Development of flow cytometry for detection and quantitation of red cell bound immunoglobulin G in autoimmune hemolytic anemia with negative direct Coombs test.
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| Abstract |
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.
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| Authors | Anchalee Thedsawad, Orathai Taka, Wanchai Wanachiwanawin |
| Journal | Asian Pacific journal of allergy and immunology (Asian Pac J Allergy Immunol) Vol. 29 Issue 4 Pg. 364-7 (Dec 2011) ISSN: 0125-877X [Print] Thailand |
| PMID | 22299318 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
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