PATOLOGI KIMIA Head of Unit : Dr Nur Shafini Binti Che Rahim
Phone : +60326155555 ext 5284 1. INTRODUCTION
Chemical Pathology unit provides diagnostic and consultative services to Hospital Kuala Lumpur and also serves as referral center for hospitals and clinics in Malaysia. Our services cover analysis and interpretation of results for screening, diagnostics and monitoring of diseases.
2. SERVICES
Chemical Pathology Unit offers specialized biochemical testing. The list of services include:
i. Endocrine Thyroid Function Test, Fertility Test, Serum and Urinary Cortisol, Urine Catecholamine, Extended Hormone (Dehydroepiandrosterone sulphate (DHEAS), Insulin, C-peptide, Growth Hormone, Adrenocorticotrophic Hormone (ACTH)) and Parathyroid Hormone (PTH), Thyroglobulin, Anti-thyroglobulin, Anti-thyroid specific peroxidase and Anti-thyroid stimulating hormone receptor.
ii. Metabolic HbA1c, Gamma Glutamyl transferase (GGT) and Cholinesterase (CHE)
iii. Protein and Proteomic Serum and Urine Protein Electrophoresis, Specific Protein (Ceruloplasmin, Immunoglobulin IgG, Immunoglobulin IgA, Immunoglobulin IgM, Transferrin, Haptoglobin, Alpha -1-antitrypsin)
iv. Hematological Biochemistry Iron,UIBC, Ferritin,Folate, Vitamin B12
3. SERVICE HOURS
Operating hours: 7.30 am – 5.30 pm (Monday to Friday)
URGENT request: Arrangement should be made by contacting Pathologist, Medical Officer or Scientific Officer in-charge.
4. REQUEST FORMS
All specimens must be accompany with PER.PAT 301 form. Forms must be filled LEGIBLY and COMPLETELY with the following information:
Patient`s details: Name, IC number, sex, age and ward/hospital name
Patient`s clinical and test details: relevant clinical history, diagnosis, test required, type of sample, time and date of sample collection.
Requesting doctors details: name, stamp and signature.
5. SAMPLE COLLECTION
5.1 BLOOD
Most of tests in Chemical Pathology require serum sample that need to be collected in plain tube. Special requirements are require for certain tests: HbA1c: require whole blood sample that need to be collected in EDTA tube. Request less than 3 months from previous result will be rejected. Morning serum cortisol: between 8 to 10 am; midnight serum cortisol: between 10 to 12 pm. Fertility tests: Sample for progesterone should be collected at day 21 of menstrual cycle, while sample for estradiol, FSH and LH should be collected at day 2 to 5 of menstrual cycle. Certain tests require to be sending in ice such as ACTH. Serum and Urine Protein Electrophoresis should be send as paired sample for better interpretation of test results
5.2 URINE
24 hours Urine Collection 24 hours urine collection require due to certain test which effected by circadian rhythmic changes. Procedure of collection: On the day of collection, the first urine voided must be thrown away. Time of first urine voided is the start of the timing for the 24 hours collection. Collect the second and subsequent voided urine for 24 hours into the 24 hours urine container until completed. For male patient, it is advisable NOT to void the urine directly into the 24 hours urine container. This is to avoid possible chemical burns. Refrigerate urine sample if possible. Label the bottle as directed and send immediately to the laboratory. eg. Tests include 24-hours urine cortisol and 24-hours urine catecholamines.
24-hours Urine Catecholamine
Please refer to procedure 24 hours urine collection to collect urine for 24 hours urine catecholamines. For adult minimum 750ml of urine should be collected. For paediatric samples urine creatinine are perform for every request. Please note that, 10ml of 6M HCl (preservative) is added into the bottle to preserve the analytes. It is important for the requesting physician to advise the patient NOT to discard the preservative. Instruction on patient preparation and specimen collection
o Certain drugs or their metabolites are a source of possible interference with catecholamines quantification. List of drugs that may interference with catecholamines quantification: a) Acetaminophen (paracetamol) b) Cimetidine (Tagamet®) c) Alpha-methyldopa (Aldomet®) d) Isoproterenol e) Labetalol f) Mandelamine g) Metoclopramide o Please advise patient to avoid stress, exercise, smoking and pain prior to and during urine collection.
24-hours Urine Cortisol Please refer to procedure 24 hours urine collection to collect urine for 24 hours urine cortisol. Minimum of 500 ml of urine should be collected.
6. RECEIPT OF SPECIMEN
Specimens are receive at the main counter (Kaunter Penerimaan Utama Unit Makmal Teras).
