Prevention is better than Treatment

R&D DIAGNOSTICS LTD 
SQ
MMR500 & SQMMR1250

INSTRUCTIONS FOR USE (PACKAGE INSERT)

A SCREENING KIT FOR DRIED BLOOD SPOTS & WHOLE BLOOD SAMPLES
This kit is particularly suitable for screening for G-6-pd deficiency in newborns.
KIT SIZE: 10 x 50 TESTS & 5 x 250 TESTS

Manufactured by :
R&D  DIAGNOSTICS LTD.
41, El. Venizelou str.,
15561 Holargos
Greece
www.rddiagnostics.com

Catalog No : SQMMR500 & SQMMR1250

Procedure
The assay procedure is according to reaction described by Beutler (1,2).
The enzyme determined is glucose-6-phosphate dehydrogenase which is abbreviated either as G-6-PD or G-6-PDH.

Test principle
                                            G-6-PD
Glucose-6-P + NADP+   ------->   gluconate-6-P + NADPH + H+
The NADPH produced in the reaction fluoresces under long-wave UV-light. If there is a marked deficiency of this enzyme, or if G-6-PDH is lacking entirely, no fluorescence will be observed.

Sample material
See noted # 1 and 2.

Apply a drop of blood to absorbent paper ("Gurthie Test Paper", Schleicher & Schuell No. 2992) and let dry completely (atable for one week at 20-25oC).

Whole blood may be used instead of dried blood. Heparin, citrate, oxalate and EDTA are suitable anticoagulants. The blood specimens are stable for seven days at most. Use 0,005 ml (5 microliters) for the assay.

Reagents
Contents of solution  Concentration in the test
Glucose-6-phosphate  1 mmol/l
NADP    0,75 mmol/l
GSSG (oxidized glutathione)  0,8 mmol/l
Saponin    0,2%
Tris(hydroxymethyl)-
aminomethane   225 mmol/l, pH 7.8

Preparation and stability of reagent solution
Dissolve the contents of the vial containing the freeze dried powder (Reagent vial; code RD7002) with 5 ml from a Dilution buffer vial (code RD7001). Please note that the Dilution buffer vial contains more than 5 ml buffer. Please also note that the Reagent vial has been sealed under vacuum to allow for a better stability.
Stable for four weeks at +4oC
                two months at -20oC

Sample preparation
Punch out a disk of blood-stained paper of 5 mm diameter (3 mm can also be used).

Procedure (see chart)

Evaluation
When the filter paper is dry (approximately after 1 hour), view under a long-wave UV-lamp in a darkened room (see note # 3). Samples obtained from normal or slightly reduced G-6-PDH activity will show strong fluorescence. Failure to fluoresce after a 10 minute incubation suggest a total or marked deficiency of G-6-PDH.

Please note

  1. In some forms of G-6-PDH deficiency, young erythrocytes manifest normal enzyme activity. Blood from patients who have just experienced a hemolytic crisis must first be treated by the procedure described by Herz et al  (4) to separate the older erythrocytes from the prevailing population of young ones. Use 0,005 ml of the suspension so obtained for the assay.

  2. If the patient has received a blood transfusion, this test is clinically significant only after 30 days have elapsed, because the donor's erythrocytes generally manifest a normal G-6-PDH activity and can thus bias the result before the expiration of this time.

  3. Any commercially available UV-lamp emitting long-wave UV-light is adequate for the evaluation.

Warning: Dilution buffer contains sodium azide as preservative. Do not swallow. Avoid contact with the skin and mucous membranes.

References

  1. Beutler E. Drug-induced hemolytic anemia and non-spherocytic hemolytic anemia. In Glucose-6-Phosphate Dehydrogenase (Yoshida A. and Beutler E., Eds) pp. 3-12, Academic, Orlando.

  2. Beutler E. A series of new screening procedures for pyruvate kinase deficiency, glucose - 6 - phosphate dehydrogenase deficiency and glutathione reductase deficiency. Blood 1966;28:553-562.

  3. Dow PA, Petteway MB, Alperin JB. Simplified method for G6PD screening using blood collected on filter paper. Am J Pathol 1974;61:333-336.

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Page last edited on 05/07/2005