Approach Considerations
Indications for testing for glucose-6-phosphate dehydrogenase (G6PD) deficiency include the following [24] :
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Development of hemolysis after taking medications or experiencing conditions that can induce oxidant stress
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Unexplained or prolonged neonatal hyperbilirubinemia
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Non-spherocytic hemolytic anemia (since the underlying cause might be severe G6PD deficiency and chronic hemolysis)
Tests to diagnose hemolysis include the following:
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Complete blood cell count (CBC) and reticulocyte count
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Lactate dehydrogenase (LDH) level
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Indirect and direct bilirubin level
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Serum haptoglobin level
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Urinalysis for hematuria
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Urinary hemosiderin
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Peripheral blood smear
Increased reticulocyte counts indicate increased bone marrow response to anemia. Increased indirect bilirubin and LDH levels indicate increased RBC destruction.
Decreased haptoglobin levels, hematuria, and presence of urinary hemosiderin indicate severe intravascular hemolysis. On the peripheral smear, routine staining may reveal polychromasia, representing increased RBC production. So-called bite cells caused by the splenic removal of denatured hemoglobin may be seen.
Heinz bodies (denatured hemoglobin) can be seen on the peripheral smear in G6PD deficiency. See the image below. Heinz bodies are not revealed by routine staining but are visualized by using a supravital stain (Heinz body prep). Heinz bodies are also seen in patients with unstable forms of hemoglobin, such as hemoglobin Köln. Heat stability and/or heat denaturation and high-performance liquid chromatography can be used to identify unstable hemoglobin and thereby rule out G6PD deficiency.
G6PD deficiency: Heinz bodies in a peripheral smear stained with a supravital stain. Heinz bodies are denatured hemoglobin, which occurs in G6PD deficiencies and in unstable hemoglobin disorders.
Abdominal ultrasound may be useful in assessing for splenomegaly and gallstones. These complications are typically limited to patients with severe chronic hemolysis.
Tests to diagnose G6PD deficiency
Screening for G6PD deficiency is indicated in patients with a suggestive family history or in geographical areas with a high prevalence of the disorder. Screening tests are described by Minucci et al. [24] Positive screening results should be confirmed by quantitative tests. The molecular analysis may be useful for population screening, family studies, females, and prenatal diagnosis. [11] Diagnosis of G6PD may be difficult in females, who may be hemizygous or have skewed X chromosome inactivation or G6PD gene mosaicism. [24]
Standard tests include the Beutler test and a quantitative assay of GPD activity. [10, 11, 24, 28] The Beutler test is a semi-quantitative rapid fluorescent spot test that detects the generation of nicotinamide adenine dinucleotide phosphate (NADPH) from nicotinamide adenine dinucleotide phosphate (NADP); the test is positive if the blood spot fails to fluoresce under ultraviolet light. The Beutler test is not reliable in females. [4]
A spectrophotometric analysis of G6PD activity in a leukocyte-depleted sample is the standard quantitative test. Testing for enzyme activity should be performed when patients are in remission, as results may be falsely negative during acute hemolysis. The reason is that older erythrocytes have been destroyed, because their diminished G6PD levels leaves them vulnerable to hemolysis, while there is a compensatory increase of immature erythrocytes and reticulocytes that have increased G6PD levels.
However, spectrophotometric analysis may fail to detect G6PD deficiency in hemizygous patients. Also, spectrophotometric quantitation may fail to detect deficiency in heterozygous females due to residual activity in G6PD-sufficient cells. The identification of G6PD-deficient as well as G6PD-sufficient cells by a cytochemical method or cytofluorometry is more sensitive in the testing for G6PD deficiency in females. Chromate inhibition is a test that is more sensitive than spectrophotometric quantitation for heterozygous G6PD deficiency in females. [29, 30]
A number of rapid point-of-care diagnostic tests for determining G6PD deficiency status have been developed. [31, 32, 33, 34] These have a potential role in malaria-endemic areas for permitting safe use of primaquine, which can provoke hemolysis in persons with G6PD deficiency. [35, 36] Newer versions of these tests are quantitative and can be used in males, females, and neonates.
G6PD activity is higher in premature infants than in term infants. This should be considered when testing for G6PD deficiency in infants. [37]
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G6PD deficiency: Heinz bodies in a peripheral smear stained with a supravital stain. Heinz bodies are denatured hemoglobin, which occurs in G6PD deficiencies and in unstable hemoglobin disorders.
