ERRORS IN THE DETECTION AND IDENTIFICATION OF HEMOGLOBIN VARIENTS

1. ERRORS IN THE DETECTION AND IDENTIFICATION OF HEMOGLOBIN VARIENTS Peter J. Howanitz MD Professor and Vice Chair Department of Pathology SUNY Downstate, Brooklyn NY, USA (Peter.Howanitz@downstate.edu)

2. GOALS AND OBJECTIVES • Describe Measurements Of Hemoglobins • Introduce Role of HPLC • Case Studies • New Finding--Only A1C Detects Variant • Questions And Answers

3. REASONS FOR HEMOGLOBIN ID AND QUANTIFICATION • Newborn Screening • Prenatal Screening • Follow-up Newborn Screening • Diagnosis Cause of Microcytosis • Anemia, Polycythemia, Chronic Hemolysis • Hemoglobinopathy Blood Replacement • Unexplained A1c Results

5. WHY USE HPLC? • Advantages • Throughput 11 Specimens/hour, 24 Hr Cal. • Analytic Sensitivity @ Low Concentrations • Improved Precision • Better Separation • Less Referrals For ID • Disadvantages • More Complex→ Higher Skill Level • Co-elution Of Hemoglobins

6. Hemoglobin Electrophoresis Patterns

7. STRUCTURE HEMOGLOBINS

8. COMMON HEMOGLOBIN POINT MUTATIONS • Alpha Chain Variants • G Philadelphia (α68 Asn→Lys) • Beta Chain Variants • S (β6 Glu→Val) • C (β6 Glu→Lys) • E (β26 Glu→Lys) • D Los Angeles (β22 Glu→Gln) • Delta Chain Variants -- A2’ (δ16 Gly→Arg)

9. INTERPRETATION OF HPLC RESULTS • Hemoglobin Retention Time • Variant Hemoglobin Percentage* • A2 Percentage* • Number of Variants* • CBC Indices* • Transfusion History • Age • Clinical Course* • * Changed By Thalassemia

10. BIO-RAD VARIANT WINDOWS

11. INTREPRATION OF RESULTS

14. INTREPRATION OF RESULTS • Hemoglobin F • >2-80% Babies • 90-100% Homozygous Hereditary Persistence Fetal Hemoglobin,β0, δβ0-Thal • 15-40% Heterozygous HPFH • 10-25% SS, Hydroxyurea Treated • 3-10% Homozygous Hemoglobinopathies, Anemias, Leukemias, Malignancies, • < 5% β-Thal, Lepore

16. INTREPRATION OF RESULTS • Hemoglobin A • Increased P2-? Diabetes (↑A1C>7%) • Increased P3-(>P2) Old Specimen • Inverse of Other Hemoglobins • Focus on Abnormal Hemoglobins

18. HEMOGLOBIN A2’ • Elutes in S Window • Δ16 Gly→Arg • Characteristic Low A2 Percentage (1.0-2.5%) • Most Common In Blacks (2%) • CBC Normal • Little Consequence, Except β-Thal (add A2)

19. INTREPRATION OF RESULTS • Hemoglobin A2 • Increased • 4.0-7.0% Β-Thalassemia, Sβ+Thal • 3.5-4.5% Hb AS, AC, SC, SS, CC • 6.5-14.0% HbLepore • 25-30% Hb E • Decreased • 1.3-1.7% Iron Deficiency, Sideroblastic, Aplastic Anemias • 1.5-2.3% δ Chain Variant (A2’), α Chain Variant

21. HEMOGLOBIN E • Found in SE Asia, β26Glu→Lys • Most Common Hemoglobinopathy Worldwide • Complicated by Iron Def, Thalassemia, A2 Elution • Trait (Hb AE) • Asymtomatic, No CBC Abnormalities • Disease (Hb EE) • Mild Anemia, Target Cells, ↓RBC Survival • ↓Osmotic Fragility • +Beta Thal = Severe, As Homozygous β-Thal • +Alpha Thal=↓Hb E

