VII. Calculation of results

The results can be calculated by microtiter plate spectrophotometer reader or manual evaluation. If a microtiter plate spectrophotometer reader with data calculation program is used, refer to the plate reader and create a program using the concentration of each of the G6PD standards in (U/gHb). For manual evaluation, a standard curve is constructed by plotting the absorbance (A) values obtained for each G6PD standard against the corresponding G6PD concentrations in U/gHb . The unknown U/gHb concentration, can then be read from the standard curve using the absorbance value of each patient specimen.

Example of calculation:

The values shown below are examples and must not be used in place of experimental data.

Specimen Absorbance G6PD Activity (U/gHb)

In order to confirm borderline results, it is recommended to repeat the semi-quantitative test or perform other laboratory methods e.g. quantitative test or molecular assay.

THE PADTAN ELM ENZYMATIC KIT FOR 96 TESTS

Neonatal G6PD

Intended use: Semi-Quantitative determination of G6PD levels in dried blood samples

For in vitro diagnostic use

I. Introduction Glucose-6-phosphate dehydrogenese (G6PD) is a cytoplasmic enzyme that catalyses the oxidation of glucose-6-phosphate to 6-phosphogluconolactone wi th simultaneous raduction of nicotinamide adenine dinucleotide phosphate (NADP⁺) to NADPH. These reactions are the first step in the pentose phosphate

(1)pathway .Glucose-6-phosphate dehydrogenese (G6PD) functions throughout the body, but its deficiency is predominantly seen in its effects on the red blood cells. G6PD protects RBCs from oxidative stress.G6PD deficiency is the most common genetic erythrocyte enzyme deficiency in humans. This X-linked recessive hereditary deficiency affects 400 milion people around the world. The clinical manifestations associated with this deficiency are neonatal jaundice, infections and acute drug induced hemolysis, favism, and chronic hemolytic

(2-4)anemia. Infants with G6PD deficiency may be at increased risk for pathological newborn jaundice and may warrant close monitoring for associated complications during the newborn period. It is therefore imperative that the deficiency is diagnosed early in the neonatal period.(4)The Padtan Elm G6PD microwell enzyme assay is useful for semi-quantitative determination of this deficiency in newborn blood samples dried on filter paper .

II. Principle of the test

The Padtan Elm Neonatal G6PD kit is a semi-quantitative test which is based on microwell enzymatic colorimetric assay. During the course of the assay, a 5mm disk of standards, controls and infant samples is punched off the blood spots collected on Whatman’s filter paper 903. The disk is then placed into the microplate wells and D.W is added. During the incubation, G6PD present in the samples is extracted from dried blood spots. After extraction, the eluted sample is transferred into new wells and reaction buffer is added to each well. Eluated G6PD oxidizes glucose-6-phosphate to 6-phosphogluconolactone and reducing NADP⁺ to NADPH. NADH produced reacts with tetrazolium salt to form a colored end-product with an absorbance maximum at 492nm. The intensity of the color is proportional to the amount of G6PD present in the sample. Standard curves are constructed for each assay by plotting absorbance value against the concentration of each standard. The G6PD concentrations of patient samples are then read from the standard curve.

III. Kit contents

The reagents provided with the Neonatal G6PD Kits are sufficient for 96 wells.The expiry date of each reagent is shown on the vial label.

Store the Kit at 2-8° C.

1. Microplate wells: 2×96 uncoated wells.2. Standards: 4 Blood Spots dried on Whatman’s filter paper 903 containing a known amount of G6PD which is printed beside the spots. 3. Control: One Blood Spot dried on Whatman’s filter paper 903 containing a known amount of G6PD which is printed beside the spots. 4. Reagent Buffer: One vial (25 ml) of reagent buffer containing Tetrazolium salt, PBS and preservative.5. Co-Enzyme Solution: One vial (0.6ml) of co-enzyme

+solution containing NADP and preservative.

