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HS CEofix™ HbA2 kit for Beckman Coulter P/ACE™ 5000 series - GB: Instructions for use


100 tests per kit

1. Date: 7-May-03-Revision: 01

2. Intended use:

The kit is intended for the quantitative determination of human hemoglobin A2 (HbA2), determination of hemoglobin F and hemoglobin variants. For hemoglobin variants the kit should be used in combination with CEofix™ HbA1c kit.

3. Principle of the test:

The hemoglobins are separated inside a capillary under the influence of an electrical field. Differences in charge to mass ratio give different migration times.

A UV detector is used and detects the hemoglobins at 415nm.

The kit consists of buffers and method. The buffers use the patented dynamic double coating technique.

The % HbA2 is defined as the percent of the HbA2 fraction in relation to the sum of all hemoglobin fractions.

4. Content:

Rinse:1x100mL aqua bidistallata (PN 10-004183)

Conditioner:1x100mL NaOH 0.2 M (PN 10-004181)

Initiator:1x 2.5 mL Aqueous solution (PN 10-004194)

Buffer:4 x 4.2 mL Taurine Arginine pH 8.9 (PN 10-004192)

Hemolyser:1 x 5.2 mLArginine (PN 10-004195)

Micro vials:130

5. Configuration of instrument:

-BeckmanCoulter P/ACE 5000

-UV detector with 415 nm filter (PN: 10-004709)

-P/ACE station software version 1.0.

6. Other material needed:

- Vials (PN 358807)

- caps (PN 359079)

- spring (PN338488)

- Cartridge 100x800 µm ( PN 727610)

- Pipettes

- Capillary 25µM ID x 20cm to the detector, 31 cm total length (PN 10-004188/844111023)

7. Training:

The operator should be familiar with instrument (P/ACE 5000) operation and maintenance.

8. Reagent preparation and storage:

Reagents are ready to use. Store kit and buffers, in closed container, between 8°C and 30°C until expiration date.

Between operation buffer vials should be capped to avoid evaporation.

If the kit arrived damaged a replacement of the kit should be requested.

The kit contains no hazardous components.

9. Type of specimen:

Biological fluids should be collected in the manner normally used for any clinical laboratory test.

Whole blood is the preferred specimen.

10. Instrument preparation:

Load the instrument as follow

figure 1

Instrument programming

figure 2

Or download program HbA2.met

Mounting of capillary:

figure 3

Burn the capillary ends to eliminate the polyimide coating on 2mm.

11. Sample preparation:

Sample: 50 µL hemolyser + 10 µL of well-mixed whole blood in a microvial and mix.

figure 4

12. Operations:

Every day operation:

- clean instrument and interface manifold

- put new rinse, conditioner, initiator buffer vials- put new waste vial - put clean and dry caps on buffer vials

- run  cond1.met when system was down for several days

- prepare samples

- place sample in instrument

- run  HbA2.met method

End of day:

- remove samples

- cap buffer vials

Placing new capillary

-When the capillary is broken or when the capillary is not operating properly replace the capillary: according the instrument manual. -run  cond1.met for conditioning

13. Calibration:

No calibration is required for the kit.

Instrument should be maintained and calibrated according to Beckman Coulter specifications.

14. Analysis of results:

Click to see examples.

15. Calculation:

% HbA2 = corrected area of HbA2 fraction in relation with the sum of corrected areas of all hemoglobin fractions.

16. Imprecision:


HbA2 HbA2 (2.9%) MT A2 (4.7 min)
Within-assay (N=20) %CV 1.4 2.3
Between-run (N=20 days) %CV 1.8 2.9

Analis May 1998


figure 5

Fig. Comparison of Hb A2 quantification.
CZE and MAEC results (n 5 57); ( B), CZE and HPLC results (n 5 57)
Hb A2 CZE (%) = 1.233 Hb A2 HPLC - 0.2

figure 6

Fig. Comparison of Hb F values obtained with HPLC and CZE.
Hb FCZE (%) = 1.118 Hb FHPLC + 0.4

F. Cotton et al.: Clinical Chemistry 45, No. 2, 1999

17. Reference range:

Each laboratory should establish its own reference range to conform to patient population.


figure 7


figure 8

18. Interference:

- The presence of hemoglobin variants will limit the determination of HbA2.

