Development of Flow Cytometry for Detection and Quantitation of Red Cell Bound Immunoglobulin G in Autoimmune Hemolytic Anemia With Negative Direct Coombs Test

Asian Pac J Allergy Immunol. 2011 Dec;29(4):364-7.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anemia, Hemolytic, Autoimmune / blood
  • Anemia, Hemolytic, Autoimmune / diagnosis*
  • Anemia, Hemolytic, Autoimmune / immunology
  • Cell Separation / methods*
  • Coombs Test
  • Erythrocytes / immunology*
  • Flow Cytometry / methods*
  • Humans
  • Immunoglobulin G / analysis*
  • Immunoglobulin G / blood
  • Immunoglobulin G / immunology


  • Immunoglobulin G