Role of gel test and flow cytometry in diagnosis of Coombs’ negative autoimmune haemolytic anaemia

Abstract

This study evaluated the use of both gel test (GT) and flow cytometry (FC) techniques in the detection of red blood cell-bound immunoglobulin G (IgG) and, hence, the diagnosis of autoimmune haemolytic anaemia (AIHA) in cases that were negative by the conventional tube technique (CTT) direct antiglobulin test (DAT). The study enrolled 50 clinically diagnosed AIHA patients with CTT Coombs' negative test and 55 control subjects. GT and FC were performed on both groups. Ninety-two percent (46/50) of patients were positive by GT. As for FC assay, the Receiver Operating Characteristic curve determined that a cut-off of 17.5% fluorescence was the best value for interpreting FC-DAT positively in the group of patients with haemolytic anaemia (100% sensitivity and specificity), while the best cut-off for mean fluorescence intensity (MFI) was 1.74, with 76% specificity and 96% sensitivity. Both mean percent fluorescence and MFI were significantly higher among patients when compared to controls (P < 0.001). FC assay results showed no statistically significant correlations with patients' laboratory data or GT grades (P > 0.05). Flow cytometry is more sensitive than GT for assessing CTT-DAT-negative AIHA. We propose that FC percent fluorescence cut-off values should be employed to determine the Coombs' negative AIHA cases.