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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 ). GOALS AND OBJECTIVES. Describe Measurements Of Hemoglobins Introduce Role of HPLC
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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)
GOALS AND OBJECTIVES • Describe Measurements Of Hemoglobins • Introduce Role of HPLC • Case Studies • New Finding--Only A1C Detects Variant • Questions And Answers
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
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
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)
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
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
INTREPRATION OF RESULTS • Hemoglobin A • Increased P2-? Diabetes (↑A1C>7%) • Increased P3-(>P2) Old Specimen • Inverse of Other Hemoglobins • Focus on Abnormal Hemoglobins
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)
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
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
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
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)
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)
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
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.
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.
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
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.
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
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
CONCLUSIONS • HPLC Valuable Laboratory Technique • Discussed Common Variants • Interpreted Chromatograms–Case Studies • New-Hemoglobin G-Georgia Not Noted • Important To ID A1c Variants • Questions?