HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. G6PD HUSM/HEMA-UPT/STM-R6 VERSION NO. VERSION DATE.

1 24.03.2011

APPROVED BY:

... ASSOC PROF DR ROSLINE HASSAN HEAD OF HAEMATOLOGY DEPARTMENT

CONTROLLED COPY NO: 1 REGISTERED HOLDER HAEMATOLOGY LABORATORY

RECORD OF REVIEW/AMMENDMENT DATE VERSION NO. DETAIL OF AMMENDMENT BY

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.

PREPARED BY DESIGNATION CHECKED BY DESIGNATION AUTHORISED BY DESIGNATION

G6PD HUSM/HEMA-UPT/STM-R6

VERSION NO. VERSION DATE.

1 24.03.2011

: AHMAD ZAKWAN MUSTAFA : DEPUTY DOC CON / SAFETY OFFICER / SCIENTIFIC OFFICER : DR ROSNAH BAHAR : HAEMATOLOGIST : ASSOC PROF DR ROSLINE HASSAN : HAEMATOLOGIST/LAB DIRECTOR

1. OBJECTIVE
Detection of G6PD deficiency in inherited G6PD deficiency disorder.

2. METHOD
2.1. UV (ultraviolet light) fluorescent spot-test method (Beutler, 1966) with modified GSSG

(White, 1972).

3. PRINCIPLE
3.1. In normal patient, NADPH generated by G6PD present in a lysate of blood cells,

fluoresces under long-wave ultraviolet (UV) light.

3.2. In G6PD deficiency, there is inability to produce sufficient NADPH. This results in a lack of

fluorescence (Dacie and Lewis, 2001).

3.3. The pathway can be simplified by the diagram below:

UV light

Fluorescent

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. G6PD HUSM/HEMA-UPT/STM-R6 VERSION NO. VERSION DATE. 1 24.03.2011

4. REQUIREMENTS
4.1. EQUIPMENT

4.1.1 Filter paper 4.1.2 Hole Puncher 4.1.3 Test tube (12 x 75mm) 4.1.4 Test tube rack 4.1.5 Water bath / Oven (37C) 4.1.6 Capillary tube (plain) 4.1.7 Adjustable pipette (100 - 1000 ul) & (10 - 100 ul) 4.1.8 Pipette Tips 4.1.9 UV viewing system box 4.1.10 Test tube (15 ml) 4.1.11 Dryer
4.2. REAGENT REAGENT -Nicotinamide Adenosine Dinucleotide Phosphate (NADP)
C21H27N7NaO17P3 xH2O (m.w=765.39)

CODE NUMBER N0505 (Sigma Aldrich) G7250 (Sigma Aldrich) G4376 (Sigma Aldrich) T1378 (Sigma Aldrich)

D-Glucose-6-Phosphate
C6H11Na2O9P xH2O (m.w=304.10)

Oxidized Glutathione
C20H32N6O12S2 (m.w=612.63)

Tris-HCL buffer
NH2C(CH2OH)3 (m.w=121.14)

4.3. SPECIMEN

4.3.1

Dried cord blood on filter paper

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. G6PD HUSM/HEMA-UPT/STM-R6 VERSION NO. VERSION DATE. 1 24.03.2011

5. PROCEDURE
NO. 5.1 ACTIVITY BUFFER STOCK PREPARATION 5.1.1 Tris-HCL Buffer Preparation
a. Dissolve 90.86g Tris HCL Buffer in 900ml distilled water. b. Adjust buffer pH to 7.8 by adding HCL (drop by drop) before diluting it with 1L distilled water. c. Label the preparation date. d. Store the buffer stock in 2 to 6C and is stable for 1 year

RESPONSIBILITY

MLT

5.2

WORKING REAGENT PREPARATION (A), (B), (C) 5.2.1 Oxidized Glutathione, (A)
a. Dissolve 0.50g of Oxidized Glutathione in the 100mL Stock Buffer. b. Label the preparation date, Store the working stock in 2 to 6C and amber bottle. The stock stable for 1 month

