G6PD Deficiency Workup

Updated: Mar 13, 2018
  • Author: Lawrence C Wolfe, MD; Chief Editor: George T Griffing, MD  more...
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Workup

Approach Considerations

Indications for testing for glucose-6-phosphatase dehydrogenase (G6PD) deficiency include the following:

  • Development of hemolysis after taking medications or experiencing conditions that  induce oxidative stress especially in patients of African, Mediterranean, or Asian descent.
  • Unexplained severe or prolonged neonatal hyperbilirubinemia with poor response to phototherapy.
  • Family history suggestive of G6PD deficiency especially among males.
  • Recurrent jaundice, splenomegaly, or cholelithiasis in patients of African, Mediterranean, or Asian descent. [4]  
  • Nonspherocytic hemolytic anemia (since the underlying cause might be severe G6PD deficiency and chronic hemolysis)
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Laboratory Studies

Hemolysis

Tests to diagnose hemolysis include the following:

  • Complete blood cell count (CBC) and reticulocyte count
  • Peripheral blood smear
  • Lactate dehydrogenase (LDH) level
  • Indirect and direct bilirubin level
  • Serum haptoglobin level
  • Urinalysis for hemoglobinuria
  • Urine hemosiderin 

Other causes of hemolysis and hemoglobinuria

Tests to rule out other causes of hemolysis and hemoglobinuria include the following:

Complete blood cell count will show mild to severe anemia depending on the G6PD variant and the type of oxidant stress. Increase in reticulocyte count represents bone marrow response to hemolysis by producing young red cells. Increase in indirect serum bilirubin and LDH indicate hemolysis. Low or absent haptoglobin levels, hemoglobinemia, hemoglobinuria, and presence of urinary hemosiderin indicate severe intravascular hemolysis, which is the main contributor to pathophysiology and diagnosis of G6PD deficiency. A part of hemolysis can be extracellular where damaged red cells are recognized as abnormal and undergo extravascular hemolysis by reticulo-enothelial system. [20]

On the peripheral smear, routine staining may reveal polychromasia, representing increased red blood cell production. Another typical feature is the presence of “hemighosts,” red cells that appear to have unevenly distributed hemoglobin, and  “bite cells” or “blister cells,” red cells that appear to have a portion of them bitten away. Blister cells are characteristic of acute hemolysis induced by oxidative stress. [22]

Denatured hemoglobin can be visualized as Heinz bodies in peripheral blood smears processed with supravital staining. Heinz bodies are shown in the figure below.

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

Semi-quantitative tests:

  • Fluorescent spot test: This is a direct test that measures the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) from nicotinamide adenine dinucleotide phosphate (NADP+); the test is positive if the blood spot fails to show fluorescence under ultraviolet light. It is rapid, simple, sensitive, and inexpensive. [6, 7, 8]  A variant of the spot test that can be interpreted by simple color change with naked eye examination is used for screening large populations in tropical areas and before starting treatment with antimalarial drugs, such as primaquine, in countries where G6PD deficiency and malaria are both endemic. The test is not reliable in heterozygous females.
  • Methemoglobin reduction test: This is a rapid indirect test that measures the reduced methemoglobin levels produced after NADPH oxidation. G6PD activity is assessed by first treating red blood cells with nitrite (converting oxyhemoglobin [red] to methemoglobin [brown]), and then examining the rate of NADPH-dependent methemoglobin reduction in the presence of an appropriate redox catalyst (Nile blue or methylene blue) and substrate (glucose). [6]
  • Cytofluorimetric method: This is a cytochemical typing assay that provides a fluorometric readout of the classic methemoglobin reduction test at the level of an individual red blood cell. This assay represents a useful addition to the screening and research toolkit for G6PD deficiency, especially in malaria-endemic areas. [7]

Quantitative test:

  • Spectrophotometric assay: Quantitative tests for G6PD activity are considered the criterion standard. The rate of NADPH generation is spectrophotometrically measured at a wavelength of 340 nm. The G6PD activity is finally expressed as G6PD IU/red blood cell and G6PD IU/hemoglobin ratios. In normal red blood cells, the G6PD activity ranges from 7-10 IU/g Hb, when measured at 30 C. [6, 7, 8]  Testing for enzyme activity should not be performed during episodes of acute hemolysis, as results may be falsely negative. Senescent red blood cells are more vulnerable to hemolysis due to their diminished G6PD levels. Compensatory increase of immature young red cells with increased G6PD levels usually occurs in state of acute hemolysis, and hence results could be altered.

A study by Peters et al indicated that in the detection of heterozygously G6PD-deficient females, spectrophotometry, cytofluorometry, and chromate inhibition have a sensitivity of 0.52, 0.85, and 0.96, respectively, and a specificity of 1.00, 0.88, and 0.98, respectively. The investigators stated that although routine means of assessing total G6PD activity can miss heterozygously G6PD-deficient females in whom a larger percentage of red blood cells is G6PD-sufficient, chromate inhibition and cytofluorometry can detect most of these cases. [37]

Screening for G6PD deficiency

A semi-quantitative test is usually indicated in patients with a suggestive family history or in geographical areas with a high prevalence of the disorder. Positive screening results should be confirmed by quantitative tests. Diagnosis of G6PD may be difficult in females, who may be hemizygous or have skewed X chromosome inactivation or G6PD gene mosaicism.

G6PD activity is higher in premature infants than in term infants. This should be considered when testing for G6PD deficiency in infants.

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Imaging Studies

Abdominal ultrasound may be useful in assessing for splenomegaly and gallstones. These complications are typically limited to patients with severe chronic hemolysis.

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Other Tests

Genetic testing consists of DNA-based genotyping and sequencing, which helps in the identification of hundreds of mutations associated with G6PD deficiency worldwide, including many region-specific common variants. The molecular analysis may be useful for population screening, family studies, females, and prenatal diagnosis.

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