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Unraveling Hemoglobinopathies with
Capillary Electrophoresis
David F. Keren, M.D.
Professor of Pathology
Division Director, Clinical Pathology
The University of Michigan
Session Number 2002
Financial Disclosure Information
In the past 12 months, I have not had a significant
financial interest or other relationship with the
manufacturer(s) of the product(s) or provider(s) of the
service(s) that will be discussed in my presentation.
Hemoglobin Structure
e Gg Ag b d b
Beta Globin Gene Cluster
( Short arm of Chromosome 11)
5
’
3’
LCRB
z z1 a1 a1 a2 a1
5
’
3’
LCRA
(HS-40)
Alpha Globin Gene Cluster
( Short arm of Chromosome 16)
40 kb
Hb A (a2b2) 95% of adult Hb Hb F (a2g2) 70% neonate <2% adult Hb A2 (a2d2) 2.2-3.4% of adult Hb
Hb Gower I (z2e2) Hb Gower II (a2e2) Hb Portland (z2g2)
Embryonic Hemoglobins Fetal & Adult Hemoglobins
Nomenclature
• Alphabetical – Hb A
• Hb A2 (minor fraction seen on Electro in 1955)
– Hb B
– Hb C
– Hb D
– Hb E
– Hb F (fetal)
– →→→→→→→→→→→→→→→→→Hb Q-India
• Location: e.g. Hb Ann Arbor
?????????????????? = Hb S
Hemoglobin Shorthand
Hb Ann Arbor = a80Leu→Arg
• a refers to the abnormal chain
• 80 is the position with a substitution
• Leucine is the normal amino acid
• Arginine is the substituted amino acid
~1,000 Variant Hemoglobins
• Most Variants are Asymptomatic
• Structural Variants
– Alpha: Hb G Philadelphia
– Beta: Hb S, Hb C, Hb D, Hb O
– Delta: Hb A2‘
• Structural & Thalassemia
– Constant Spring (alpha variant)
– HbE (beta variant)
– Lepore (delta-beta fusion protein)
Malaria Distribution parallels
Major Hemoglobinopathies &
Thalassemias
Distribution
of malaria
Harteveld and Higgs Orphanet Journal of Rare Diseases
2010, 5:13
http://www.ojrd.com/content/5/1/13
Investigation of Hemoglobin
• Clinical: age, transfusion, race, therapy
• Routine: RBC, MCV, MCH, RDW, sickle test
• Analytical
– Alkaline & Acid electrophoresis
– HPLC—(Hb A2 & Hb F)
• Cationic exchange: several types
– Capillary Electrophoresis (CE)
• High pH (10.0)
• Confirm Variant: two methods
• Referral: Mass spectrometry/Molecular
Gel Electrophoresis
Alkaline conditions pH 8.6
• Densitometry for fractionation
(inadequate for Hb A2 and Hb F)
• Cannot differentiate:
Hb A2, C, O, or E
Hb S, D, G
Acid conditions pH 6.5
• Differentiates:
D & G from S (but can’t tell D from G)
E & O from C
Alkaline Gel Acid Gel
( ) denotes low concentration
F A S C
F A S
F S
F Köln /A
F S C
F (C)
(F) E/A
(F) E/A
(F) A G/S
(F) A S
F A/J
(F) A/Chicago
(F) A S
(A) S C
(F) A S
Carbonic Anhydrase
+ A
node
+ A
node
C S F A
A2 S F A
S F
Köln F A
C S F
(C) F
E A
E A
SG S/G A
A2 S A
F A J
A2 A/Chicago
A2 S (F) A
C S (A)
A2 S A
High Performance Liquid
Chromatography (HPLC)
• Improved Sensitivity over gels
• Accurate measurement of Hb A2 and Hb F
• Complex patterns for interpretation
• Hb H difficult to measure
– Does not separate from A1c on some
– Elutes prior to routine measurement on some
• Bilirubin interferes with Hb Bart’s detection
• Hb S & Hb C adducts interfere with Hb A2
• Cannot separate Hb A2’ from Hb S
• Cannot separate Hb A2 from Hb E on most
Biorad Variant I HPLC
Bilirubin &
degradation products
Glycated & Aged Hb A
Hb A
Hb A2
Bio-Rad Variant-II HPLC
Degradation products
Gly
cate
d H
bA
Hb A2
Hb A A
gin
g H
bA
Nl <5%
Nl 1.7-3.1
Peak 1 (glycated A1a,A1b &
degradation products)
Peak 2 (glycated A1c)
Peak 3 (glycated A1d & degradation products)
Peak 4 (Hb A) Peak 5 (Hb A2)
Biotech-
Trinity Ultra
HPLC
Capillary Electrophoresis
Sample
Positive buffer ions (+) flow to cathode
- Cathode (+)
Hb A
Hb F
Hb S
Hb A2
Detector
+Anode
415nm
Sebia Capillarys
Hb SC-What to do with no HbA?
