James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Neonatal Anemia: Recognizing Thalassemia and Hemoglobin Variants James H. Nichols, PhD, DABCC, FACB Professor of Pathology, Microbiology, and Immunology Medical Director, Clinical Chemistry Associate Medical Director of Clinical Operations Vanderbilt University School of Medicine Nashville, TN 37232‐5310 [email protected] Objectives • Describe hemoglobin genetics • Interpret hemoglobin chromatograms and IEF • Recognize common hemoglobin variants Case • 4 mo male, African American, abnormal newborn screen, seen for follow‐up testing • Newborn screen shows hemoglobin FS at birth 1 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN HbF = 33.8% HbA = <1% HbA2 = 2.7% HbS = 62.5% SickleDex = Positive C S F A NB Audience Poll • What do these results indicate? A. Normal profile B. Abnormal amounts of hemoglobin F C. Sickle cell disease D. Sickle cell trait Hemoglobin Tetramer 2 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Chromosomal Organization of Globin Genes Normal Hemoglobins in Adults Globin Chain Expression Reasons for Requesting Hemoglobin Variant Analysis • Follow‐up to abnormal newborn screen • Adoption • Prenatal screening –patients of ethnic origin • Anemia of unknown origin in ethnic patient • Athletic exam for competitive sports 3 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Hemoglobinopathies 1. Structural – substitution, addition or deletion of one or more AAs in the globin chain i.e HbS, HbC, HbE, HbD, HbO, etc… 2. Thalassemia‐ quantitative defect in globin chain production i.e. alpha and Beta Thalassemia 3. Combination of 1 and 2 4. Asymptomatic disorders –i.e. Hereditary persistence of fetal Hemoglobin Sickle Cell Disease • Disease diagnosis based upon presence of a specific variant gene, the sickle gene – Mutation = Glu6Val substitution in the β-globin protein •Onebs gene = Sickle trait • 2 variant genes (bs or other) = disease • Others include Hb C, D, O and b-Thal 4 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Symptoms of Sickle Sell Disease: • Pallor • Delayed growth & • Pain Crises puberty • Jaundice • Priapism • Hand‐foot syndrome • Infections • Eye problems • Gallstones • Stroke • Sores (ulcers) on the • Acute chest syndrome legs (chronic) • • Weakness, general Spleen dysfunction Severity Depends on Genotype (i.e. S/C = mild, vs S/S and S/OArab = severe) Laboratory Diagnosis of Sickle Cell Disease • Sickle Chex (Dex) – Qualitative Solubility Kit ‐red cells lysed, hemoglobin released – reduced HbS insoluble – cloudy/turbid suspension‐detects homo and heterozygous HbS • HPLC –BioRad Variant II ‐thal program • Electrophoretic Separation Technique – Isoelectric focusing (IEF) – Citrate Agar Electrophoresis (Acid) – Cellulose Acetate Electrophoresis (Alkaline) • DNA Sequencing of ‐globin gene High Performance Liquid Chromatography (HPLC) BioRad Variant II -Thal Short Program Peak Retention time, Hb A1c Hb E name min Hb D F window 0.98–1.20 A0 window 1.90–3.10 A2 window 3.30–3.90 D window 3.90–4.30 S window 4.30–4.70 C window 4.90–5.30 5 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Isoelectric Focusing (IEF) Application + - NB A E AD H/Barts AFS C Treatment of Sickle Cell Disease Hydroxyurea • Increases Hb F levels in RBCs • Decreases neutrophil counts – 4‐12 weeks after initiation – SS neutrophils have enhanced binding to fibronectin and are more prone to activation – Modest neutropenia may be beneficial • Increases the water content of RBCs • Alters the adhesion of RBCs to the endothelium • Increases the flexibility of sickled cells 6 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Hydroxyurea Therapy Case • 1 day male, Hispanic/Latino. Normal newborn screen. Here for follow‐up testing. HbF = 77.4% HbA = 22.6% HbS = <2% SickleDex = Negative Audience Poll • What is the most appropriate interpretation of these results? A. Hereditary persistence of F B. Normal profile C. Sickle cell disease D. Beta thalassemia 7 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Hemoglobin F in Health and Disease • Normal • Hb Disorders – adult <2% – Hb S <20% – newborn ~80% – Unstable Hb <10% – 10‐week‐old ~50% – ‐thal <30% – 6‐month‐old ~2% – ‐thal 30 ‐ 95% – pregnancy 3 ‐ 15% – S/‐thal 10 ‐ 30% • Anemias – ‐thal 25 – 35% – Aplastic 5 ‐ 25% – ‐thal <1% – Iron def. 2 ‐ 8% • Malignancies • Hereditary Persistence – Leukemias 2 ‐ 20% – HPHF 10 ‐ 100% Hereditary Persistence of F (HPFH) • Molecular studies have identified two groups of disorders where expression of the globin gene of Hb F persists