IV. Materials required (but not supplied with the kit)

1. Microtiter plate spectrophotometer reader with a wavelength of 492 nm (with reference wavelength at 630 nm) and absorbance range of 0 to 3.0.2. Precision 25,200 and 1000 μl micropipettes.3. 5mm Hole puncher4. ELISA shaker capable of 1500rpm5. Deionized or distilled water

V. Specimen collection and preparation

For Infant G6PD screening, a heel stick sample collected on Whatman’s filter paper 903, 24 to 72 hours postpartum is suggested. The approved method of Blood collection for Newborn Screening programs according to NCCLS, LA4-LA2 publication is as following:(5)Clean the heel of the infant with soap and wipe dry. Use alcohol on the area and allow to air dry. Puncture infant’s heel with sterile lancet and wipe away the first drop of blood. Allow a second, large drop of blood to form and lightly touch filter paper to this large drop of blood. Allow blood to soak through and completely fill the preprinted circle with a single application to this large blood drop.The blood volume must be enough to completely fill circles on the card. Place the filter paper card horizontally on a clean surface and allow to air dry thoroughly for at least 3 hours at ambient temperature. Avoid direct sunlight. After 3 hours of drying, place each specimen in its own paper envelope with silica gel and store at 4°C up to 2 days. At -20°C the stability of samples increases up to 2 weeks.

VI. Assay procedures

All reagents should be brought to room temperature prior to use.

1.Punch 5 mm diameter disks off the infant’s blood spots, standards and controls dried on filter paper using a 5 mm puncher. Place them into the wells.2.Add 300µl deionized or distilled water to each well. Mix them by gentle tapping the plate 30 seconds. 3.Incubate the microplate for 1 hour at 2-8°C .4.After incubation mix hemolysed solution 5 times.5.Transfer 25µl of the eluted present in the primary wells (wells containing blood spots) to the newly prepared wells. 6.Add 200 μl of Reaction buffer to each well. In order to prepare Reaction buffer, mix 2 ml of Reagent buffer with 50 μl Co-enzyme solution. This amount is sufficient for 8 wells. The Reaction buffer is stable up to 30 minutes after preparation. 7.Incubate the plate for 1 hour at room temperature with constant shaking (Do not cover the wells).8.Read absorbance at 492 nm (with reference wavelength at 630 nm) in a microtiter plate reader.

SUMMARY OF ASSAY PROCEDURE

Punch 5 mm diameter disks off the standards , controls or infant’s boold spots

Pipette 300µl D.Water

Incubate 1 hour at at 2-8°C

Pipette 25µl eluted samples into the new wells

Pipette 200µl Reaction buffer

Incubate 1 hour at RT on shaker

Read at 492/630nm

0.0540.2880.5361.1800.305

Standard AStandard BStandard CStandard DBlood Spot A

048

204.3

Abso

rbance

(Concentration U/gHb)

0 5 10 15 20 25

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

X. References

1.Yoshida,A. & Beutler,E.(1986). Glucose-6-Phosphate Dehydrogenase.n.p.:Academic Press,Inc.

2.Turan,Y.(2006).”Prevalence of Erytrocyte Glucose-6-Phosphate Dehydrogenase Deficiency in the population of western Turkey.Archive of Medical Research:880-882.

3. Scriver, C.R., et al. (1995). "Glucose-6-Phosphate Dehydrogenase Deficiency." In: The metabolic and molecular bases of inherited disease. 7th ed. n.p.: McGraw- Hill, Inc.:3367-98.

4. S.Behjati-Ardakani.(2007).”The association between G6PD Deficiency and total serum Bilirubin Level in Icteric Neonates”. Acta Medica Iranica:233-235.

5.National Committee for Clinical Laboratory Standards: Blood collection on filter paper for neonatal screening programs, approved standard, 4th ed. NCCLS Document LA4-A4 vol. 23, no. 21 (2003).

6..RZ. Azma.(2010).”G6PD Enzyme Activity in Normal Term Malaysia Neonates and Adults using a Osmmar 200-D kit with HB Normalization.”Vol41,No.4.