7. REPORTING OF RESULTS
Results are validated by Chemical Pathologist/Medical Officer/Scientific Officer according to the test following laboratory turnaround time.
Reference ranges are provided for all results. These may be subject to variation differentiated by age and sex where important / available.
Reports are dispatched to the respective pigeon hole or posted via mail for external samples. 8. ENQUIRY OF RESULTS
Enquiry of test results can be made using: 1) Form for tracing laboratory results i.e Borang Mendapatkan Keputusan Ujian Patologi, HKL/JP/PA/AK-05-01 at Kaunter Penerimaan Utama Unit Makmal Teras in Pathology Department. 2) Labviewer in respective ward or clinic 3) Phone extension 5284,
For external clients tracing can be made via official letters to:
Head of Department, Pathology Department, Jalan Pahang, 50586 Hospital Kuala Lumpur.
9. SERVICES AFTER OFFICE HOURS
If test needed after working hours, consultation and agreement from Chemical Pathologist on-call are required.
10. PROTOCOLS FOR INVESTIGATION OF ENDOCRINE DISORDERS
The protocols listed below are only as guide and are subjected to changes according to clinician requirement. These protocols are mainly for adult.
10.1 PITUITARY DISORDERS
Assessment of Anterior pituitary Hormone
1. Pituitary Hormone Insufficiency
Anterior pituitary hormones include Growth Hormone (GH), Prolactin, Thyroid Stimulating Hormone (TSH), Follicle Stimulating Hormone (FSH), Luteinizing Hormone
(LH) and Adrenocorticotrophic Hormone (ACTH). Main abnormalities to look for are Corticotroph deficiency, Thyrotroph deficiency, Gonadotroph deficiency or Somatotroph deficiency.
Assessment of Anterior Pituitary Reserve a) Initial assessment
• Morning serum Cortisol and ACTH or Short Synachten Test • Thyroid Function Test (TSH, FT4) • Prolactin, LH, FSH • GH • Testosterone for man Estradiol for woman
b) Combine Anterior Pituitary Stimulation Test (Insulin Stress Test + Gonadotrophin Stimulation Test)
Procedures:
• Fast the patient overnight. • Insert intravenous catheter or intravenous line. • Rest patient for 30 minutes. Take samples for glucose, growth hormone, cortisol, LH, FSH and TSH (as baseline investigation). • Give insulin 0.1-0.15 unit/kg body weight, 200µg TRH and GnRH 100ug intravenously. • Collect samples into plain tubes and Glucose tubes and label as follows:
Time Tests Tube 0 min (basal) Glucose, Glucose tube Cortisol,FSH,LH 1 plain tube TSH,GH
15 min Glucose Glucose tube 20 min FSH, LH, TSH 1 plain tube
30 min Glucose Glucose tube Cortisol, GH, 1 plain tube 45 min Glucose Glucose tube
60 min Glucose Glucose tube Cortisol, FSH, LH, 1 plain tube TSH,GH 90 min Glucose, Glucose tube Cortisol, GH 1 plain tube 120 min Glucose, Glucose tube Cortisol, GH 1 plain tube
* GH-Growth hormone, LH-Luteinizing hormone, FSH- Follicular stimulating hormone, TSH – Thyroid Stimulating Hormone Adapted from Clinical Chemistry Sixth Edition, William J Marshall & Stephen K Bangert, Mosby, 2008, pg 140. • Label specimens according to sampling time. • Send all samples after test is completed to main counter, Pathology Department.
Notes: • Plasma glucose level must fall below 2.2 mmol/L and/or clinical signs and symptoms of hypoglycaemia (sweating, tachycardia etc) must be observed. • Additional intravenous insulin may be given if this does not occur by 30 min and sampling should be prolonged by another 30 min. • Physician should be in attendance throughout the tests and 50% i.v. dextrose should be kept by bed side if severe hypoglycemia is documented. • Giving glucose for severe hypoglycemia does not invalidate the test results. • Test is contraindicated for patient with seizure, ischeamic heart disease or cardiovascular insufficiency and in young children. • Normal ECG is mandatory.
c) Insulin Hypoglycaemic Test:
• Similar as Combine Anterior Pituitary Stimulation Test but without GnRH injection.
