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ANALIS

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

QUOTATION
INFORMATION
QUOTATION
INFORMATION
DESCRIPTION DOWNLOADS
DESCRIPTION

100 tests per kit

1. Date: 17-Dec-03 - Revision: 01

figure 1

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:

For Lot 050315 and higher

Rinse:
1 x 45 mL aqua bidistillata (PN 10-004713)
Conditioner:
1x 20 mL NaOH 0.2 M (PN 10-004711)
Initiator:
1x 15 mL Malic acid, Arginine pH 4.6 (PN 10-004714)
Buffer:
1x45 mL Taurine/Arginine pH 8.9 (PN 10-004712)
Hemolyser:
1 x 15 mL Taurine/Arginine pH 9.0 (PN 10-004715)

For Lot 031205

Rinse:
1 x 10 mL aqua bidistillata (PN 10-004713)
Conditioner:
1x 10 mL NaOH 0.2 M (PN 10-004711)
Initiator:
1x 3 mL Malic acid, Arginine pH 4.6 (PN 10-004714)
Buffer:
1x10 mL Taurine/Arginine pH 8.9 (PN 10-004712)
Hemolyser:
1 x 15 mL Taurine/Arginine pH 9.0 (PN 10-004715)

5. Configuration of instrument:


- BeckmanCoulter 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)

6. Other material needed:


- Vials 2 mL (PN 144980)

  • 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)


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 8°C and 30°C until expiration date.

Put every day fresh rinse, conditioner, initiator and buffer in clean vials.

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 2

Instrument programming

figure 3

Or download program: HbA2_meth_96well_plate.met

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

11. Sample preparation:

Sample: 150 µL hemolyser + 30 µL of well-mixed whole blood in a microtiterplate and mix.

figure 4

12. Operations:

Every day operation:

  • clean instrument and interface manifold
  • put rinse, conditioner, initiator buffer vials
  • put clean and dry caps on buffer vials
  • run cond.met
    when system was down for several days

figure 5

- prepare samples

- place sample in instrument

- run  HbA2_meth_96well_plate.met method

End of day:

- remove samples

- remove 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.

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 Migration Time HbA2
mean: 6.08 min
Migration Time HbA
mean: 6.60
% HbA2
mean 2.82
Within-run assay (N=46) %CV

0.74 0.73 3.34

17. Reference range:

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

figure 6

HbF < 1%

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
1999;45:237-43
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
Bibliography
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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