Use these
measurements
Overlay with AFSC Controls
Use these
measurements
Mix 1:1 with Normal Sample
Do NOT use these
measurements
• Patterns much less complex than HPLC
• Accurate quantification of Hb A2, F, and S
• No interference of Hb S adducts with Hb A2
• HbA2’ visible in the presence of Hb S
• Clear separation of Hb D, G &E from Hb A2
• Detects and measures Hb H and Hb Bart’s
• Bilirubin does not interfere with Hb Bart’s
Capillary Electrophoresis
51 y/o man
HPLC Pattern
Position Hb A2 Hb ?
RBC 6.14 4.4-5.7
Hgb 13.5 13.5-17
Hct 42.8 40-50
MCV 78.1 79-99
MCH 21.9 27-32
RDW 18.1 11.5-15.0
Hb A
Ultra HPLC Relative Retention
G Philadelphia
D Los Angeles
0.88-0.91
0.91-0.95
Alpha
Beta
RT/S
Hb A2 0.85-0.91 Delta
51 y/o man
Position for Hb G
0.88-0.92
(Cannot separate
HbA2 included)
Hb G2
RBC 6.14 4.4-5.7
Hgb 13.5 13.5-17
Hct 42.8 40-50
MCV 78.1 79-99
MCH 21.9 27-32
RDW 18.1 11.5-15.0
Hb A 59.6 >95
Hb A2 ? 1.7-3.1
Hb F 0 <2.0
Hb G+A2 27.4 36-40*
Hb A 72.6 >95
Hb A2 ? 1.7-3.1
Hb F 0 <2.0
Hb G+A2 27.4 36-40*
Hb G2 ?
Capillary Electrophoresis
HbG Philadelphia Trait
Hb G Philadelphia
Hb A2
Hb A
Hb G2
b g d
A
G
Hb A Hb F Hb A2
Hb G Hb F Hb G2
a
a G
Clinically benign, even with Hb S
Associated with a Thalassemia
0 a deletions = 25% Hb G (& G2 relative to A2)
1 a deletion = 33%
2 a deletions = 50%
G2 band is always present
Hb G (Philadelphia a68Asn→Lys)
& a Thalassemia
A Hb A Hb F Hb A2 a
A Hb A Hb F Hb A2 a
32 y/o woman
HbA2)
Hb A
Hb ??
RBC 4.14 3.9-5.0
Hgb 12.4 12.0-16.0
Hct 36.5 36-48
MCV 88.1 79-99
MCH 30.1 27-32
RDW 12.9 11.5-15.0
Hb ??
Ultra HPLC Relative Retention
G Philadelphia
D Los Angeles
0.88-0.91
0.91-0.95
Alpha
Beta
RT/S
Hb A2 0.85-0.91 Delta
32 y/o woman
Hb D trait
b variant
Normal MCV
Normal RBC
No “G2”
Hb D (Cannot separate HbA2)
Hb A
Hb D
RBC 4.14 3.9-5.0
Hgb 12.4 12.0-16.0
Hct 36.5 36-48
MCV 88.1 79-99
MCH 30.1 27-32
RDW 12.9 11.5-15.0
Hb A 58.1 >95
Hb D + A2 41.9
Hb F 0 <2.0
Capillary
HbD Trait
Hb D Punjab
Hb A2
Hb A
Hb F
Hb A 51.9 >95
Hb A2 3.2 1.7-3.1
Hb F 0.7 <2.0
Hb D 44.2
Hemoglobin D (Los Angeles or
Punjab) b121 glu→gln
Innocuous as Hb D Trait or Homozygote
Difficult to distinguish from Hb G by gels
Distinction is important:
Hb SG behaves like sickle trait
Hb SD moderate sickling disorder
Hb D with b-thalassemia gives Thalassemia
Intermedia or even Thalassemia Major picture
Hb SD & a-thalassemia gives microcytosis
Patel et al. Intl J Lab Hematol 2014;36:444-50.
Comparison of CE to HPLC
• Easier pattern to interpret
• No glycated products to deal with
• But what about Precision in separating
variants?
