Methods Evaluated for Autoimmune Hemolytic Anemia Diagnosis

The use of both the gel test (GT) and flow cytometry (FC) techniques in the detection of red blood cell-bound immunoglobulin G (IgG) has been assessed.

The two tests are important in the diagnosis of autoimmune hemolytic anemia (AIHA) in cases that were negative by the conventional tube technique (CTT) or direct antiglobulin test (DAT).

Hematologists at Ain Shams University (Cairo, Egypt) conducted a case-control study between August 2008 and October 2010. The study included 50 patients, who were clinically diagnosed to have Warm Antibody AIHA with negative CTT-DAT. Fifty-five healthy volunteers with negative polyspecific CTT-DAT were also enrolled as control subjects.

Blood samples were tested by the Direct Antiglobulin Test by CTT and GT and by FC technique. In the FC assay, the results were expressed as percent fluorescence, which is the percentage of erythrocytes expressing the anti-human IgG at levels above those of the isotype control, and mean fluorescence intensity (MFI). For the GT assay, a positive test is where agglutinated cells formed a red line on gel surface or dispersed agglutinates in the gel and were graded from G1-G4. For the CTT, agglutination was detected macroscopically and confirmed by microscopic examination.

The results of the study showed that 46 of 50 (92%) patients were positive by GT. The Receiver Operating Characteristic curve for FC assay determined that a cut-off of 17.5% fluorescence was the best value for interpreting FC-DAT positively in the group of patients with hemolytic anemia, with 100% sensitivity and specificity. The best cut-off for 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. The FC assay was performed using the Coulter Epics XL 3-colour flow cytometer (Beckman Coulter Inc., Brea, CA, USA).

The authors concluded that GT and FC are sensitive tools for assessing CTT-DAT-negative AIHA. However, FC is more precise and so should be utilized when a strong clinical suspicion of AIHA is associated with dissonant CTT and GT results. They propose that FC percent fluorescence cut-off values should be employed to determine the Coombs’ negative AIHA cases. However, they do imply that larger studies are needed to define the best cut-off value for FC IgG DAT positivity in such cases. The study was published in the June 2012 issue of the International Journal of Laboratory Hematology.

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Ain Shams University
Beckman Coulter Inc.