↑ ↑↑ ↑ ↑↑ ↑ ↓↓ ↓ ↑ ↑ VV VNoNoNoNo N or Insulin Proinsulin C-Peptide of Hyperglycemia mutation γ Increased in 7% of late onset type 1 patients.1 type onset late Increased in 7% of Prior to any insulin therapy. to any insulin Prior Insulin resistance, Insulin resistance, B type Type 2 diabetesGestational or N Nor MODY Insulin resistance, A type Rabson-Mendenhall syndrome Leprechaunism N or Mutant insulin Lipoatrophic diabetes N or gene Mitochondrial mutations PPAR polycystic young; PCOD, the of diabetes onset maturity MODY, * † Permanent neonatal Permanent neonatal diabetes Type 1 Monogenic forms of forms Monogenic diabetes Diabetes mellitus Table 5. Assessment Table Disorder
245 Test Application and Interpretation
Table 6. Diagnostic Thresholds for Diabetes and Lesser Degrees of Impaired Glucose Regulation* Fasting Plasma Glucose 2-h Plasma Glucose mg/dL mg/dL Normal <100 <140 IFG† 100-125 Not applicable IGT† Not applicable 140-199 Diabetes‡ ≥126 ≥200 *Diabetes Care. 2006;29(Suppl 1):S4-S48. †When both tests are performed, IFG or IGT should be diagnosed only if diabetes is not diagnosed by the other test. ‡A diagnosis of diabetes needs to be confirmed on a separate day.
Table 7. Pediatric Testing Criteria for Type 2 Diabetes Mellitus* Overweight… • BMI >85th percentile for age and sex • Weight >85th percentile for height • Weight >120% of ideal for height …plus any 2 of the following: • Family history of type 2 diabetes in first or second degree relative • Race/ethnicity (Native American, African American, Latino, Asian American, Pacific Islander) • Signs of insulin resistance or associated conditions (acanthosis nigricans, hyper- tension, dyslipidemia, PCOS) • Maternal history of diabetes or GDM BMI, body mass index; PCOS, polycystic ovary syndrome; GDM, gestation diabetes mellitus. *Consider testing at 10 years of age or at onset of puberty if puberty occurs <10 years; frequency: every 2 years; FPG test preferred. From Diabetes Care. 2006;29(Suppl 1):S4-S48.
Table 8. Criteria for Diabetes Testing in Asymptomatic Adults • Age >45 years, at 3-year testing interval • Any age if BMI >25 kg/m2 plus one or more of the following: Inactivity First degree relative with diabetes Race/ethnicity (Native American, African American, Latino, Asian American, Pacific Islander) Delivered baby >9 lb or diagnosis of GDM Hypertensive (>140/90 mm Hg) HDL cholesterol <35 mg/dL and/or triglyceride >250 mg/dL PCOS diagnosis IGT or IFG on previous test Features of insulin resistance (eg, acanthosis nigricans) History of vascular disease Diabetes Care. 2006;29(Suppl 1):S4-S48. BMI, body mass index; GDM, gestational diabetes mellitus; PCOS, polycystic ovary syndrome; IGT, impaired glucose tolerance; IFG, impaired fasting glucose.
246 Test Application and Interpretation
• Total cholesterol, HDL cholesterol, triglycerides, LDL cholesterol • Liver function tests; if abnormal, add tests for fatty liver or hepatitis • Microalbuminuria