Hb electrophoresis by CE

Samples processed on CE and HPLC

Heterozygous A/C

Hb A2

Hb A Hb C

N

Anh. Car.

A

C+A2

Alc.

N

A0

A1A1

C

A0

Ac.

Heterozygous A/D-PunjabAFSC control overlaying

Hb A Hb D

Hb A2

Anh. Car.

A

A2

Alc.

N

D

A0

A1

Ac.

N

D1+A1

D0+A0

Heterozygous A/E

Hb A

Hb A2

Hb E

Anh. Car.

A

E+A2

Alc.

N

A0

A1A1+E1

Ac.

N

A0+E

Heterozygous S/C

S

N

C

Alc.

Anh. Car.

N

Ac.

A0

A1F(traces)

SC

AFSC control overlaying

Hb CHb S

Hb F

Hb A2

Beta thalassemia

Hb A

Hb A2

Anarh. C.

A0+A2A1

Alk. Ac.

N N

A2

A

Alpha thalassemia with Hb H

Hb A

Hb A2Hb H

Hb Bart’s (Baby’s blood)

Hb A

Hb F

Hb Bart’s

Alk.

N

A

Anh. Car.

H

A2

Alk Ac

Hb Bart’s

Hb Bart’sHb F

Hb F

Alpha Globin mutation

300 points with 15 zones Curve

Hemoglobin Analysis by Capillary2 Electrophoresis (Sebia)

Thalassemia Research Center

Institute of Science and Technology for Research and Development 

Mahidol University 

Nakhonpathom, Thailand.

Wantana C. Artittaya I. Thongperm M.

Oct 31’ 2006

Hb type Cases

Normal 45

Beta-thal heterozygote 39

Hb E- heterozygote 45

Hb E- heterozygote/ Alpha thal-1 heterozygote ๒๘

Hb E- heterozygote/ Hb H disease (EA Bart’s disease) ๑๔

Beta-thal/HbE disease ๓๕

Hb H disease 25

Hb H-CS disease ๙

Hb CS homozygote 8

Hb CS heterozygote 6

Alpha thal-1 heterozygote 36

Alpha thal-2 heterozygote 30

Total Samples Analyses

Hb CS : Hb Constant Spring (wait for DNA Analysis 20 cases)

Normal Subject

Alpha thal 1 heterozygote

Alpha Thal2 heterozygote

Beta thal heterozygote

Hb E heterozygote

Hb E heterozygote/ alpha thal1 heterozygote (‐‐SEA type)

Homozygous Hb E

Beta thal/E Disease

Hb H –CS Disease

CSEA Bart’s Disease

Hb Constant Spring heterozygote

Hb Constant Spring homozygote

Normal E trait Homo E b-trait Beta/E

Genotype

2.0

4.0

6.0

8.0

% Hb

A2_

Sebia

HbA2 levels in cases with beta‐thalassemia disorders

Normal, Beta‐Trait, Alpha1‐Trait, Alpha2‐Trait

E‐Trait

Levels of HbA in normal and all heterozygotes

Normal, Beta‐Trait, Alpha1‐Trait, Alpha2‐Trait

Hb E ‐Trait

HbF levels in normal and thalassemia‐trait

Levels of HbF in all thalassemic diseases

Hb H‐Disease, Hb H‐CS Disease

Homo E

Beta/E‐Disease

HbA2 and HbE in normal and thalassemia‐trait

E‐Trait

Beta‐Trait

Normal, Alpha1‐Trait, Alpha2‐Trait

HbA2 and Hb E in all disease cases

Homo E

Beta/E‐Disease

Hb H Disease, Hb H‐CS Disease

Cord Blood

CAPILLARYS / HPLC 

COMPARISON

PATTERN

CAPILLARYS HPLC

Clarity of the pattern +++ -

Normal sample

HPLC CAPILLARYS

GLYCATED FORMS

CAPILLARYS HPLC

Glycated formsThey are included with

the correspondingHemoglobin

All glycated fractions are separated

Interferences of glycated forms

No interferences for the quantification

Interference of glycated HbS on HbA2

value

Peak quantification

More accurate.All subfractions are included in the main

peak

All main peaks do not include the glycated

forms. (Normally all subfractions have to be included

in each corresponding main peak).

VALUES

CAPILLARYS HPLC

HbF value on normal samples

< 0.5 %

Higher than CAPILLARYS value due to glycated form

of HbA

HbA2 on normal samples = =

HbE Separated from HbA2Not separated from

HbA2.

