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
([email protected])
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
Hemoglobin
Electrophoresis
Patterns
STRUCTURE HEMOGLOBINS
Hemoglobi
n
Globin Chain
22
A2
2 2
2 2
Adult Level
A >95%
2-3%
F< 2.0%
COMMON HEMOGLOBIN
POINT
MUTATIONS
Alpha Chain Variants
G Philadelphia (68 AsnLys)
S (6 GluVal)
C (6 GluLys)
E (26 GluLys)
D Los Angeles (22 GluGln)
Beta Chain Variants
Delta Chain Variants
-- A2 (16 GlyArg)
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
BIO-RAD VARIANT
WINDOWS
PEAK NAME RETENTIO PEAK NAME RETENTIO
N TIME
N TIME
(MIN)
(MIN)
F Window 0.981.20
P2
Window
P3
Window
A0
Window
1.241.40
1.401.90
1.903.10
A2
3.30-3.90
Window
D Window 3.90-4.30
S Window 4.30-4.90
C Window 4.90-5.30
INTREPRATION OF RESULTS
# Abnormal
Peaks (%)
A%
A2%
VARIANT
1 (25-40) 50-60 3.5-4.5 -Chain
2 (25, 1.0) 70-80 1.5-Chain
2.2*
2 (50,45) 0
3.5-4.5 2 Chain
3
40-50 2.0*
1 -,1 -,
1(12,
Chain
20,14)
EXAMPL
E
AS, AC
AG-Phil
SC
ASGPhilly
INTREPRATION OF RESULTS
Hemoglobin F
>2-80% Babies
90-100% Homozygous Hereditary Persistence
Fetal Hemoglobin,0, 0-Thal
15-40% Heterozygous HPFH
10-25% SS, Hydroxyurea Treated
3-10% Homozygous Hemoglobinopathies,
Anemias, Leukemias, Malignancies,
< 5% -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
16 GlyArg
Characteristic Low A2 Percentage (1.0-2.5%)
Most Common In Blacks (2%)
CBC Normal
Little Consequence, Except -Thal (add A2)
INTREPRATION OF RESULTS
Hemoglobin A2
Increased
4.0-7.0% -Thalassemia, S+ 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% Chain Variant (A2), Chain Variant
HEMOGLOBIN E
Found in SE Asia, 26GluLys
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 -Thal
+Alpha Thal=Hb E
HEMOGLOBIN D
D Window On Bio-Rad Variant
121GluGln
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
68AsnLys of Hb A and A2
Heterozygote-CBC Normal
Most Common Chain Variant In Blacks,
Italians (25%), Chinese
Associated With -Thal (30%, 45%G)
Association With S or C Common
(Double Heterozygote)
HEMOGLOBIN S
S Trait (Hemoglobin AS) 6GluVal
Common In Blacks; Other Populations
Asymptomatic, Blood Sickles in Vitro
Protective Against Malaria
S Disease (Hemoglobin SS)
Severe Symptoms, Sickling in Vivo
Hydroxy Urea TreatmentInduces F
CrisesBone Pain, Hemolysis, Stroke, etc
Similar Symptoms Other Double Heterozygotes (SC)
HEMOGLOBIN C
Prevalent in West Africa, 3% U.S Blacks
Trait (Hb AC) 6GluLys
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 A1CCHROMATOGRAM
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 patients 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 ChromatogramsCase Studies
New-Hemoglobin G-Georgia Not Noted
Important To ID A1c Variants
Questions?