-For determination of hemoglobin variants the kit should be used in combination with CEofix HbA1c kit.

19. Quality Control:

Pooled control or commercially available control should be included in each run.

20. References:

1 Cotton F, Lin C, Fontaine B, Gulbis B, Janssens J, Vertongen F. Evaluation of a Capillary

Electrophoresis Method for Routine Determination of Hemoglobins A2 and F. Clin Chem


2 Lin C, Cotton F, Fontaine B, Gulbis B, Janssens J, Vertongen F. Capillary zone electrophoresis: an

additional technique for identification of hemoglobin variants. Hemoglobin 1999;23:97-109

3 Gerritsma J, Sinnige D, Drieze C, Sittrop B, Houtsma P, Hulshorst-Jansen N and Huisman W.

Quantitative and qualitative analysis of haemoglobin variants using capillary zone

electrophoresis. Ann Clin Biochem 2000;37:380-9

4 Mario N, Baudin B, Bruneel A, Janssens J, Vaubourdolle M. Capillary Zone Electrophoresis for

the Diagnosis of Congenital Hemoglobinopathies. Clin Chem 1999;45:285-8


1 Castagnola M, Messana I, Cassiano L, Rabino R, Rossetti D, Giardina B. The use of capillary

electrophoresis for the determination of hemoglobin variants. Electrophoresis 1995;16:1492-8

2 Hempe J, Craver R. Quantification of hemoglobin variants by capillary isoelectric focusing. Clin

Chem 1994;40:2288-95

3 Hempe J, Granger J, Craver R. Capillary isoelectric focusing of hemoglobin variants in the

pediatric clinical laboratory. Electrophoresis 1997;18:1785-95

4 Hempe J, Granger J, Warrier R, Craver R. Analysis of hemoglobin variants by capillary

isoelectric focusing. J Cap Elec 1997;4:131-5

5 Hempe J, Craver R. Separation of hemoglobin variants with similar charge by capillary

isoelectric focusing : Value of isoelectric point for identification of common and uncommon

hemoglobin variants. Electrophoresis 2000;21:743-48

6 Jenkins M, Hendy J, Smith I. Evaluation of hemoglobin A2 quantification assay and hemoglobin

variant screening by capillary electrophoresis. J Cap Elec 1997;004:137-43

7 Landers J. Clinical capillary electrophoresis. Clin Chem 1995;41:495-509

8 Lin C, Gulbis B, Delobbe E, Cotton F, Vertongen F. Separation of human globin chains by

micellar electrokinetic capillary chromatography. J Chromatogr B 1998;719:47-54

9 Lukens J, Lee R. Hereditary disorders affecting hemoglobin structure and synthesis. Wintrobe's

Clinical Hematology, Ninth Edition 1993;1:1023-145

10 Mario N, Baudin B, Aussel C, Giboudeau J. Capillary isoelectric focusing and high-performance

cation-exchange chromatography compared for qualitative and quantitative analysis of

hemoglobin variants. Clin Chem 1997;43:2137-42

11 Mohammad A et al. Clinical application of capillary isoelectric focusing on fused silica capillary

for determination of hemoglobin variants. Clin Chem 1997;43:1798-9

12 Molteni S, Frischknecht H, Thormann W. Application of dynamic capillary isoelectric focusing to

the analysis of human hemoglobin variants. Electrophoresis 1994;15:22-30

13 Oda R, Clark R, Katzmann J, Landers J. Capillary electrophoresis as a clinical tool for the

analysis of protein in serum and other body fluids. Electrophoresis 1997;18:1715-23

14 Ong C, Liau L, Ong H. Separation of globins using free zone capillary electrophoresis. J

Chromatogr 1992;576:346-50

15 Shihabi Z, Hinsdale M, Daugherty H. Hemoglobin A2 quantification by capillary zone

electrophoresis. Electrophoresis 2000;21:749-52

16 Sugano M et al. Analysis of hemoglobin and globin chain variants by a commonly used capillary

isoelectric focusing method. Electrophoresis 2000;21:3016-9.

17 Weykamp C, Penders T, Muskiet F, van der Slik W. Influence of hemoglobin variants and

derivatives on glycohemoglobin determinations, as investigated by 102 laboratories using 16

methods. Clin Chem 1993;39:1717-23

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