Heterozygous HbE + heterozygous Constant Spring

HPLC CAPILLARYS

VARIANTS, Hb BART’s & HbH

CAPILLARYS(more than 50 variants

identified)

HPLC(about 45 variants

identified)

Main variants All detected. All detected

Rare variantsPossible

superimposition withthe main peaks

Possible superimposition with

the main peaks

Constant Spring Well detected Sometimes difficult to detect

Hb Bart’sand HbH

Detected and quantified

Detected but not quantified due to overevaluation by

glycated form of HbA

Hb H –CS Disease

CHARACTERISTICS

CAPILLARYS HPLC

Throughput 34 samples / hour 10 samples / hour

Primary tubeYes,

after sedimentationand plasma removal.

Not possible to test whole blood

Yes,on whole blood

Plasma Protein interferences

No, analysis on red blood cells only

Possible interferences, eg

Bilirubin at the level of Hb Bart’s

Reproducibility Very Good Very Good

Cap Piercing No Yes

Case comparison between LPLC, HPLC and CE

Meditop – Sebia Distributor MeetingBangkok – November 2007

Based on our experience in Thailand, we can say that the advantages of Capillary Electrophoresis (CE) vs Low Pressure Liquid Chromatography (LPLC) and High Performance Liquid Chromatography (HPLC) are:

1. CE can separate and quantify the various types of alpha-thalassemia and beta-thalassemia: it gives % Hb H, % Hb Bart’s and % Hb CS.

LPLC and HPLC cannot separate Hb H from Hb Bart’sand cannot show their percentage, so they can be used only to analyse Beta-thalassemia.

Chromatogram of EA Bart’s diseaseLPLC vs CE

Chromatogram of Beta thal / Hb E diseaseLPLC vs CE

Chromatogram of Beta thal / Hb E diseaseHPLC vs CE

Chromatogram of Hb H diseaseLPLC vs CE

Chromatogram of Hb H disease (case with blood transfusion history) HPLC vs CE

Chromatogram of Hb Bart’s hydrop fetalisHPLC vs CE

Chromatogram of Hb H disease (newborn)HPLC vs CE

2. CE can separate exactly Hb E from Hb A2 and allows to quantify the percentage of Hb E, which is important in case of blood transfusion.

CE can distinguish Homozygous Hb E disease from Beta-thalassemia with Hb Edisease, which is especially useful in cord blood analysis, whereas LPLC and HPLC cannot separate Hb E from Hb A2 nor quantify them.

Chromatogram separating Hb E and Hb A2 (HPLC vs CE)

Chromatogram separating Hb E & Hb A2 (case with blood transfusion history) (LPLC vs CE)

3. CE can distinguish between Hb A๒ and other Hb A๒ variants as well as quantify their concentrations, whereas LPLC and HPLC cannot always either separate or quantify them.

LPLC and HPLC can show misleading results: a high Hb A2 concentration in Alpha and Delta-thalassemia (actually normal Beta-globin gene) leads to a false positive of Beta-thalassemia.

Chromatogram separating Hb A2 variants(HPLC vs CE)

4. CE has high sensitivity and specificity: it detects Hb Constant Spring as effectively as DNA analysis.

CE can quantify Hb CS even at concentrations of less than ๐.๑%, whereas HPLC and LPLC cannot quantify it at all.

Chromatogram of Hb CS heterozygoteLPLC vs CE

Chromatogram of Hb E trait / Hb CSHPLC vs CE

5. Using CE, the slight difference of migration between Hb S and Hb D allows the user to easily distinguish between them by overlaying the electropherogram with a reference curve memorized in the CE software.

Chromatogram of Hb S heterozygoteLPLC vs CE

6. CE throughput is 34 samples/hr for Hb typing, whereas LPLC throughput is 6 samples/hr and HPLC throughput is 9 samples/hr. So, CE analyzer does the work of 6 LPLC analyzers or 4 HPLC analyzers, and does it much better.

Capillarys II allows:

-Throughput of up to 34 samples/hr for Hb typing

-up to 150,000 graphs and results stored on the hard disk

-Quality control features (L-J chart, statistics)

- Bi-directional and network capabilities

-Customized reports

Feature comparison between Capillarys II, Hb Gold, Variant I and Variant II

Features Capillarys II Hb Gold Variant I Variant II

Throughput 34 samples/hr

6 samples/hr

9 samples/hr

9 samples/hr

Data storage capacity

150,000 samples

99 samples No storagecapacity

Previous &Current sample

Ability to separate & quantify Hb E and Hb A2

Yes No No No

Ability to quantify Hb H, Hb Bart’s & Hb CS

Yes No No No

Feature comparison between Capillarys II, Hb Gold, Variant I and Variant II

Features Capillarys II Hb Gold Variant I Variant II

Q.C., L-J chart, statistics

Yes No No No

Analytical capability

1.Protein electrophoresis2.Immunotyping3.CDT (Carbohydrate deficient transferrin)4.HR (High resolution protein electrophoresis)

HbA1C HbA1C HbA1C

Network capabilities

Yes No No No