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G-6-PDH (5 + 1 Reagent)

"liquid"
Quantitative Enzymatic Determination
of Glucose-6-phosphate Dehydrogenase
in Whole Blood
Cat.No.
143 100

ASSAY PROCEDURE ANALYZERS

Package Size
4 x 50 mL R1 / 2 x 20 mL R2

incl. Calibrator

TEST PRINCIPLE
G6PDH catalyzes the oxidation of Glucose-6-phosphate (G-6-P)
to 6-phosphogluconate (6-PG) and reduction of NADP to
NADPH. The increase of absorbance of NADPH is proportional
to the activity of the G-6-PDH in the sample and is measured
kinetically.

Ask for individual procedures for special Analyzers:


The following procedure describes the basic parameters.
Note the use of Lysing Reagent see Note 3
--------------------------------------------------------------------------------------- Wavelength:
Cuvette:
Reading:
Temperature:

340 nm (second 700nm)


1 cm
against air or dist. water
37C

REAGENTS
Components and concentrations (in the test) :
Goods Buffer
G-6-P
NADP
Activators, Stabilizers

>20 mmol/L
>0.1 g/L
>0.19 mmol/L

Reagent R1 and Reagent R2 are ready to use


Stability:
R1 and R2, stored at 2-8C, in the original vials, are stable up to
the expiry date printed on the labels, provided that contamination
is avoided.
After opening R1 and R2 are at 2-8C stable for at least 4weeks,
if contamination is avoided
Close vials immediately after use in order to avoid any
contamination!

Cal/Con
Reagent R1
200 L
Calibrator/Control
8 L
Sample
---Mix carefully and incubate 3 to 5 min at 37C
Reagent 2
40 L
After 4 min read A1 of Cal/Con and Samples.
Repeat readings 2 - 5 min later for A2.

S
200 L
---8 L
40 L

CALCULATION
G6PDH (U/L 37C) =

As/min
Cal. Value x ----------------------ACAL/min

Before testing G6PDH determine (A) or (B) :


Calibrator (values see the vial-labels)
Dissolve the lyophilized materials by adding 1ml of dist. water to
the lyophilisate vials.
Stability:
Stored at 2-8C : up to 1 week
Stored at < -20C: at least 1 month:

SAMPLES
Whole blood collected with EDTA, heparine or ACD (AcidCitrate-Dextrose), is diluted 1+9 with G6PDH lysing reagent.
Mix well and use immediately.
Red cell G6PDH is stable in whole blood for 1 week at 2-8C,
but unstable in Red Cell hemolysate.
Freezing of blood is not recommended (see 2,4)

ASSAY PROCEDURE MANUAL

(A) number of erythrocytes (RBC) expressing G-6-PDH


activity as
U/10^12 erythrocytes (RBC), or
(B) conc. of Hemoglobin in g/dL, expressing G-6-PDH
activity as
U/g of Hemoglobin

NOTES
- The most accurate red cell counts are received with erythrocytes in ADC, due to increased stability of erythocytes in that
medium .
- For heparin samples best results are reported in terms of
hemoglobin concentration .

CALCULATION
G6PDH (U/10^12 RBC)

Wavelength:
340 nm
Cuvette:
1 cm
Reading:
against air or dist. water
Temperature:
37C
Let reagent reach working temperature
Pipette into test tubes or cuvettes:
S = Sample, Cal = Calibrator, Con = Control :
Cal/Con
S
Reagent R1
1000 L
1000 L
Calibrator/Control
40 L
---Sample (RBC hemolysate)
---40 L
Mix carefully and incubate 3 to 5 min at 37C, then add
Reagent 2
200 L
200 L
Mix well, and after 4 min read A1 of Cal/Con and Samples.
Repeat readings exactly 3 min later for A2.
Determine Absorbance / min (A/min) for Calibrator, Control
and Samples , calculating

As/min x

Cal. Value
-------------------ACAL/min x N

N = Red Cell Count divided by 10^6

or
Cal. Value
G6PDH (U/g Hemoglobin) = As/min x --------------------------ACAL/min x Hb (g/dL)
Hb (g/dL) = Hemoglobin conc. determined for each sample

REFERENCE VALUES
Activity at 37C
U / 1012 RBC
221 570
U / g Hb
7.0 20.5
Note 1 : Values for Newborns can be somewhat higher
Note 2 : Each laboratory should establish its own ref.values!

(SECOND readings FIRST readings) / 3

Greiner Diagnostic GmbH - Unter Gereuth 10 D-79353 Bahlingen Germany


www.greiner-diagnostic.com

NOTES
1. Attention must be given to interfering substances: copper and
sulfate are strong inhibitors, other substances are able to
influence levels of G6PDH
2. Reticulocytes have higher G-6-PDH levels than mature Red
Cells; it is not recommended to run the assay after a severe
hemolytic crisis, since G-6-PDH may appear falsely elevated.
3. As supplementary reagent Greiner offers
G6PDH-Lysing Reagent

SYMBOLS USED
For in vitro diagnostic medical use

Batch Code

Use by

BIBLIOGRAPHY
1. Beutler E. et al., Brit. J. Haem. 43, 469 (1979)
2. Stiene EA, Am.J.Med.Tech. 38, 454 (1972)
3. Young D.S. et al., Clin. Chem. 21, 302D (1975)
4. Textbook of Clinical Chemistry, Ed. by N.W. Tietz,
W.B. Saunders Co., Philadelphia (1999).
5. Lowe M.L. et al., Clin. Chem. 18,440 (1972)
6. Kornberg A., et al., Methods in Enzymology 323 (1955)

Temperature limitation

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Greiner Diagnostic GmbH - Unter Gereuth 10 D-79353 Bahlingen Germany


www.greiner-diagnostic.com