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CEofix™ HbA1c kit for Beckman Coulter P/ACE™ MDQ - GB: Instructions for use


series 400 tests

EURO PN 844111042 PN 10-004700

100 tests

EURO PN A08251 PN 10-004730

1. Date: 26-10-04 - Revision: 01

figure 1

2. Intended use:

The kit is intended for the quantitative determination of human hemoglobin A1c (HbA1c). For hemoglobin variants the kit should be used in combination with CEofix™ HbA2 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 % HbA1c is defined as the percent of the HbA1c fraction in relation to the sum of all hemoglobin fractions.

4. Content:
Conditioner: 1x60mL NaOH 0.2 M (PN 10-004701)
Initiator: 1x 25 mL Malic acid, Arginine pH 4.6 (PN 10-004704)
Buffer: 3 x 25 mL Malic acid, Arginine pH 4.6 (PN 10-004702)
Hemolyser: 400: 2x 50 mL Malic acid, Arginine pH 5.9 (PN 10-004705)
100: 1x 25 mL Malic acid, Arginine pH 5.9 (PN 10-004735)
Post-Injection: 1 x 60 mL Malic acid, Arginine pH 5.8 (PN 10-004707)

5. Configuration of instrument:

-Beckman Coulter P/ACE MDQ

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

- Sample cooling option

-P/ACE 32 Karat software version 5.0

(use configuration without CAESAR integration)

-Large volume buffer reservoir kit (PN 144824)


6. Other material needed:

- Vials 2 mL (PN 144980)

- Bi-distilled water

- caps (PN 144648)

- microtiterplate

- Blank Cartridge assembly 100 x 800 µm (PN 144738)

- Pipettes

- Capillary 25 µM ID x 10 cm to the detector 31 cm total length (PN 10-004748/844111023)

- Filter 415nm (PN 10-004709)

(Note: injection and separation is performed at the short end side.)

7. Training:

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

8. Reagent preparation and storage:

Reagents are ready to use. Store kit and buffers, in closed container, between 18°C and 26°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 hemolysate is the preferred specimen.



10. Instrument preparation:

Load the instrument as follow

figure 2Instrument programming

figure 3

Or download program: HbA1c_mdq.met. Burn the capillary ends to eliminate the polyimide coating on 2mm.

11. Sample preparation: Sample: 240 µL hemolyser + 80 µL of well-mixed whole blood in a microtiterplate 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  cond.met when system was down for several days - prepare samples - place sample in instrument - run  HbA1c_mdq.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  cond.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: See table for position of fractions.

15. Calculation:

% HbA1c = corrected area of HbA1c fraction in relation with the sum of corrected areas of all hemoglobin fractions.

16. Imprecision:

Imprecision: Three samples Low, Medium and H High were run in duplicate twice a day over 3 days in a sequence.

N=12 Low Medium High
Mean HbA1c% 2.64 6.39 9.40
% CV 2.97 2.68 3.89

Reference: NCCLS EP5 Preliminary precision.

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

figure 5

18. Interference:

-.The presence of hemoglobin variants will limit the determination of HbA1c.

- For determination of hemoglobin variants the kit should be used in combination with CEofix HbA2 kit.


19. Quality Control:

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

20. References:

1. Doelman C, Siebelder C, Nijhof W, Weykamp C, Janssens J, Penders T. Capillary electrophoresis system for

hemoglobin A1c determinations evaluated. Clin Chem 1997;43:644-8

2. Siren H, Laitinen P, Turpeinen U, Karppinen P. Direct monitoring of glycohemoglobin A1c in the blood samples of

diabetic patients by capillary electrophoresis, comparison with an immunoassay method. J of Chromatogr A


3. Weykamp C, Miedema K, de Haan T, Doelman C. Carbamylated Hemoglobin Interference in Glycohemoglobin

Assays. Clin Chem 1999;45:438-40

Selected Bibliography

1. Abraham E C. Glycosylated hemoglobins, methods of analysis and clinical applications. Marcel Dekker Inc 1985

2. Allen D W et al. Observations on the chromatographic heterogeneity of normal adult and fetal human

hemoglobin. J Am Chem Soc 1958;80:1628-34

3. Bissé E, Huaman-Guillen P, Wieland H. Chromatographic evaluation of minor hemoglobins: clinical significance of

hemoglobin A1d, comparison with hemoglobin A1c, and possible interferences. Clin Chem 1995;41:658-63

4. Eckfeldt J, Bruns D. Another Step Toward Standardization of Methods for Measuring HbA1c . Clin Chem


5. Finke A, Kobold U, Hoelzel W, Weycamp C, Miedema K, Jeppsson J-O. Preparation of a Candidate Primary

Reference Material for the International Standardisation of HbA1c Determinations. Clin Chem Lab Med


6. Goldstein D, Little R et al. Is glycohemoglobin testing useful in diabetes mellitus? Lessons from the Diabetes

Control and Complications Trial. Clin Chem 1994;40:1637-40

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


8. Kobold U, Jeppsson J-O, Dülffer T, Finke A, Hoelzel W, Miedema K. Candidate reference methods for hemoglobin

A1c based on peptide mapping. Clin Chem 1997;43:1944-51

9. Kwan J, Carr E, Bending M, Barron J. Determination of carbamylated hemoglobin by high-performance liquid

chromatography. Clin Chem 1990;36:607-10

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

Hematology, Ninth Edition 1993;1:1023-145

11. Molteni S, Frischknecht H, Thormann W. Application of dynamic capillary isoelectric focusing to the analysis of

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

12. Mosca A et al. Commutability of control materials in glycohemoglobin determinations. Clin Chem 1998;44:632-8

13. Nathan D, Francis T, Palmer J. Effect of aspirin on determinations of glycosylated hemoglobin. Clin Chem


14. Tarif et al. Carbamylated hemoglobin in hemodialysis patients. Am J Kidney Diseases 1997;3:361-5

15. Turpeinen U, Stenman U, Roine R. Liquid-chromatographic determination of acetylated hemoglobin. Clin Chem


16. 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-


17. Weykamp C, Penders T, Siebelder C, Muskiet F, van der Slik W. Interference of carbamylated and acetylated

hemoglobins in assays of glycohemoglobin by HPLC, electrophoresis, affinity chromatography and enzyme

immunoassay. Clin Chem 1993;39:138-42

18. Weykamp C, Penders T, Baadenhuijsen H, Muskiet F, Martina W, van der Slik W. Vitamin C and Glycohemoglobin.

Clin Chem 1995;41:713-6


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