Blood samples are taken at 0 minute (basal), 30 minutes and 60 minutes after insulin injection for glucose, cortisol and growth hormone (GH) as follows:
Time Tests Tube 0 min (basal) Glucose Glucose tube 1 Cortisol, GH plain tube 30 min Glucose Glucose tube 1 Cortisol, GH plain tube 60 min Glucose Glucose tube 1 Cortisol, GH plain tube • Label specimens according to sampling time. • Send all samples after test is completed to main counter, Pathology Department. d) Gonadotrophin- Releasing Hormones Stimulation Test:
• Collect samples into plain tubes for LH and FSH (basal sample). • Give 100 ug GnRH. • Collect samples into plain tubes at 15 minutes, 30 minutes, 60 minutes and 90 minutes after GnRH injection for Luteinizing Hormone (LH) and Follicular Stimulating Hormone (FSH).
Time Tests Tube
0 min (basal) FSH, LH 1 plain tube
15 min FSH, LH 1 plain tube
30 min FSH, LH 1 plain tube 60 min FSH, LH 1 plain tube
90 min FSH, LH 1 plain tube • Label specimens according to sampling time. • Send all samples to main counter Pathology Department
2. Pituitary surgery assessment a) Pre-operative assessment
• Morning serum cortisol • Thyroid Function Test (TSH, FT4) • Prolactin, LH, FSH • GH • Testosterone for man • Estradiol for woman b) Post-operative assessment (2-4 days after surgery)
• Steroid coverage with hydrocortisone is administered immediately before, during and after surgery. • If adrenal function was normal before surgery, hydrocortisone is stopped on second or third post-operative day. • 24 hours after stopping – take morning blood for cortisol
c) Follow up assessment (one month after surgery)
• FT4 • Testosterone for man • Estradiol for woman • Cortisol and ACTH at 9.00 am and Short Synacthen test, even if function is subnormal after surgery. ACTH deficiency after surgery is often transient. After pituitary irradiation, patient should be evaluated at least once per year with measurement of FT4, estradiol (if female), testosterone (if male), FSH, LH, prolactin, cortisol, ACTH and Short Synacthen Test.
3. Acromegaly a) Screening and biochemical diagnosis • 2 tests must be done to attain biochemical diagnosis of active acromegaly. • Measure IGF 1 level according to age-adjusted reference. • Perform oral glucose tolerance test with 75g oral glucose after at least 8 hours of overnight fasting. • Active acromegaly is indicated by elevated IGF 1 and failure of GH to be suppressed below 1 ng/ml (3 mIu/L) • GH may not be suppressed in poorly controlled diabetes mellitus, severe illness, chronic liver disease and chronic kidney disease.
b) Other biochemical tests (Anterior Pituitary hormones and metabolic Screening) • Serum prolactin • ACTH and cortisol (morning sample) • TFT • LH, FSH, testosterone (male), estradiol (female) – morning sample • Fasting serum lipids • RP, uric acid • LFT, calcium, phosphate • Urine FEME
Assessment of Posterior Pituitary Hormone
Posterior pituitary secretes vasopressin (ADH) and oxytocin. These hormones are synthesized in hypothalamus and pass down nerve axons into the posterior pituitary and released into the circulation.
1. Diabetes Insipidus (DI)
• Lack of ADH caused by pituitary/hypothalamic disease (cranial DI) or failure of kidney to respond to ADH (nephrogenic DI)
• Presented with polyuria –urine volume >3 L/day • common causes of polyuria such as diabetes mellitus , hypokalemia , hypercalcemia and diuretic therapy have been excluded • Measure serum and urine osmolality and sodium If serum osmolality > 295 mOsm/kg, urine osmolality is < 300 mOsm/kg and sodium >145 mmol/l - Diagnosis of Diabetes Insipidus is likely and not to do Fluid Deprivation test
• If diagnosis is in doubt; perform Fluid Deprivation test
a) Protocol for Fluid Deprivation Test
Procedure
(**Ensure adrenal and thyroid function normal before contemplating the test)
• Allow fluids overnight before test and give light breakfast with no fluid; no smoking permitted • Weigh patient • Allow no fluid for 8hours; patient must be under constant supervision • Every 2 hours - Weigh patient (stop test is weight falls by > 5% initial body weight) - Measure urine volume and osmolality - Measure serum osmolality ( stop test if osmolality >300 )
After 8 hours - Allow patient to drink ( no more than twice urine volume of period of fluid
deprivation, to avoid acute hyponatraemia) and give 2 µg desmopressin i.m Measure urine osmolality every 4 hours for further 16 hours
Interpretation:
Algorithm for the investigation of polyuria.
