What are the direct & indirect lab. methods for diagnosing G6PD deficiency?
Here is a recent review paper which includes this description for G6PD diagnosis:
"The definitive diagnosis of G6PD deficiency is based on the estimation of enzyme activity, by quantitative spectrophotometric analysis of the rate of NADPH production from NADP. For rapid population screening, several semiquantitative methods have been applied, such as the dye-decolouration test developed by Motulsky in 1961, and fluorescent spot tests, which indicate G6PD deficiency when the blood spot fails to fluoresce under ultraviolet light. Other semiquantitative tests have been used, but require definitive testing to confirm an abnormal result. and "
Here is some elaboration from Wikipedia:
"The diagnosis is generally suspected when patients from certain ethnic groups (see below) develop anemia, jaundice and symptoms of hemolysis after challenge to any of the above causes, especially when there is a positive family history.
Generally, tests will include:
- Complete blood count and reticulocyte count; in active G6PD, Heinz bodies can be seen in red blood cells on a blood film;
- Liver enzymes (to exclude other causes of jaundice);
- Haptoglobin (decreased in hemolysis);
- A "direct antiglobulin test" (Coombs' test) - this should be negative, as hemolysis in G6PD is not immune-mediated;
- TSH measurement.
When there are sufficient grounds to suspect G6PD, a direct test for G6PD is the "Beutler fluorescent spot test", which has largely replaced an older test (the Motulsky dye-decolouration test). Other possibilities are direct DNA testing and/or sequencing of the G6PD gene.
The Beutler fluorescent spot test is a rapid and inexpensive test that visually identifies NADPH produced by G6PD under ultraviolet light. When the blood spot does not fluoresce, the test is positive; it can be false-positive in patients who are actively hemolysing. It can therefore only be done several weeks after a hemolytic episode.
When a macrophage in the spleen "sees" an RBC with a Heinz body, it removes the precipitate and a small piece of the membrane, leading to characteristic "bite cells". However, if a large number of Heinz bodies are produced, as in the case of G6PD deficiency, some Heinz bodies will nonetheless be visible when viewing RBCs that have been stained with crystal violet. This easy and inexpensive test can lead to an initial presumption of G6PD deficiency, which can be confirmed with the other tests."
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