• Looked at separation of two closely migrating
variants:
– 43 consecutive cases of Hb D and HbG traits
Ultra HPLC Relative Retention
G Philadelphia
D Los Angeles
0.88-0.91
0.91-0.95
Alpha
Beta
RT/S
HPLC (Ultra)-Elution Time
30 of 43
samples
overlap.
Keren et al. Am J Clin Pathol 2012;137:660-4.
Capillarys-Migration Position
25 of 43
samples
overlap.
Keren et al. Am J Clin Pathol 2012;137:660-4.
HPLC (Ultra)-Elution Time/Hb S
Only 2 of
43 samples
overlap.
Keren et al. Am J Clin Pathol 2012;137:660-4.
Capillarys-Migration/Hb A2
9
7 7
1 1
2 3
11
Keren et al. AJCP 2012
0 of 43
samples
overlap.
Chromosome 11
Beta Thalassemia Trait (Minor)
• >200 b+ vs b0 Mutations (deletions uncommon)
• Lose 30-50% b globin
• Key is elevated hemoglobin A2 (a2d2) (>3.5%)
• Low MCV & MCH nl RDW, usually nl hgb, ↑
RBCs
e Gg Ag b d b
5’ 3’
LCRB
e Gg Ag b d b
5’ 3’
LCRB
X
Hb A2 on Variant II HPLC
University of Leiden
Hb H Disease
a Thalassemia trait
a Thalassemia trait
a Thalassemia trait
Fe Deficiency
Normal Range is Method Dependent Normal
“Normal” Hb A2 b Thalassemia carriers
High HbA2 b Thalassemia carriers
b/a Thalassemia combinations
d/b Thalassemia
HbA2 reduced in d Thalassemia & varriant carriers
Specificity and overlap of Hb A2 values in different cohorts of patients
Beta Thal Trait Method HPLC
Van Delft et al. Intl J Lab Hematol 2009;31:484-95.
Precision of Hb A2 CAP Survey 2010
Results on Normal Samples
Survey # Gel-1 Gel-2 HPLC CE
HG-01 27.4* 26.7 7.5 6.5
HG-02 23.4 21.6 5.6 5.6
HB-03 22.6 21.0 6.8 4.0
* Data is Coefficient of Variation (%)
Precision of Hb A2
Method Instrument Hb A2 <3.5 Hb A2≥3.5
HPLC BioRad I 2.7 4.4
BioRad II 1.6 2.0
Menarini HA 8160 0.5 0.5
Tosoh G7 2.8 1.5
Tosoh G8 1.1 0.8
Capillary Beckman MDQ 4.4 3.2
Beckman PA800 3.3 1.6
Sebia Capillarys II 2.0 1.2
Paleari et al. Intl J Lab Hematol 2012: 1-7
• Samples (duplicates) run at 2 institutions:
• 40 healthy, 29 beta thalassemia & 11 low Hb A2
Beta Thalassemia
Trait
Ref Range
A2 1.7-3.1
RBC 5.0 3.9-5.0
Hgb 10.6 12.0-16.0
Hct 33.9 36-48
MCV 68 79-99
MCH 21.3 27-32
RDW 15.0 11.5-15.0
56 y/o female
Hb A 94.4 >95
Hb A2 4.6 1.7-3.1
Hb F 1.0 <2.0
Same Case
Beta Thalassemia Trait
95-97
<2.0
2.2-3.2
94.1
1.0
4.9
Hb A
Hb F
Hb A2
Fractions % Ref. %
Hb A 94.1 >95
Hb A2 4.9 1.7-3.1
Hb F 1.0 <2.0
Hb A2 (Delta)Variants
A2 1.3
A2’ 1.3
95-97
<2.0
2.2-3.2
97.5
1.3
1.2
Hb A
Hb A2
Hb A2’
Fractions % Ref. %
Hb A2’
• Most Common Delta Variant
• Present in ~1% of African Americans
• Migrates in the same position as Hb S by
HPLC (but not by Capillary Electrophoresis)
• When present need to add to Hb A2 to assess
the complete delta component in:
– Beta Thalassemia
– Alpha Thalassemia
– Iron Deficiency
A2 1.6
A2v 0.7
95-97
2.2-3.2
97.5
1.8
0.7
Hb A
Hb A2
Hb A2’
Fractions % Ref. %
95-97
2.2-3.2
97.5
1.8
0.7
Hb A
Hb A2
Hb A2v
Fractions % Ref. %
Delta Thalassemia
• Clinically Silent trait
• Decrease in normally migrating Hb A2
– Structurally normal delta
– Suspect with nl CBC & decreased Hb A2
– May give falsely ―normal‖ value in patient with
beta thalassemia
• Decrease in Hb A2 + Hb A2v
– Similar to Hb E a beta variant that is produced in
decreased amount
A2 1.1
95-97
<2.0
2.2-3.2
97.5
6.9
1.3
1.2
Hb A
Hb F
Hb A2v
Hb A2
Fractions % Ref. %
Hidden Delta Variant
