G6PDH Quantitative Kinetic 12

Description

About:

• AutoZyme NEW G6PDH (Qualitative-Dye) is a reagent set for qualitative determination (screening) of G6PDH deficiency in erythrocytes by decolorization method.
• AutoZyme NEW G6PDH (Qualitative-Dye) is a reagent set with simple procedure and easy technique.
• AutoZyme NEW G6PDH (Qualitative-Dye) is available in convenient pack-size of 12 tests.

Principle:

Glucose-6-Phosphate Dehydrogenase (G6PDH) present in hemolysate acts on substrates, Glucose-6-Phosphate and NAD, giving NADPH, which in presence of PMS (Phenazine methosulfate) decolorizes blue coloured indophenol dye (DCPIP) leaving behind colour only due to hemolysate.

The rate of reaction is proportional to G6PDH present, hence time required for decolorization is inversely proportional to G6PDH activity in the hemolysate.

Procedure:

Reaction type……..Kinetic
• Reaction direction..Increasing
• Wavelength ……..340 nm
• Flowcell temp………..30°C/37°C
• Zero setting with…..Distilled water
• Delay time……180 seconds
• No. of readings………. …0000. 4
• Interval……60 seconds
• Sample volume …………..0.025 ml (25 pl)
• Working solution volume ……….. 1.0 ml (1000 ul)

STEP I : PREPARATION OF RED CELL HAEMOLYSATE
Wash 0.1 ml of whole blood with 2 ml of physiological saline (0.9% NaCI) 3 times and suspend the washed, packed and centrifuged erythrocytes in pre-cooled 0.5 ml of G6PDH R3 (Lysing Reagent). Mix well and keep in the refrigerator (2 – 8°C) for at least 15 minutes and maximum for 2 hours. Centrifuge the lysate at 3000 r.p.m. for 5 minutes prior to use as given below:

STEP II: MANUAL ASSAY PROCEDURE
Reagents Test
Working Solution 1.0 ml
Haemolysate 25 ul

Mix and aspirate. After the initial delay of 180 seconds, record the absorbance of the test at an interval of one minute for the next 3 minutes at 340 nm. Determine the mean change in absorbance per minute and calculate test results.

Quality Control:

To ensure adequate quality control, it is recommended that each batch should include normal and abnormal reference control. It should be realised that the use of quality control material checks both instrument and reagent functions together. Factors which might affect the performance of this test include proper instrument function, temperature control, cleanliness of glassware and accuracy of pipetting.

If the G6PDH activity is very low, measure the absorbance change for 5 minutes after addition of buffered substrate and divide by 5 to obtain A A/min. and calculate the test results.