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Detecting the Deficiency with G6PD Screening

Screening means testing individuals to see if they have the deficiency. This is done by taking a sample of blood and then using one or more special tests to detect either a variant (mutation) in their DNA or a measurable deficiency in the activity of the enzyme in their red blood cells.

An important aspect of the different screening methods is the Sensitivity and Accuracy of the tests.The ideal test will have few or no false negatives — identifying a sample as normal when it is from a deficient subject — and few false positives — identifying a normal sample as deficient. It is usually better to err on the side of sensitivity since a positive test can always be rechecked with a new test, perhaps using a different screening method.

In the screening of individuals for G6PD deficiency, a number of problems must be solved that arise from the unusual natural history of the condition. In the first place, it seems that most deficiencies affect the stability of the enzyme more than its intrinsic activity. This means that young red cells have significantly higher measured levels than older cells. Thus if a patient has just suffered an acute hemolytic attack, their red cell G6PD level might appear quite normal because the older red cells have been removed from the circulation. A subsequent test 2 or 3 weeks later will likely show the deficiency.

Of even greater significance is the problem of diagnosis of the heterozygous female. A female with a G6PD mutation on just one of her X chromosomes is a heterozygote. In a process called Lyonization or Mosaicism, in any given cell just one of those X chromosomes will be active. Thus, a proportion of her cells will be normal, while the others are G6PD deficient. The proportion is unpredictable and apparently variable so that, at any time, heterozygous females can have red blood cell G6PD activities that range from near normal to activity levels usually associated with the classification of deficient. But even when the activity level are near normal, there may be a sufficient number of compromised red cells to trigger acute hemolysis, or hyperbilirubinemia in a newborn. As a result, a major consideration when evaluating screening procedures is how successfully they can identify heterozygous females.

Methods for screening:

Quantitative Biochemical methods:
Enzyme Assay
The quantitative enzyme assay is simply a measure of the activity of the enzyme in the subject’s red blood cells. Commercial kits are available and in principle it is the definitive objective test of the functioning of the enzyme.

Issues: Enzyme activities can be tricky to measure and are very sensitive to a number of factors, such as the exact composition of the solution and the temperature. Normal controls must be used for comparison and the results expressed relative to the amount of hemoglobin or the number of red cells. Other enzymes in the red cells can cause some interference, which must be accounted for in the test procedure. For these reasons, the assay requires a properly equipped laboratory and well-trained staff. It is also noteworthy that research laboratories have reported only moderately strong correlations between different test kits when compared on the same populations

The measured level of G6PD activity must be referred to a reference population of presumed normal subjects. There is a wide range of activities in the normal population, so care must be taken to define a level of activity below which a test result is considered deficient. The result of these considerations is to define three groups of subjects: normal, deficient and intermediate. Generally, males will fall into the normal or deficient range but females can fall into any of the groups. The difficulty lies in defining the boundaries of the intermediate range so as to accurately identify heterozygotes. Thus the test, although quantitative, may not always be definitive.

Commercial clinical laboratories (e.g. Walk-In-lab) now offer the enzyme assay at a price below $100 and a turn around time of 2 – 3 days.

A double enzyme assay has been suggested as being more reliable — it measures the ratio of Glucose-6-Phosphate Dehydrogenase activity to that of the enzyme 6-Phosphogluconate Dehydrogenase activity. No commercial kits exist for this, however, and it remains a research tool.

Cytochemical Assay
Cytochemical assays use chemical reactions that can indicate the activity of the G6PD enzyme in individual red blood cells. Thus the numbers of cells with normal vs. deficient enzyme levels in a population of cells from an individual can be determined. An instrument called a flow cytometer can be used to automatically determine the ratio of these two types of cells.

A method for doing this has been developed but, as in the case of the enzyme assays, requires (even more) expensive equipment and, as a multistage assay process, also requires well-trained laboratory staff. It is thus still an assay that would be performed in a research setting rather than as a routine clinical assay.

Semi-quantitative methods:
Methods for measuring the activity of the enzyme in blood samples have been derived that can be incorporated into simple test kits that can be used to quickly determine if the sample has a markedly low level of G6PD activity. These are based on either a colorimetric method that can be read with the naked eye under ambient light or a fluorometric method that is read under a UV lamp.

