Comparison of Spectrophotometry, Chromate Inhibition, and Cytofluorometry Versus Gene Sequencing for Detection of Heterozygously Glucose-6-Phosphate Dehydrogenase-Deficient Females

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide. Detection of heterozygously deficient females can be difficult as residual activity in G6PD-sufficient red blood cells (RBCs) can mask deficiency. In this study, we compared accuracy of 4 methods for detection of G6PD deficiency in females. Blood samples from females more than 3 months of age were used for spectrophotometric measurement of G6PD activity and for determination of the percentage G6PD-negative RBCs by cytofluorometry. An additional sample from females suspected to have G6PD deficiency based on the spectrophotometric G6PD activity was used for measuring chromate inhibition and sequencing of the G6PD gene. Of 165 included females, 114 were suspected to have heterozygous deficiency. From 75 females, an extra sample was obtained. In this group, mutation analysis detected 27 heterozygously deficient females. The sensitivity of spectrophotometry, cytofluorometry, and chromate inhibition was calculated to be 0.52 (confidence interval [CI]: 0.32-0.71), 0.85 (CI: 0.66-0.96), and 0.96 (CI: 0.71-1.00, respectively, and the specificity was 1.00 (CI: 0.93-1.00), 0.88 (CI: 0.75-0.95), and 0.98 (CI: 0.89-1.00), respectively. Heterozygously G6PD-deficient females with a larger percentage of G6PD-sufficient RBCs are missed by routine methods measuring total G6PD activity. However, the majority of these females can be detected with both chromate inhibition and cytofluorometry.

Keywords: chrome inhibition; cytofluorometry; females; gene sequencing; glucose-6-phosphate dehydrogenase deficiency; spectrophotometry.

Figure 1.

(A) Fluorescence distribution patterns after cytofluorometric analysis of G6PD activity in RBCs of a homozygous or hemizygous G6PD-deficient individual. All RBCs have a low fluorescence intensity (region P2). (B) Heterozygous G6PD deficiency. One population of RBCs has a low fluorescence intensity (region P2), and another one has a high fluorescence intensity (region P3). (C) Healthy individual without G6PD deficiency. The majority of RBCs have a high fluorescence intensity (region P3). Only a few old RBCs with low G6PD activity are found in region P2. (D) Healthy individual with an asymmetric population of RBCs with a high fluorescence intensity (intensity in region P3 and a shoulder in region P2). A substantial amount of RBCs with low fluorescence intensity are localized in region P2. Without proper visual inspection of the figure, this patient is falsely classified as heterozygously G6PD deficient. Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; RBCs, red blood cells.

Figure 2.

(A) Relationship between spectrophotometrically detected G6PD activity in RBC lysates and the percentage negative cells as determined with the cytofluorometric assay. The dashed horizontal line indicates the threshold for the percentage G6PD-negative cells in healthy controls (10.15%). The dashed vertical line indicates the lower threshold for G6PD activity (3.8 IU g/Hb) in healthy controls. Black dots represent patients without G6PD deficiency, and gray dots represent patients with heterozygous G6PD deficiency as determined by genetic analysis. Note that 4 females with normal G6PD activity and without mutations in G6PD showed a significant number of negative RBCs as measured by flow cytometry. In all these cases, the negative population appeared as a shoulder of the positive populations and should be interpreted as inconclusive. (B) Relationship between G6PD:GR ratio and the percentage negative cells as determined with the cytofluorometric assay. Additional testing for heterozygous G6PD deficiency was recommended for females with a G6PD:GR ratio between 0.46 and 0.9 (dashed vertical lines). The dashed horizontal line indicates the threshold for the percentage G6PD-negative cells in healthy controls (10.15%). Black dots represent patients without G6PD deficiency, and gray dots represent patients with G6PD deficiency as determined by the reference standard, genetic analysis. (C) Relationship between chromate-inhibited spectrophotometric determination of GR activity and the percentage negative cells as determined with cytofluorometry. The dashed horizontal line indicates the threshold for the percentage G6PD-negative cells in healthy controls (10.15%). The dashed vertical lines indicate the range from 1.7 to 2.0, which was considered to represent an inconclusive result of chromate inhibition. Chromate-inhibited activity <1.7 was considered to indicate absence of G6PD deficiency. Chromate-inhibited activity >2.0 was considered to indicate G6PD deficiency. Black dots represent patients without G6PD deficiency, and gray dots represent patients with G6PD deficiency as determined by genetic analysis. In all 4 females without a mutation in G6PD that showed a significant number of negative RBCs as measured by flow cytometry, the negative population appeared as a shoulder of the positive peak and should be interpreted as inconclusive. Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; RBC, red blood cell; IU g/Hb, international units per gram hemoglobin; GR, glutathione reductase.

Figure 3.

(A) Results of G6PD gene sequencing, chromate inhibition, cytofluorometry, and spectrophotometric determination of G6PD activity and the G6PD:GR activity ratio to detect heterozygous G6PD deficiency. The Venn diagram indicates the number of females with a positive result in the tests, which implies G6PD deficiency. For example, 14 females had a positive result in all tests whereas 31 patients had a positive result of the G6PD:GR ratio determination only. (B) Results of G6PD gene sequencing, chromate inhibition, cytofluorometry, and spectrophotometric determination of G6PD activity and the G6PD:GR ratio to detect heterozygous G6PD deficiency. The Venn diagram indicates the number of females with a negative result in the tests, which implies normal G6PD function. For example, 9 females had a negative result in all tests whereas 9 patients only had a negative result in the G6PD activity determination. Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; GR, glutathione reductase.