G6PD/6PGD

for the diagnosis of G6PD deficiency

                                                                                                  

 

1)     INTRODUCTION    

 

G6PD deficiency is caused by numerous mutations that have been selected by malaria due to the protection conferred by the enzyme deficiency.

Both  homozygous and heterozygous G6PD deficiency can result in chronic anemia or severe hemolytic crisis after ingestion of pro-oxidants drugs or food (fava beans). Due to intermediate levels in heterozygous subjects and in some genetic variants, the diagnosis requires a quantitative measurement of the enzyme. The measure is anyway affected by a variety of situations that may determine erroneous results:

a) Individual variability of the enzyme levels: it is closely related to the average age of erythrocytes (G6PD decreases during the aging of the erythrocyte). An increase of the reticulocytes causes a marked increase in G6PD.

b) Thalassemias, unstable hemoglobins, erythrocyte membrane defects determining an increase of reticulocytes are often associated to a substantial increase of the G6PD.

c) Additive measurement of 6PGD: The enzyme 6-phosphogluconate dehydrogenase (6PGD) uses the product of the reaction of G6PD as substrate. The addictive activity of 6PGD may lead to false negative results unless specific solutions are adopted.

d)Leukocytes: The presence of leukocytes causes an increase of G6PD due to their elevated enzyme content. On the other hand, the enzyme is very susceptible to the action of proteases, after the hemolysis of the sample, G6PD is rapidly degraded unless specific solutions are adopted.

e) Poor stability of the enzyme: The enzyme decays rapidly in hemolyzed blood  requiring an adequate stabilization.

f) Inconsistence of the reference system: In addition to the variables shown above, the parameter used to normalize the G6PD may largely contribute to the error. The use of Hb concentration to normalize G6PD values,  in case of slight anemia, falsely increases the enzyme levels and it is the major cause of missed diagnosis of approxinmately 50% of the heterozygous G6PD deficient women in some geographic areas .

 

 

Figure 1

 

 

 

 

 

2) INTENDED USE

 

Diagnosis of G6PD deficiency in blood anticoagulated with EDTA or eparin.

 

3) PRINCIPLE OF THE TEST

 

The test derives from a study carried out in approximately 20.000 subjects in an area with high frequency of G6PD deficiency, thalassemias and unstable hemoglobins.   A series of solutions have been adopted to optimize the method in order to obtain the diagnosis in the heterozygous females irrespectively of mutations affecting erythrocytes and various causes of anemia. G6PD deficiency was confirmed by genetic analysis.

a) G6PD is measured as the difference between the activity of G6PD and 6PGD in order to subtract the activity of 6PGD

b) 6PGD is used as a reference of G6PD. The 6PGD constitutes a dependent reference varying in parallel with the G6PD: all factors that increase G6PD

c) (reticulocytosis, leukocytosis, thalassemias and unstable hemoglobins) also determine an increase of 6PGD. This method is not dependent on the levels of Hb eliminating this source of error, moreover it eliminates the necessity and the additional cost related to the measurement ofHb.

d) a series of solutions have been introduced to stabilize the enzyme allowing its measurement in whole blood, in presence of leukocytes

e) the test has been adapted and validated in all major clinical chemistry instruments to increase the throughput and to reduce the cost. All validated instruments automatically calculate the final result preventing any human error. 

The ratio between the two enzymes is not affected by the Hb concentration, the number of erythrocytes, the number of reticulocytes and/or leukocytes. The distribution of the G6PD activity displays an efficient separation in heterozygous women compared to normal subjects (Fig. 2A). On the contrary, if Hb was used as reference (Fig. 2B), a large overlap between heterozygous and normal subjets was observed.
The analysis of results revealed that the G6PD/6PGD test allows the diagnosis in 97% of heterozygous and 100% of homozygous subjects. The method is also effective for the diagnosis of heterozygosity in neonates and in patients carriers of thalassemias if the cut of values were adequately changed. In contrast, the enzyme normalized to the concentration of Hb allowed the diagnosis in only 50% of heterozygous subjects. The use of G6PD/6PGD test avoided the measurement of the Hb accelerating the execution and decreasing costs. A further advantage of the method is the possibility to perform the measurement with whole blood (eliminating the removal of leukocytes by a column of cellulose as indicated in the original G6PD assay). For this purpose in the Hemolysis Solution (HS) provided in the kit, different protease inhibitors, high concentration of magnesium and NADP were introduced. The mixture of detergents contained in HS  has been studied to ensure the structural stability of the enzyme and to minimize the release of leukocyte proteases.

 

Figure 2A

                                                                                                                                                                                               

 

 

Figure 2B

 

 

 

 4) MATERIAL SUPPLIED WITH THE KIT

 

 

COD.

SOLUTION A

SOLUTION B

HEMOLYSING SOLUTION (HS)

SUBSTRATE A

SUBSTRATE  B

G 201

   G 201K1

1x100 ml

1x100 ml

1x200 ml

25 mg

38 mg

G 202

1x50 ml

1x50 ml

1x100 ml

13 mg

19 mg

 

 

COD.