These methods are called semi-quantitative since they will readily indicate that a sample has 10% or less of normal G6PD activity but cannot reliably tell if a sample is normal or has activities around 50% of normal. Thus normal and deficient (hemizygous) males are easily identified, with low numbers of false positives and no false negatives. The fluorometric test has some ability to identify intermediate levels of activity but cannot be considered very accurate at distinguishing heterozygous from normal females. The currently available colorimetric tests (BinaxNOW and CareStart) are characterized as qualitative tests that can only identify samples as deficient or non-deficient and have been mostly tested in males.

Molecular Screening:
Instead of attempting to measure the activity of the enzyme, it is possible to screen subjects for the presence of mutations in the G6PD gene itself. This might seem to be a definitive test for G6PD deficiency but DNA screening tests are designed to look for specific known mutations and are thus configured to look for those mutations thought to be most common in the target population. Thus, while most deficient subjects including heterozygous females will be correctly identified, individuals with less common mutations could be missed, even though a biochemical test might quickly identify low G6PD activity. The tests also require highly sophisticated laboratories and results are not available quickly. This technology is undergoing rapid advancement, however.

Who should be screened?

Obviously, for anyone who has shown symptoms of acute hemolytic anemia, G6PD assays should be considered after the episode has resolved. The medical literature also describes two populations for whom preemptive screening for G6PD deficiency is particularly recommended.

The first group is the populations of areas where malaria is endemic and efforts are being made to eliminate the disease by mass treatment with primaquine. Since this is one of the classic triggers of hemolytic anemia in G6PD deficient people, of which there is a high incidence in just those areas, the development of robust screening methods has been considered a high priority.

The second group is neonatal infants, who are at risk of jaundice, hyperbilirubinemia, and kernicterus. Later in life, but especially in childhood, they may be susceptible to episodes of acute hemolytic anemia.

The World Health Organization has recommended that wherever the male population has an incidence of G6PD deficiency of greater than 3-5%, universal screening of newborns should be carried out using the fluorescent spot test. Routine neonatal screening has proven very successful in preventing cases of Favism on Sardinia and a comprehensive screening program in Singapore is credited with contributing to the absence of any cases of kernicterus in several decades.

The United States has has an overall incidence of G6PD deficiency estimated to be between 4 and 7% but this number masks the heterogeneity of the US population. African-Americans have an incidence of G6PD A – of 12-13%, and there are substantial immigrant populations from Southern Europe and South-East Asia where there are high frequencies of G6PD-Mediterranean and G6PD-Canton, among other fairly common mutations.

Despite these considerations, neonatal screening for G6PD deficiency is not currently routinely performed in the United States although there are programs in Pennsylvania and the District of Columbia. These, however, are genetic screening programs, and the results are not usually available until the infant is 10–14 days old and past the usual time when life-threatening hyperbilirubinemia occurs. A pilot program in Cleveland screened all male infants with an ethnicity that included high risk groups using the fluorescent spot test. Test results were reported within 48 hours, i.e before discharge in most cases, but the effect of this program on reducing the incidence of hyperbilirubinemia was not reported.

In considering the question of routine screening in order to reduce the incidence of dangerous hyperbilirubinemia, several factors must be taken into account. The test must be able to be performed, and the results obtained, within 48 hours of birth and ideally it should be sensitive enough to identify female heterozygotes as well as homozygous males. It is not clear that such a practice is feasible in most birth institutions in the US. In addition, the highest association of risk factors with hyperbilirubinemia is with breastfeeding; G6PD deficiency is second. Thus, even proponents of routine screening feel that more study is needed to determine if routine G6PD deficiency screening is more effective than enhanced procedures for evaluating serum bilirubin levels before discharge. A recent report on the Cleveland study implies that a positive outcome for their G6PD screening was an enhanced awareness of the pediatric providers of the dangers of hyperbilirubinemia in general and G6PD deficiency in particular.

Brody's Battle

Brody’s Battle happened in the first ten days of his life and nearly ended it, leaving him with terrible brain damage caused by a condition called Kernicterus. Our foundation was started to spare other newborns such life-threatening events by raising awareness of how undiagnosed G6PD deficiency can cause Jaundice. A Kernicterus event can occur if neonatal Jaundice is not properly managed.

This story does not have to happen to another family...