at high levels in adult erythroid cells with normal RBC indices and morphology • Form of ‐thalassemia Pancellular forms • clearly increased Hb F in heterozygotes (15 – 35%) • usually due to major deletions of the globin gene cluster, including the gene silencers • Evenly distributed among RBCs Heterocellular forms • modest elevations in Hb F (1 - 4 %) distributed in an uneven fashion among the F cells • molecular lesions include promoter mutations in the globin genes and mutations distant from the globin cluster, including a determinant on chromosome 6q in some families • HbF expression is not evenly distributed among RBCs HPFH Type % Hb F GA Hb F Cellular Distribution African GA Heterozygous 13 - 31 2:3 pancellular Homozygous 100 variable pancellular African G Heterozygous 15 - 20 Gonly pancellular Swiss Heterozygous 1 - 3 variable heterocellular Normal neonate 50 - 80 3:1 heterocellular Normal adult <2 2:3 heterocellular 8 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Case • 23 mo female, African American. Abnormal newborn screen showing HbFS (84% F, 16% S) HbF = 27.3% HbA = <1% NB HbA2 = 3.2% HbS = 68.5% A F S SickleDex = Positive C Audience Poll • What is the most appropriate interpretation of these results? A. Normal profile B. Hereditary persistence of HbF C. Sickle cell disease D. Sickle cell disease with hereditary persistence HbF Case • 33 mo male, unknown ethnicity with anemia (9.7 Hgb), microcytosis (small cells), anisocytosis (unequal size). Mentzer’s index = 13.4 (indeterminate) HbF = 9.6% HbA = 85.5% HbA2 = 4.9% SickleDex = Negative C S F A NB 9 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Audience Poll • What is the most appropriate interpretation for these results? A. Normal profile B. Sickle cell disease C. Suggestive of beta thalassemia D. Hereditary persistence of HbF Mentzer’s Index • If CBC shows microcytic anemia, ratio of MCV/RBC can distinguish iron deficiency from thalassemia. • < 13 beta thalassemia more likely (thalassemia is a disorder of globin synthesis, normal amount of cells, but cells produced are smaller and more fragile, RBC normal, but MCV down, so ratio is low) • >13 iron deficiency more likely (in iron deficiency bone marrow can’t produce as many cells and cells are small, both MCV and RBC down) • Not reliable, iron deficiency and beta thalassemia can coexist, and ferritin more reliable measure of iron deficiency Beta Thalassemia • A group of genetic disorders resulting in dimished (or absent 0‐chain synthesis • β‐thalassemia is commonly associated with decreased Hb A and increased HbA2 in heterozygotes • Heterozygous thal is asymptomatic • Homozygous thal is a severe disorder associated with transfusion dependent hemolytic anemia • Homozygous + thal is a heterogenous disorder with severity depending on mutation and % of HbA (the more HbA, the less severe the disease) 10 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Expected Hgb chain distributions in β‐thalassemia HbA HbA2 HbF 0) present 4 –8% 0 –5% β0/ β0none1‐6% >94% β+/ β+present2.4‐8.7% 20‐90% β0/ β+present0.6‐3.4% >75% δβ0/ δβ0none0%70‐92% Case • 13 y/o female, Asian, 9 yrs status post bone marrow transplant. On chronic transfusions for anemia, premature deliver at 28 wks to prevent hydrops (in utero transfusions) HbF = <1%% HbA = 67.3% HbA2 = 2.8% HbH = 28.9% A SickleDex = Negative F Hb= 10.0 (low) S Ferritin = 962 on C exjade for chronic transfusions Audience Poll • What is the most appropriate interpretation of these results? A. Normal profile B. Suggestive of beta thalassemia C. Alpha thalassemia D. Sickle cell trait 11 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School of Medicine Nashville, TN Alpha Thalassemia • Our patient has alpha thalassemia major. • A group of genetic disorders associated with defective ‐chain synthesis • Difficult to diagnose after neonatal period • Characteristic HPLC profile –HbBarts (4 chains –most common in neonate) and HbH (4 ‐ chains). • Clinical symptoms range from mild microcytosis –severe hemolytic anemia depending on the number of mutations Alpha Thalassemia Alpha Thalassemia (AA) α α / α α Normal -Thal silent (-/) ~ 28% -Thal-1 mutation (--/) is virtually non-existent - α / α α Heterozygous Consequently, incidence of (--/- and Silent Carrier --/--) is extremely rare in this group --/ α α Homozygous Thalassemia minor1 - α /- α Homozygous Thalassemia minor2 --/ α - Hgb H disease Alpha Thal Intermedia - - / - - Hydrops Fetalis Alpha Thal Major Alpha Thalassemia 12 James H. Nichols, Ph.D., DABCC, FACB Vanderbilt University School