Positive control

Negative control

GK20

8X0.5 ml

8x0.5 ml

 

 1G201K: G201+GK20

 

5) MATERIAL AND REAGENTS REQUIRED BUT NOT PROVIDED

 

- A set of micro-pipettes or diluter to hemolyze the sample

 

6) TECHNICAL AND PERFORMANCE

 

The method allows the diagnosis of heterozygosis for G6PD deficiency in a percentage higher than 95%. In case of total deficiency sensitivity and specificity are 100%

 

7) STORAGE OF REAGENTS

 

- At -18/-20°C, the kit is stable until the expiration date

- Solution A and Solution B are stable for 10 days at 2-8°C after thawing and reconstitution

- HS is stable for 10 days at 2-8°C after thawing

- Solution A and Solution B are stable for 3 months at -18/-20°C after reconstitution and they can be used only once after thawing

 

8) WARNINGS AND PRECAUTIONS

 

- Only for in vitro diagnostic use. Read carefully the instructions before use.

- The sample are potentially infectious. Adopt the standard procedures for the protection of operators.  Follow the regulations regarding the disposal of infectious biological materials.

- Avoid contact with skin, eyes and mucous membranes with all reagents. In case of accidental contact, wash with plenty of water. Carefully consult the safety data sheet for information on the toxicity and safety measures/treatment in case of contamination. The harmful materials or that represent a biological risk are labeled in accordance with the 1999/45/EC.

- Use only the reagents provided in the kit. Do not use the kit after the expiration date. Do not mix reagents from different kits. Store all reagents at specified temperatures. The improper storage of the reagents may alter the results of the tests.

 

 

9) PREPARATION AND STORAGE OF SAMPLES

 

- Sample: whole blood collected in heparin or EDTA

- The minimum amount required for a test: 50 ul of blood

- The sample can be stored for  4-6 hours at room temperature and for 72 hours at 2-8° C.

 

 

10) PROCEDURE

 

- Thaw and reconstitute the solutions A and B with the respective substrates, mix carefully and store at 2-8 °C

- HS is ready for use after its thawing

- Before analysis bring the solutions at room temperature

- Lyse the erythrocytes diluting the blood 1: 20 in HS (for example: 20 ul of whole blood + 400 ul of HS). Shake and wait for 10 minutes at room temperature to complete the hemolysis. The final volume of hemolyzed blood depends on the amount required by the instrument used to run he test 

- Measure the enzyme at 37°C, in a manual or automated spectrophotometer as follow:

 

 

hemolyzed

BLOOD

SOLUTION A

(6PGD)

SOLUTION B

(G6PD+6PGD)

10 ul

500 ul

 

 

 Read the increase in absorbance for minute at  340nm:  A

10 ul

 

500 ul

 Read the increase in absorbance for minute at  340nm:  B

 

 

Calculation of results: R = (B / A) – 1

 

 

Volumes can vary according to the used instrument, for specific applications to different  instruments, see point 12

 

11) INTERPRETATION OF THE RESULTS

 

 

 

 

       G6PD/6PGD

           G6PD(%)

   G6PD (UI/gHb)

NORMAL SUBJECTS

 

< 0.10 complete deficiency

0.10 - 0.85  intermediate deficiency

> 0.85  normal levels

 

<10% complete deficiency

10% -85% intermediate deficiency

>85%  normal levels

<1 complete deficiency

 

1-8 likely intermediate deficiency

 

>8 likely normal levels

 

Marked Microcitemia

 

reticulocytosis > 5%

 

leukocytosis  > 20.000 WBC

0.00-0.10 complete deficiency

 

0.10 - 0.95 intermediate deficiency

>0.95  normal levels         

<10 complete deficiency

 

 

10% - 95% intermediate deficiency

>95%  normal levels

NEWBORNS

 

< 0.10 complete deficiency

0.10 - 1.10  intermediate deficiency

> 1.10  normal levels

 

<10% complete deficiency

10% - 110%  intermediate deficiency

>110%  normal levels

 

 

 

It must be noticed that the diagnosis made measuring the enzyme activity  is phenotypic. A fraction of heterozygous females may then present levels of G6PD overlapping to the levels measured in G6PD deficient or in normal subjects.

 

 

12) VALIDATED INSTRUMENTS

 

The kit G6PD/6PGD has been validated and CE-IVD labeled for use with the following instruments:

 

ABBOTT, models C4000,  C8000,  C16000

BECKMAN, Analyzers Serie AU400 - AU640 - AU2700 - AU5400 - AU480 - AU680 - AU5800

BECKMAN, Analyzers i Serie Synchron Lx e Synchron CX 

BIOTECNICA INSTRUMENT , models TARGA BT 3000 PLUS, BT 3500 PLUS

BPC, models KUADRO - KEYLAB 

DADE BEHRING , models Dimension AR-RXL-EXPAND

ORTHO CLINICAL DIAGNOSTICS (JOHNSON & JOHNSON) models Vitros 5.1/FS - 5600

ROCHE, Serie COBAS 4000 (c311), COBAS 6000 (c501), COBAS 8000 (c502-c702), COBAS Integra 400

SIEMENS, models Advia 1200 -1650 - 1800 - 2400

SIEMENS, Analyzers Serie Dimension Vista 1500-500