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

Introduction: 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). Methods: 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. Results: 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). Conclusion: 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.