polyuria measure: abnormal diagnosis blood glucose plasma creatinine potassium
calcium
normal
fluid deprivati on test
Urine osmolality (mmol/kg) after: Cranial diabetes 8 h fluid desmopressin insipidus deprivation Nephrogenic <300 >750 diabetes insipidus
<300 <300 Primary polydipsia
>750 >750 Non-diagnostic
300-750 <750
Adapted from Clinical Chemistry Sixth Edition, William J Marshall & Stephen K Bangert, Mosby, 2012, pg 135 ADRENAL DISORDERS
Disorders of Adrenal Cortex
1. Adrenal hypofunction (Addison’s Disease)
measure AM Cortisol
<50 nmol/L – diagnostic of adrenal failure
>550 nmol/L – excludes adrenal failure
50-550 nmol/L – ACTH stimulation test (Short synacten test) a. Short Synacthen Test
• High index of suspicion is required to diagnose adrenal insufficiency. • Indications for screening: – Unexplained hyponatremia. – Prolonged corticosteroid or traditional medication ingestion. • Screening is by doing short synacthen test.
Procedure:
• Take blood sample for baseline cortisol level (0 minutes). • Give 250ug cosyntropin (synthetic ACTH) intramuscularly or intravenously. • Take samples at 30 minutes and 60 minutes after injection for cortisol level.
Time Tests Tube
0 min (basal) Cortisol 1 plain tube
30 min Cortisol 1 plain tube
60 min Cortisol 1 plain tube
Interpretation:
• Normal response is cortisol increment of 200nmol/L with peak of >550nm/l • Patient with atrophy of adrenal cortex (exogenous steroid / pituitary or hypothalamic disease) shows slight rise in serum cortisol.
2. Adrenal Hyperfunction (Cushing’s syndrome)
Screening tests should be done in patients:
– With multiple and progressive features of Cushing syndrome – With adrenal incidentaloma. – After excluding exogenous steroid intake.
• Screening tests are: i. 24-hours urine free cortisol: if level is 3-4 times greater than upper limit normal, suggestive of Cushing syndrome.If less than 300 nmol/day, Cushing syndrome is excluded ii. Overnight Dexamethasone Suppression Test (OLDDST)
Procedure:
- Give 1 mg dexamethasone orally at 2300 or 2400 hours. - Fill up the request form complete with clinical summary and request test mentioned above. - Collect blood at 8.00 am the next morning for determination of serum cortisol and send to main counter, Pathology Department.
Interpretation: In normal subjects, serum cortisol is suppressed to less than 50 nmol/l. Serum cortisol level of more than 50 nmol/l can also be seen in cases of stress, obesity, infection, acute or chronic illness, alcohol abuse, severe depression, oral contraceptive, pregnancy, estrogen therapy, failure to take dexamethasone, or treatment with diphenylhydantoin or phenobarbital (enhancement of dexamethasone metabolism). iii. Low Dose Dexamethasone Suppression Test
Procedure: - At 9.00am on 1st day of test, collect blood for serum cortisol (basal) and request test mentioned above. - Immediately after sampling, give 0.5mg dexamethasone orally every 6 hrs for 2 days (8 times). - Collect blood for serum cortisol 6 hours after last dose of 0.5mg dexamethasone and send to main counter Pathology Department.
Note:
• Ensure the times are followed strictly and with full compliance.
Day 1 Day 2 Day 3
Sample taken for 0900 am (basal) 0900 am - serum cortisol.
Drug given: 0.5mg 0900 am 0300 am 0300 am (last dexamethasone dose) every 6 hours (8 times) 1500 pm 0900 am -
2100 pm 1500 pm -
- 2100 pm -
Interpretation:
In normal subjects, serum cortisol will be suppressed to <50nmol/l.
• Localization Test: • After 2 concordantly positive screening tests, localization tests are recommended, which include: iv. Plasma ACTH Procedure: – Blood should be taken together with serum cortisol at 9am. – Keep the tube in ice water bath and send to lab for centrifuged and frozen as soon as possible to avoid falsely low result.
Interpretation: – ACTH < 5 ng/L (<1 pmol/L): ACTH independent Cushing → proceed with CT scan of adrenals. – ACTH >15 ng/L (>3 pmol/L): ACTH dependent Cushing → proceed with MRI pituitary/ CXR.
v. Bilateral inferior petrosal sinus sampling: For localization of pituitary tumour (Cushing disease).
11. REFERENCES
• Special Endocrinology Test Protocols for Adults ,Endocrinology Unit, Department of Medicine Hospital Putrajaya, 2010
• Clinical Chemistry Sixth Edition, William J Marshall & Stephen K Bangert, Mosby, 2008