Oral Presentation Indian Ocean Rim Laboratory Haematology Congress 2019

A cytofluorometric assay for G6PD-deficient RBCs provides a quantitative assessment in heterozygous female carriers (#15)

Prashant Sharma 1 , Alpeshkumar B Kapadia 1 , Karuna Jain 1 , Namrata Singh 1 , Man Updesh S Sachdeva 1 , Amanjit Bal 2 , Alka R Khadwal 3 , Reena Das 1 , Neelam Varma 1
  1. Hematology Department, Postgraduate Institute of Medical Education and Research, Chandigarh, India
  2. Histopathology Department, Postgraduate Institute of Medical Education and Research, Chandigarh, India
  3. Adult Clinical Hematology and BMT Unit, Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Introduction. Conventional tests for G6PD-deficiency, the commonest red cell enzymopathy, are disadvantaged by reticulocyte interference, technical complexity and/or ambiguous/missed diagnoses in heterozygous female carriers who can be at risk for hemolysis due to unfavourable X-inactivation. We assessed the performance of a novel cytofluorometric assay1 for RBC-level assessment of G6PD-activity.

Methods. The methemoglobin-reduction (MR)-test, a quantitative enzyme activity (Enzopak™, Reckon Diagnostics) and the cytofluorometric assay (flow cytometric+fluorescent microscopic readouts) were done in 3 subject groups: 20 G6PD-deficient males, 22 heterozygous females (including 20 mothers of the deficient males) and, 20 G6PD-replete controls. Loco-regional G6PD-activity cut-offs were determined as per WHO.2 Spectral properties, performance characteristics and cost of the cytofluorometric assay were calculated.

Results. The %bright cells correlated significantly with MR-test results (p<0.01) but only moderately with the quantitative enzyme assay (r=0.6, p<.00001). Flow cytometry correctly classified all deficient (<5% bright cells) and normal cases (>95% bright cells). G6PD-replete (bright) vs. deficient (negative) RBCs could be readily distinguished in heterozygous women as 2 peaks on flow or bright/dim cells on fluorescent microscopy.

The processed red cells showed absorption and emission maxima at 410 and 480 nm respectively with best separation in the 425–475 nm (Pacific Blue) detection channel. Limit of detection for deficient RBCs was <5%, limit of blank (unstained “deficient” control) was <1% and between run coefficient of variation was 5.1% overall and 0.29% in G6PD-replete controls. Means (ranges) of the %bright cells (G6PD-replete erythrocytes) were 0.76 (0.2-2.6) in deficient males, 52.15 (0.7-99.1) in heterozygous females and 98.35 (95.3-99.8) in G6PD-replete groups (p<0.0001).

Reagent costs per sample were USD 0.038 U for the MR-test, USD 0.087 for cytofluorometric assay and USD 0.95 for the spectrophotometric enzyme activity assay.

Conclusions. The low-cost, technically simple and robust cytofluorometric test confirms/exclude G6PD deficiency accurately in males and provides a quantitative assessment of %bright red cells in heterozygous females.

  1. Shah SS, Diakite SAS, Traore K et al. A novel cytofluorometric assay for the detection and quantification of glucose-6-phosphate dehydrogenase deficiency. Sci Rep 2012;2:299.
  2. Technical Specifications Series for submission to WHO prequalification – diagnostic assessment: in vitro diagnostics medical devices to identify Glucose-6-phosphate dehydrogenase (G6PD) activity. Geneva: World Health Organization; 2016. Licence: CC BY-NC-SA 3.0 IGO. (Accessed on June 28, 2019 at https://apps.who.int/iris/bitstream/handle/10665/252628/9789241511865-eng.pdf?sequence=1.)