4. Recovery and dilution: Recovery is defined as the increase in concentration seen when a known concentration of an analyte is added to a sample. Several citrated blood samples with high G6PD activity were mixed with samples containing low G6PD activity. G6PD activity of the samples on Whatman’s filter paper 903 were then analyzed by Padtan Elm G6PD kit. The percentage recovery is calculated as:

[(Measured Enzyme activity (U/gHb)/ Expected Enzyme activity (U/gHb)] X100

The percentage recovery ranged from 95.5% to 104.5%.

Recovery test

Added Expected Enzyme Measured Enzyme %Recovery Volume (µl) activity (U/gHb) activity (U/gHb)

For dilution test, several blood samples containing elevated G6PD activity were tested after serially diluting with the zero standard, then were spotted on 903 papers and analysed with the kit for G6PD activity. The results of one of these samples are shown in the following table. The percentage recovery ranged from 100% to 106%.

Dilution test

Dilution Expected Measured %Recovery activity (U/gHb) activity (U/gHb)

5. Comparison of correlation coefficient the Padtan Elm quantitative and semi-quantitative G6PD kit: G6PD activity of 60 blood samples was measured by both methods. The correlation coefficient was 0.99.

WARNING!

This kit contains toxic materials, animal and human sourced components. Since no method can completely rule out the presence of blood-borne disease(e.g. HIV,HCV and HBV) therefore,all human sourced material must be considered potentially infectious.In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective gloves and protective glasses where necessary. Never pipette by month. Do not eat, smoke or apply cosmetics in the laboratory.

Rev: Jul.2015

No.26, Shahid Sarparast Ave.West Taleghani Ave.Tehran 14168-IRAN

Tel & Fax: (+98-21) 63481Email: office@padtan.com

PADTAN ELM Co.

IVD2

8 C

EC REP

Kemco SARL11 Allee des marronniers95390 Saint-PrixFrance

VIII. Normal values

78 infant and 86 adults whole blood samples were analyzed by Padtan Elm G6PD kit and normal values of G6PD activity has been determined. According to the results obtained, the normal means for G6PD activity for neonates and adults were 12.4±3.8 U/gHb and 9.7±2.5U/gHb, respectively. Any person with an activity below 2.4U/gHb should be regarded as G6PD severe deficient and within range of 2.5-5.3U/gHb

(6)reported as partial deficient .

IX. Specific assay characteristics

1. Sensitivity: The sensitivity of this kit was calculated based upon the standard curve and expressed as the minimal activity of G6PD showing a significant difference from the "zero" standard. This minimal detectable activity of G6PD for the present kit is 0.5 U/gHb.

2. Precision: Precision was evaluated for inter and intra-assay variability. Intra-assay reproducibility was determined by measurement of 10 replicates of 3 samples in a single run. Inter-assay reproducibility was determined by replicate measurement of 3 samples in 10 separate runs. The results of standard deviation(SD) and coefficient variation(CV) are shown in the table.

Reproduciblity

Intra-assay Inter-assay

Enzyme activity SD %CV Enzyme activity SD %CV Mean (U/gHb) Mean (U/gHb)

3. Accuracy: To assess the accuracy of Padtan Elm neonatal G6PD kit, G6PD activity of 180 random blood samples (normal & abnormal) were measured by Zentech Neonatal G6PD Screening Assay (Enzymatic Colorimetric method) and compared with Padtan Elm results. The correlation coefficient of test was 0.91.

2.79

6.44

11.1

0.11

0.41

0.37

3.94

6.43

3.3

1.4

4.4

9.0

0.1

0.3

0.8

6.0

7.4

8.5

-

25

50

100

200

-

6.4

10

14.5

19.0

1.0

6.35

10.45

14.45

18.15

-

99.2

104.5

99.7

95.5

-

1:2

1:4

1:8

1:16

-

10.0

5.0

2.5

1.25

20

10.6

5.0

2.65

1.25

-

106

100

106

100