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) Peter.Howanitz@downstate.edu

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

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

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

6. Hemoglobin Electrophoresis Patterns Hemoglobin Electrophoresis Patterns

7. STRUCTURE HEMOGLOBINS STRUCTURE HEMOGLOBINS Hemoglobin Globin Chain Globin Chain Adult Level Adult Level A α2β2 α2β2 A >95% A >95% A 2 A 2 α2δ2 α2δ2 2-3% 2-3% F α2γ2 α2γ2 F< 2.0% F< 2.0%

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

9. INTERPRETATION OF HPLC RESULTS INTERPRETATION OF HPLC RESULTS Hemoglobin Retention Time Hemoglobin Retention Time Variant Hemoglobin Percentage* Variant Hemoglobin Percentage* A 2 Percentage* A 2 Percentage* Number of Variants* Number of Variants* CBC Indices* CBC Indices* Transfusion History Transfusion History Age Age Clinical Course* Clinical Course* * Changed By Thalassemia * Changed By Thalassemia

10. BIO-RAD VARIANT WINDOWS PEAK NAME RETENTION TIME (MIN) PEAK NAME RETENTION TIME (MIN) F Window 0.98-1.20 A 2 Window 3.30-3.90 P2 Window 1.24-1.40 D Window 3.90-4.30 P3 Window 1.40-1.90 S Window 4.30-4.90 A 0 Window 1.90-3.10 C Window 4.90-5.30

11. INTREPRATION OF RESULTS INTREPRATION OF RESULTS # Abnormal Peaks (%) A% A% A2% A2%VARIANTEXAMPLE 1 (25-40) 50-603.5-4.5 β-Chain AS, AC 2 (25, 1.0) 70-801.5-2.2* α-Chain AG-Phil 2 (50,45) 03.5-4.5 2 β-Chain SC 3 (12, 20,14) 40-50 2.0* 2.0* 1 α-,1 β-, 1αβ- Chain ASG- Philly

14. INTREPRATION OF RESULTS Hemoglobin F Hemoglobin F –>2-80% Babies –90-100% Homozygous Hereditary Persistence Fetal Hemoglobin,β 0, δβ 0 -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 INTREPRATION OF RESULTS Hemoglobin A Hemoglobin A –Increased P2-? Diabetes ( ↑ A 1 C>7%) –Increased P3-(>P2) Old Specimen –Inverse of Other Hemoglobins –Focus on Abnormal Hemoglobins

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

19. INTREPRATION OF RESULTS INTREPRATION OF RESULTS Hemoglobin A 2 Hemoglobin A 2 –Increased 4.0-7.0% Β-Thalassemia, Sβ + Thal 4.0-7.0% Β-Thalassemia, Sβ + Thal 3.5-4.5% Hb AS, AC, SC, SS, CC 3.5-4.5% Hb AS, AC, SC, SS, CC 6.5-14.0% Hb Lepore 6.5-14.0% Hb Lepore 25-30% Hb E 25-30% Hb E –Decreased 1.3-1.7% Iron Deficiency, Sideroblastic, Aplastic Anemias 1.3-1.7% Iron Deficiency, Sideroblastic, Aplastic Anemias 1.5-2.3% δ Chain Variant (A2’), α Chain Variant 1.5-2.3% δ Chain Variant (A2’), α Chain Variant

21. HEMOGLOBIN E HEMOGLOBIN E Found in SE Asia, β 26Glu → Lys Found in SE Asia, β 26Glu → Lys Most Common Hemoglobinopathy Worldwide Most Common Hemoglobinopathy Worldwide Complicated by Iron Def, Thalassemia, A 2 Elution Complicated by Iron Def, Thalassemia, A 2 Elution Trait (Hb AE) Trait (Hb AE) –Asymtomatic, No CBC Abnormalities Disease (Hb EE) 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 HEMOGLOBIN D D Window On Bio-Rad Variant Β 121Glu → 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 HEMOGLOBIN G PHILADELPHIA Elutes In D-Window Elutes In D-Window α68 Asn → Lys of Hb A and A 2 α68 Asn → Lys of Hb A and A 2 Heterozygote-CBC Normal 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) Association With S or C Common (Double Heterozygote)

27. HEMOGLOBIN S HEMOGLOBIN S S Trait (Hemoglobin AS) β6 Glu → 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 Hydroxy Urea Treatment → Induces F Crises → Bone Pain, Hemolysis, Stroke, etc Similar Symptoms Other Double Heterozygotes (SC)

29. HEMOGLOBIN C HEMOGLOBIN C Prevalent in West Africa, 3% U.S Blacks Prevalent in West Africa, 3% U.S Blacks Trait (Hb AC) β6 Glu → Lys Trait (Hb AC) β6 Glu → Lys –No Symptoms or Anemia, –Hypochromia, Up to 40% Target Cells Disease (Hb CC) 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 A 1C 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. 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. HEMOGLOBIN IDENTIFICATION CHROMATOGRAMS

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

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

41. HEMOGLOBIN G-GEORGIA HEMOGLOBIN G-GEORGIA Five Cases In Literature Five Cases In Literature Found In Blacks & Portuguese Found In Blacks & Portuguese Increased 0 2 Affinity, Decreased Heme- Heme Interaction Increased 0 2 Affinity, Decreased Heme- Heme Interaction No CBC Abnormalities No CBC Abnormalities Double Heterozygote With S & C Double Heterozygote With S & C

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