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
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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
Hemoglobin Electrophoresis
Patterns
STRUCTURE HEMOGLOBINS
Hemoglobin
A
Globin Chain
α2β2
A2
α2δ2
F
α2γ2
Adult Level
A >95%
2-3%
F< 2.0%
COMMON HEMOGLOBIN POINT
MUTATIONS
• Alpha Chain Variants
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G Philadelphia (α68 Asn→Lys)
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S (β6 Glu→Val)
C (β6 Glu→Lys)
E (β26 Glu→Lys)
D Los Angeles (β22 Glu→Gln)
--
A2’ (δ16 Gly→Arg)
• Beta Chain Variants
• Delta Chain Variants
INTERPRETATION OF HPLC RESULTS
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Hemoglobin Retention Time
Variant Hemoglobin Percentage*
A2 Percentage*
Number of Variants*
CBC Indices*
Transfusion History
Age
Clinical Course*
• * Changed By Thalassemia
BIO-RAD VARIANT WINDOWS
PEAK NAME
RETENTION PEAK NAME
TIME (MIN)
RETENTION
TIME (MIN)
F Window 0.98-1.20 A2 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
A0 Window 1.90-3.10 C Window
4.90-5.30
INTREPRATION OF RESULTS
# Abnormal
Peaks (%)
A%
A2%
VARIANT
EXAMPLE
1 (25-40)
50-60 3.5-4.5
β-Chain
AS, AC
2 (25, 1.0)
70-80 1.5-2.2* α-Chain
AG-Phil
2 (50,45)
0
3
40-50
(12, 20,14)
3.5-4.5
2.0*
2 β-Chain SC
1 α-,1 β-, ASG1αβPhilly
Chain
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’
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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% Hb Lepore
• 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
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Elution
Trait (Hb AE)
– Asymtomatic, No CBC Abnormalities
• Disease (Hb EE)
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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
MORE RARE VARIANTS?
BIORAD TURBO A1C-CHROMATOGRAM
BIO-RAD A1C-AS CHROMATOGRAM
BIO-RAD A1C AC CHROMATOGRAM
BIO-RAD UNKNOWN VARIANT
A1C CHROMATOGRAM TYPE 1
BIO-RAD UNKNOWN VARIANT
A1C CHROMATOGRAM TYPE 2
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
CONTROL
PATIENT 1
PATIENT 2
Capillary electrophoresis of a control specimen and the patients’ specimens. A hemoglobin variant is not identified in either
electrophoretogram
HEMOGLOBIN ELECTROPHORESIS
ALKALINE GEL
ACID 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 HemeHeme 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?