Development of flow cytometry for detection and quantitation of red cell bound immunoglobulin G in autoimmune hemolytic anemia with negative direct Coombs test.
About 2-10% of patients with warm-antibody autoimmune hemolytic anemia (WAIHA) exhibit a negative direct Coombs test (DAT), requiring more sensitive tests, including detection of RBC-bound immunoglobulins by flow cytometry, for diagnosis. In this study, the optimal conditions for detection and quantitation of RBC-bound IgG by flow cytometry were studied using blood samples from six patients with AIHA and two healthy individuals. Quantitation of RBC-bound IgG was performed using quantum simply cellular (QSC) beads coated with goat anti-mouse IgG antibodies. For detection of RBC bound IgG, a 60-minute incubation of all blood samples with 40 microl of 1:10 dilution of FITC-conjugated mouse anti-human IgG gave mean fluorescent intensity (MFI) values comparable to experiments using larger amounts or higher concentrations of the anti-human IgG. The acquired antibody binding capacity (ABC) values (or IgG molecules) for each QSC bead level, at 40 microl of 1:5 and 1:10 dilution of anti-human IgG for 60 minutes were close to the manufacturer-assigned ABC values. The IgG molecules per RBC in all six patients with positive DAT of 4+, 3+, 2+, 1+, trace and negative DAT were 31,725, 3,823, 1,753, 524, 260 and 88 respectively and in two healthy individuals with negative DAT they were 104 and 78.
Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University.
Anchalee
A
Thedsawad
Orathai
O
Taka
Wanchai
W
Wanachiwanawin
D000744:Anemia, Hemolytic, Autoimmune / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D003298:Coombs Test; D004912:Erythrocytes / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000097:blood / Q000276:immunology
D007074:Immunoglobulin G