Unraveling Hemoglobinopathies with

Capillary Electrophoresis

David F. Keren, M.D.

Professor of Pathology

Division Director, Clinical Pathology

The University of Michigan

dkeren@med.umich.edu

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

• http://globin.bx.psu.edu/html/huisman/variants/