• Clinically Silent trait
• Decrease in normally migrating Hb A2
– Structurally abnormal delta
– Suspect with nl CBC & decreased Hb A2
– May give falsely ―normal‖ value in patient with
beta thalassemia
• Repeat with a different technique
– Capillary, HPLC, Isoelectric Focusing
18 y/o female
sickledex
positive
RBC 4.2 3.9-5.0
Hgb 12.8 12.0-16.0
Hct 39.4 36-48
MCV 84.0 79-99
MCH 28.4 27-32
RDW 14.6 11.5-15.0
Hb A 59.6 >95
Hb A2 3.8 1.7-3.1
Hb F 0 <2.0
Hb S 36.6 36-40*
*Expected for Sickle Trait
Same Case: Sickle Trait
Hb A2
HPLC CE
3.8 2.9
95-97
<2.0
2.2-3.2
58.4
0
38.7
2.9
Hb A
Hb F
Hb S
Hb A2
Fractions % Ref. %
HPLC vs CE for Hb A2
Effect of Hb S on Hb A2
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
0.0 2.0 4.0 6.0 8.0
Sebia CE
Pri
mu
s H
PL
C
Hb SContainingSamplesNoStructuralVariant
Keren et al. AJCP 2008;130:824-31
Hb S Trait: HPLC vs CE Keren et al. AJCP 2008;130:824-31
0
1
2
3
4
5
6
CE CE-S HPLC HPLC-S
Hem
og
lob
in A
2 (
%)
Hb S Trait with Elevated Hb A2
• Hb S Trait: Hb S = 36-40%, normal CBC but
slight increase in Hb A2 (usually in nl range)
• Reasons for Increase Hb A2
1. d globin competes better than bs for a
globin - actual increase ~0.5%
2. HPLC artifact: Hb S breakdown products
in Hb A2 peak - false increase 1-2%
36 y/o woman
Sickledex
negative
Rel Rt = S 0.91 Hb? = S 1.01
RBC 4.87 3.9-5.0
Hgb 12.1 12.0-16.0
Hct 35.4 36-48
MCV 75.8 79-99
MCH 25.4 27-32
RDW 14.3 11.5-15.0
Ultra HPLC Relative Retention
G Philadelphia
D Los Angeles
0.88-0.91
0.91-0.95
Alpha
Beta
RT/S
Hb A2 0.85-0.91 Delta
36 y/o
woman
Hb G + A2 = 35.9% Hb G2 = 2.4%
RBC 4.87 3.9-5.0
Hgb 12.1 12.0-16.0
Hct 35.4 36-48
MCV 75.8 79-99
MCH 25.4 27-32
RDW 14.3 11.5-15.0
Hb A 60.7 >95
Hb F 1.0 <2.0
Hb G+A2 35.9
Hb G2 2.4
Hb G Philadelphia & b & a
Thalassemia
Hb A2 + G2
HPLC CE
? 5.5
95-97
<2.0
2.2-3.2
61.4
0.4
32.7
3.4
2.1
Hb A
Hb F
Hb G
Hb A2
Hb G2
Fractions % Ref. %
b d
A
G
Hb A Hb A Hb A2
Hb G Hb G Hb G2
a
a
Clinically benign, even with Hb S
Associated with a Thalassemia
0 a deletions = 25% HbG (& G2 relative to A2)
1 a deletion = 33%
2 a deletions = 50%
G2 band is always present
Hb G (Philadelphia a68Asn→Lys)
& a Thalassemia
A Hb A Hb A Hb A2 a
A Hb A Hb A Hb A2 a
b
Chromosome 16
Hemoglobin H Disease
• Severe microcytosis (MCV 55-64)
• Hb A2 low (<1.7)
• Moderate hemolytic anemia, splenomegaly
• Usually not transfusion dependent
• Hb Bart’s &/or H is found
• Can transmit Bart’s Hydrops fetalis
MCR a2 a1
MCR a2 a1
z
z
Hb H Disease
31 yr woman
Hb Bart’s
Hb H & A1c
(can’t measure HbH alone)
Ref Range
A2 1.7-3.1
RBC 5.07 3.9-5.0
Hgb 9.5 12.0-16.0
Hct 31.9 36-48
MCV 63 79-99
MCH 18.8 27-32
RDW 23.8 11.5-15.0
Hb A 89.3 >95
Hb A2 1.0 1.7-3.1
Hb H
& A1c
7.9
Hb
Bart’s
1.8
Bilirubin Masquerading as Barts
Bilirubin
Howanitz et al. AJCP 2006;125:608-14
Hb H Disease with Hb H and Barts
Hb Barts Hb A2
Hb A
Hb H
95-97
2.2-3.2
16.5
0.7
82.2
0.6
Hb H
Hb Bart’s
Hb A
Hb A2
Fractions % Ref. %
36 y/o woman
sickledex positive
RBC 4.67 3.9-5.0
Hgb 11.8 12.0-16.0
Hct 35.1 36-48
MCV 75.2 79-99
MCH 25.4 27-32
RDW 15.0 11.5-15.0
Hb A 73.0 >95
Hb A2 4.3 1.7-3.1
Hb S 32.7 35-40*
*Expected for Sickle Trait
Beta globin products in
Thalassemia with & without Hb S
Normal b Thal Hb S Trait Hb S/a Thal Hb S/b Thal
24 y/o woman
Hb A
Rel Rt = A 1.17 Hb A2 = 1.4%
?
