Role of gel test and flow cytometry in diagnosis of Coombs’ negative autoimmune haemolytic anaemia M. H. FAYEK*, A.A. SAAD*, D. G. EISSA*, L. M. TAWFIK*, G. KAMAL † INTRODUCTION Autoimmune haemolytic anaemia (AIHA) is charac- terized by shortened red blood cell (RBC) lifespan owing to autoantibodies directed against RBCs’ self-antigens (Gehrs & Friedberg, 2002). Direct anti- globulin test (DAT), which reveals RBC-bound immu- noglobulins and/or complement, is still a pivotal serological assay in the diagnosis of AIHA (Garratty, 1994). A positive DAT is often seen in conjunction with AIHA, haemolytic disease of newborn (HDN), immune-mediated haemolytic transfusion reactions and drug-induced haemolytic anaemia. In most cases of haemolytic anaemia, RBC-associated immunoglob- ulin G (IgG), C3d fragment of complement or both *Clinical Pathology Department, Ain Shams University, Cairo, Egypt † Internal Medicine Department, Ain Shams University, Cairo, Egypt Correspondence: Dr Lamis Mohamed Tawfik, Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Abbasia, Cairo, Egypt. Tel./Fax: +202 2482 4073; E-mail: lamistawfik@gmail.com doi:10.1111/j.1751-553X.2011.01397.x Received 6 April 2011; Accepted for publication 27 September 2011 Keywords Autoimmune haemolytic anaemia, Coombs’ test, flow cytometry, gel test, DAT SUMMARY 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 auto- immune 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 deter- mined 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 signifi- cant 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’ nega- tive AIHA cases. ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY Ó 2012 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2012, 34, 311–319 311 International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology