Isotype‐specific detection of ABO blood group antibodies using a novel flow cytometric method

Abstract

SummarySeveral methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABOfluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0AE870 and 0AE783, respectively (n ¼ 240; P < 0AE001) whereas the comparison of ABO-FACS with ABO-enzymelinked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.

Long-term outcomes after ABO-incompatible kidney transplantation; a single-center French study

“…It is based on semi-quantitative measurement of blood-group-specific IgM (direct agglutination) or IgG (indirect detection using antiIgG reagents) (15,50). Alternative techniques used to detect anti-A/B antibodies include flow cytometry and surface plasmon resonance, which is a cell-independent method that enables detection-reagent-independent analysis of antibody-antigen binding using immobilized blood group A or B trisaccharides (6,51,52). Many different strategies to remove anti-bloodtype antibodies for desensitization have been developed (53)(54)(55).…”

ABO-Incompatible Kidney Transplantation

“…DTT, dithiothreitol; 2ME, 2-mercaptoethanol; MFIR, mean fluorescence intensity ratio. There are several options for the measurement of isoagglutinin titers: conventional tube method, gel or bead column agglutination method, and flow cytometry (Krishnan et al, 2008;Stussi et al, 2005). These three methods are summarized in Table 1.…”

“…Human AB serum, which is further depleted by incubation with highly concentrated A and B RBCs, can be used as a negative control, and human serum of blood group O is used as a positive control. Commercially available O RBCs with information of antigen expression are also helpful for the detection of irregular antibodies (Stussi et al, 2005). Using undiluted serum, quantification of anti-A/B antibody can be determined by calculation of the geometric mean fluorescence intensity ratio (MFIR).…”

“…This value is calculated by dividing the geometric mean fluorescence intensity of test sera with that of negative control. One study reported that the correlation coefficient between MFIR using flow cytometry and isoagglutinin titer was 0.870 for IgM and 0.783 for IgG (Stussi et al, 2005). For determination of titer using flow cytometry, recipient serum is serially diluted with normal saline solution (2% bovine serum albumin, 0.1% azide).…”

Development of a Novel ELISA for Detection of Anti-A and Anti-B Antibodies in Recipients of ABO-Incompatible Living Donor Liver Grafts

“…Until now, several methods have been used to measure anti-A and anti-B Abs, including enzyme-linked immunosorbent assay (ELISA) (Rieben et al 1991;Spalter et al 1999;Galili et al 2002) and flow cytometry (Sharon and Fibach 1991;Stussi et al 2005), but these methods have not been useful in clinical practice. A new method that uses surface plasmon resonance was recently reported, but this method did not serve to measure IgM Ab (Kimura et al 2005).…”