Detection of Red Blood Cell—Bound Immunoglobulin G by Flow Cytometry and its Application in the Diagnosis of Autoimmune Hemolytic Anemia

Abstract

Detection of autoantibodies to erythrocytes is of fundamental importance in the diagnosis of autoimmune hemolytic anemia (AIHA). The routinely used direct antiglobulin test (DAT) has the disadvantage of low sensitivity. In this study, we investigated the optimal test conditions of measurement of red blood cell (RBC)-bound immunoglobulin (Ig) G by flow cytometry (FCM). We studied 64 patients with AIHA, 30 anemic patients diagnosed with other diseases, and 36 healthy individuals. In 33 AIHA patients who were found to have RBC-bound IgG, both the mean fluorescence intensity (MFI) and percentage of fluorescence-activated RBCs were remarkably increased and results of both were considered positive. In the remaining 31 AIHA patients who had positive results for RBC-bound complement C3d, the MFI and the percentage of fluorescence-activated RBCs was also increased and 17 patients (54.8%) were considered to have a positive result by this method. In anemic patients with negative DATs the results of FCM were always negative. These results could be confirmed by enzyme-linked immunosorbent assay (ELISA), and the values obtained by FCM and ELISA corresponded to titration scores of the DAT. Additionally, in 3 of the other 8 patients who were suspected to have DAT-negative AIHA, RBC-bound IgG was detected by FCM and ELISA. Our investigation demonstrates that FCM is a precise, reliable, and sensitive method of detecting RBC-bound autoantibodies and could be used as a new routine diagnostic technique for AIHA and other immune hemolytic anemias.

The Diabetes-Prone NZO/HlLt Strain. I. Immunophenotypic Comparison to the Related NZB/BlNJ and NZW/LacJ Strains

“…Flow cytometric analysis of Igϩ RBC offers greater sensitivity for the detection of antierythrocyte antibodies in humans than a direct antibody binding (Coombs) test (Wang et al, 2001). An antierythrocyte antibodies positive representative NZB sample histogram is compared with age-and sex-matched NZO and NZB samples in Figure 3.…”

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies

“…First, we adapted the DAT and IAT to a fluorescence-activated cell sorting (FACS)-based assay, since a similar strategy had previously been shown to be successful for the detection of IgG and C3 on RBC. [13][14][15][16] Secondly, we studied a method for detecting IgM based on the presence of deposited complement factors on patients' RBC. It is known that IgM activates complement more efficiently than IgG and it has been shown that if a patient's serum induces hemolysis in vitro, this is often caused by anti-RBC IgM 17 .…”

“…First we focused on a FACS-based adaptation of the IAT and DAT. IgG and C3 detection on RBC by FACS has been described in various previous studies, [13][14][15][16] but IgM detection in serum has not and on RBC hardly. In a very recent article, concomitant detection of IgG, C3d, IgM and IgA was described.…”

Anémie hémolytique chez l’adulte : principales causes et démarche diagnostique

“…En revanche, jusqu'à près de 10 % des patients hospitalisés ont un TDA positif en l'absence d'AH et ce du fait de différents facteurs non mutuellement exclusifs : hypergammaglobulinémie polyclonale, leucémie lymphoide chronique, transfusions répétées, traitement par immunoglobulines polyvalentes, prise de certains médicaments [24,26,44,46]. Chez les sujets ayant une AH, la valeur prédictive positive du TDA pour le diagnostic d'AHAI est de 83 %, alors qu'elle n'est que de 1,4 % chez le sujet sain [44] [46,47] ; le TDA par cytométrie en flux peut é galement repré senter un complé ment d'investigation pour sensibiliser la dé tection des auto-anticorps fixé s aux GR [48,49]. Interprétation du test direct à l'antiglobuline et tests complémentaires :…”