Professional Documents
Culture Documents
Identification of Hemoglobin Varients Through HPLC
Identification of Hemoglobin Varients Through HPLC
Identification of Hemoglobin Varients Through HPLC
IDENTIFICATION OF HEMOGLOBIN
VARIENTS
Peter J. Howanitz MD
Professor and Vice Chair
Department of Pathology
SUNY Downstate, Brooklyn NY, USA
(Peter.Howanitz@downstate.edu)
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
S (6 GluVal)
C (6 GluLys)
E (26 GluLys)
D Los Angeles (22 GluGln)
INTERPRETATION OF HPLC
RESULTS
* Changed By Thalassemia
BIO-RAD VARIANT
WINDOWS
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
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
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)
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)
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
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?