RBC 5.10 3.9-5.0
Hgb 13.8 12.0-16.0
Hct 40.4 36-48
MCV 78.2 79-99
MCH 27.1 27-32
RDW 13.6 11.5-15.0
Hb E 1.17-1.26
Hb E trait
Hb E Hb A2
Hb A
RBC 5.10 3.9-5.0
Hgb 13.8 12.0-16.0
Hct 40.4 36-48
MCV 78.2 79-99
MCH 27.1 27-32
RDW 13.6 11.5-15.0
Hb A 74.1 >95
Hb E&A2 22.9
Hb F 2.0 <2.0
Capillarys separates Hb E & Hb A2
Hb E
Hb A2
Hb A
Hb F
95-97
<2.0
2.2-3.2
71.0
1.9
23.8
3.3
Hb A
Hb F
Hb E
Hb A2
Fractions % Ref. %
Hb A 71 >95
Hb A2 3.3 2.2-3.2
Hb E 23.8
Hb F 1.9 <2.0
Hb E = b26glu val
Homozygotes and heterozygotes are clinically
well with mild microcytosis
The mutation activates a cryptic splice site in
Exon 1 in the beta globin gene producing a
mild b-Thalassemia
Hb E/b0Thalassemia patients are anemic (may
be as severe as Thalassemia Major) with
elevated Hb F 40% or higher
BioRad I HPLC
Hb E Homozygote
64 y/o woman
RBC 5.36 3.9-5.0
Hgb 11.4 12.0-16.0
Hct 34.8 36-48
MCV 70.5 79-99
MCH 20.9 27-32
RDW 14.6 11.5-15.0
Hb A 0 >95
Hb E &A2 95.8
Hb F 4.2 <2.0
Hb E
Hb A2
Hb F
CZE on Hb E Homozygote
Increased Hb A2
is consistent with
the b Thalassemia
seen in Hb E
Hb F 4.8 <2.0
Breakdown 1.4 NA
Hb E 89.8 0
Hb A2 4.0 2.2-3.2
Hb A2 4.0 2.2-3.2
Hb E 91.2
Hb F 4.8 <2.0
Table from Steinberg et al. Disorders of Hemoglobin, Ch 43, 2001
UM Hb E/E 11.4 70.5
Hb A2
Hb F
Hb E
Hb E/b0 Thalassemia
Hb A2 4.3 2.2-3.2
Hb E 46.5
Hb F 49.2 <2.0
Mix 1:1 with Control to see Zones
Hb F
Hb A2
Hb A
Hb E
Table from Steinberg et al. Disorders of Hemoglobin, Ch 43, 2001
UM Hb E-bo 7.1 73.5
Technique Comparison
Gels
Fair
Straightforward
Poor at low level
No interference
Cannot separate
No interference
Fair
HPLC
Excellent
Complex
Excellent
Adduct issue
Some separate
Interferes*
Excellent
Capillary
Excellent
Straightforward
Excellent
No interference
Separates
No Interference
Excellent
Parameter
Automation
Interpretation
Hb A2 Measure
Hb A2 & Hb S
Hb A2 & Hb E
Bilirubin/Barts
Separating Hbs
*Prewashing of the RBCs removes the interference