23. HEMOGLOBIN D D Window On Bio-Rad Variant Β121Glu→Gln Found In India (D-Punjab/D-Los Angeles) Most Common D In U.S. Blacks (< 0.02%) Trait Asymtomatic, No Anemia, Normal CBC Disease Asymtomatic, No Anemia/ Hemolysis D Los-AngelesS = Symptoms of Sickle Cell Disease

25. HEMOGLOBIN G PHILADELPHIA • Elutes In D-Window • α68Asn→Lys of Hb A and A2 • Heterozygote-CBC Normal • Most Common α Chain Variant In Blacks, Italians (25%), Chinese • Associated With α-Thal (30%, 45%G) • Association With S or C Common (Double Heterozygote)

27. HEMOGLOBIN S S Trait (Hemoglobin AS) β6Glu→Val Common In Blacks; Other Populations Asymptomatic, Blood Sickles in Vitro Protective Against Malaria S Disease (Hemoglobin SS) Severe Symptoms, Sickling in Vivo Hydroxy Urea Treatment→Induces F Crises→Bone Pain, Hemolysis, Stroke, etc Similar Symptoms Other Double Heterozygotes (SC)

29. HEMOGLOBIN C • Prevalent in West Africa, 3% U.S Blacks • Trait (Hb AC) β6Glu→Lys • No Symptoms or Anemia, • Hypochromia, Up to 40% Target Cells • Disease (Hb CC) • Mild Hemolytic Anemia, Spenomegly • Rod Shaped Crystals in RBCs • Normochromic, Normocytic Anemia, • 40-90% Target Cells

30. MORE RARE VARIANTS?

31. BIORAD TURBO A1C-CHROMATOGRAM

32. BIO-RAD A1C-AS CHROMATOGRAM

33. BIO-RAD A1C AC CHROMATOGRAM

34. BIO-RAD UNKNOWN VARIANT A1C CHROMATOGRAM TYPE 1

35. BIO-RAD UNKNOWN VARIANT A1C CHROMATOGRAM TYPE 2

36. HEMOGLOBIN A1C CHROMATOGRAPHS CONTROL PATIENT 1 PATIENT 2 A1C HPLC results of a control specimen and the patients’ specimens. Note the variant eluting at 0.872 & 0.853 minutes in chromatograms of patient 1 and patient 2 depicted by an arrow.

37. HEMOGLOBIN IDENTIFICATION CHROMATOGRAMS CONTROL PATIENT 1 PATIENT 2 Hemoglobin HPLC results of a control specimen and the patients’ specimens. A hemoglobin variant is not identified in either chromatogram.

38. HEMOGLOBIN IDENTIFICATION CAPILLARY ELECTROPHORETOGRAMS PATIENT 2 CONTROL PATIENT 1 Capillary electrophoresis of a control specimen and the patients’ specimens. A hemoglobin variant is not identified in either electrophoretogram

39. HEMOGLOBIN ELECTROPHORESIS ACID GEL ALKALINE GEL Hemoglobin electrophoresis on alkaline and acid gel. The patient’s specimen migrates as S on alkaline gel, and a split A band on acid gel, identified as an arrow. Electrophoresis of the specimen from the second patient was identical to the first (not shown).Controls for C, S, F and A are the top two specimens in either gel.

40. GENETIC ANALYSIS OF VARIANT • DNA Sequence Analysis • Alpha-2 Substitution • Codon 95 CCG To CTG, Pro To Leu • Hemoglobin G-Georgia • Compatible With Other Lab Findings

41. HEMOGLOBIN G-GEORGIA • Five Cases In Literature • Found In Blacks & Portuguese • Increased 02 Affinity, Decreased Heme-Heme Interaction • No CBC Abnormalities • Double Heterozygote With S & C

42. CONCLUSIONS • HPLC Valuable Laboratory Technique • Discussed Common Variants • Interpreted Chromatograms–Case Studies • New-Hemoglobin G-Georgia Not Noted • Important To ID A1c Variants • Questions?