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ANALIS

HS CEofix™ HbA1c kit for Beckman Coulter P/ACE™ 5000 series - GB: Instructions for use

QUOTATION
INFORMATION
QUOTATION
INFORMATION
DESCRIPTION
DESCRIPTION

100 tests per kit

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

figure 1

2. Intended use:

The kit is intended for the quantitative determination of human hemoglobin A1C (HbA1c). It can also been used for the determination of some hemoglobin variants in combination with CEofix™ HbA2 kit (PN 10-004190/844111026) ).


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:

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

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

Initiator:1x 2.5 mL Malic acid pH 4.6 (PN 10-004184)

Buffer:4 x 4.2 mL Malic acid pH 4.6 (PN 10-004182)

Hemolyser:1 x 5 mL(PN 10-004185)

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 17cm to the detector (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 hemolysate is the preferred specimen.

10. Instrument preparation:

Load the instrument as follow

figure 2

Instrument programming

figure 3

Or download program: HbA1c.met

Mounting of capillary:

figure 4

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 5

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  HbA1C.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:

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


16. Imprecision:

Imprecision

HbA1c Low (4.3%) Medium (7.0%) High (10.5%)
Within-assay (N=20) %CV 1.7 2.9 1.4
Between-run (N=20 days) %CV 3.7 3.3 1.9

Correlation

figure 6

CE HbA1c = -1.41 + 1.02 HPLC HbA1c (r = 0.98) N = 100

Doelman et al.: Clinical Chemistry 43, No. 4, 1997

17. Reference range:

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

figure 7

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

2002;979:201-7

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

1997;43:1811-13

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

1998;36:299-308

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

1994;40:2288-95

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

1983;29:466-9

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

1989;35:33-6

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-

23

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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