G6PD Deficiency in the Newborn Workup

Updated: Jun 25, 2020
  • 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:

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

In January 2020, the British Society for Haematology released guidelines for the laboratory diagnosis of G6PD deficiency that recommended re‐assay after a hemolytic episode of unknown cause to confirm that a G6PD-deficiency diagnosis was not missed. [38]

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Laboratory Studies

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 tests

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 hemoglobin when measured at 30ºC. [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 a state of acute hemolysis, and results could therefore 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. [9]

Guidelines

The British Society for Haematology guidelines on the laboratory diagnosis of G6PD deficiency include the following testing recommendations [38] :

  • Because the G6PD reaction is temperature‐dependent, an accurate cuvette temperature is essential
  • Controls should be run with every sample batch; it is preferable to use a normal and deficient sample obtained through a commercial company than to employ an in‐house control
  • White cells, which contain a significant amount of G6PD, ideally should be removed before assay; especially consider removal of white cells with a cellulose/”real” cotton wool column prior to assay if the count is above the lab’s reference interval upper limit
  • If performed only infrequently, carry out assays in duplicate; the duplicates’ results, on normal samples, should be within 0.5 IU/g of hemoglobin of each other
  • Employ in-house testing to establish a laboratory reference range
  • Assay absorbance should be checked to see that it increases in a linear fashion (which may take a minute or two to achieve), and, for non-kit methods, the absorbance should be measured over 10 minutes at 20-second intervals
  • Measurement of the hemoglobin concentration of the hemolysate is equal in importance to measurement of the enzyme activity, since both measurements have a comparable impact on the final result; this applies similarly to well-mixed whole blood where a kit indicates such use
  • Interpret the final G6PD activity in light of the reticulocyte count measured on the same sample
  • Participation in an accredited external quality assessment scheme is important for laboratories undertaking these screening tests and assays
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Screening for G6PD deficiency

A semi-quantitative test is usually indicated in patients with a suggestive family history or in geographic 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.

The British Society for Haematology guidelines on the laboratory diagnosis of G6PD deficiency note that screening tests should not be relied on for the diagnosis of female patients, stating that instead, G6PD activity should be measured directly by quantitative spectrophotometric assay. They also specify that in patients in general, an abnormal or borderline screening test necessitates the performance of a quantitative assay.

Genetic Testing

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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