5.2.2 Nicotinamide Adenosine Dinucleotide Phosphate (NADP), (B)

a. Dissolve 0.58g of NADP in the 100mL Stock Buffer. b. Label the preparation date, Store the working stock in 2 to 6C and amber bottle. The stock stable for 1 month

MLT

5.2.3 Glucose-6-Phosphate (G6P), (C)

a. Dissolve 0.30g of G6P in the 100mL Stock Buffer. b. Label the preparation date, Store the working stock in 2 to 6C and amber bottle. The stock stable for 1 month

5.3

TEST PREPARATION 5.3.1 Working Reaction Mix

a. Prepare equal volume of fresh mixture of A, B & C aliquots. (for example; 2ml A + 2ml B + 2ml C = 6ml working mix for 50 tests) b. The working mixture is stable for 7 hours in room temperature. c. The working mixture must be used immediately and avoid it from light.

5.3.2 Control Preparation (will be run together with sample testing)

a. b. c. d. e. f. g. h. i. a. b. c. d. e. f. g. h. Control must be run each consecutive batch of test. Obtain G6PD controls (deficient & normal). Punch from the filter paper dried blood into the test tube. Pipette 0.1ml (100ul) of working mix into the test tube. Mix well and incubate at 37C for 30 minutes. Spot the mixture using capillary tube on filter paper. Let the spot dry using dryer. Observe fluorescent spot under UV viewing box (360nm wavelength). Validate and record the control sample. Number each test tube according to the sequence of sample in the batch Punch from each filter paper dried blood sample into the test tube. Pipette 0.1ml (100ul) of working mix into each test tube. Mix well and incubate at 37C for 30 minutes. Spot the mixture using capillary tube on filter paper. Number each spot according to the sequence of sample in the batch. Let the spot dry using dryer. Examine the filter paper under UV viewing box (360nm wavelength). Report NORMAL if the spot fluorescent in UV light. Report INTERMEDIATE if the spot slightly fluorescent in UV light. Report DEFICIENT if the spot non-fluorescent in UV light. Use the appropriate result stamp to report the result. Chop the technologist name and sign the requesting form. Intermediate & deficient result will be informed to ward/clinic.

MLT

5.3.3 Sample Testing

5.4

REPORTING OF RESULTS
a. b. c. d. e. f.

MLT

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.

NO. 5.5

G6PD HUSM/HEMA-UPT/STM-R6
ACTIVITY

VERSION NO. VERSION DATE.

1 24.03.2011
RESPONSIBILITY

DISPATCHING OF RESULTS
a. Record the G6PD results in the LIS. b. Dispatch the original copy to the ward & clinic pigeonhole. c. Keep the carbonized copy in the lab.

MLT

6. LIMITATION
6.1

False-normal results if patient underwent blood transfusion recently and if there is reticulocytosis. If the test is carried out during an acute hemolytic episode, the patients blood should be retested when the reticulocyte count has return to normal. False-deficient results might occur if the sample is from severely anemic patient.

6.2

6.3

7. SOURCES OF ERROR
7.1. The dried blood on the filter paper still wet. 7.2. The spot on the filter paper still wet. 7.3. The spot left dried too long in room temperature. 7.4. Test tube contaminated with external sources of water during incubating. 7.5. Too dilute or too concentrated sample.

8. REFERENCE RANGE
Not applicable

9. RESULTS
Result will be reported as NORMAL, INTERMEDIATE or DEFICIENT.

10. REFERENCES
9.1 9.2 9.3 Dacie and Lewis; Practical Hematology (8th edition), 2001; 216-219.
G6PD Deficency, E.Beutler 1994, Blood Journal Beutler E, Blume KG, Kaplan JC, et al: International Committee for Standardization in Haematology. Recommended screening test for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. British Journal of Haematology 1979; 43:465-477.

9.4

Beutler E, Mitchell M: Special modification of the fluorescent screening method for glucose-6phosphate dehydrogenase deficiency. Blood 1968; 32:816-818.

End of Document
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