Quantitation of immunoglobulin classes and subclasses of autoantibodies bound to red cells in patients with and without hemolysis

Abstract

The concentration of IgG, IgA, and IgM, as well as IgG subclasses, was measured by an enzyme-linked immunosorbent assay in autoantibodies eluted from red cells (RBCs); the number of molecules of each isotype per RBC was calculated. Three groups were analyzed: Group 1 included 23 patients with autoimmune hemolytic anemia (AIHA) associated with warm autoantibodies of IgG class; Group 2 included 11 patients without anemia but with a positive direct antiglobulin test (DAT); Group 3 included 10 healthy DAT-negative subjects. The mean number of IgG molecules per RBC in Group 1 (920) was about three times that in Group 2 (306) and about 17 times that in Group 3 (54). The range of RBC-bound IgG showed an overlap between the two groups of patients. The mean number of IgM and IgA molecules per RBC was low in the three groups. IgG1 predominated in all groups except in two patients with AIHA, in whom IgG3 made up at least 50 percent of total IgG. The mean number of IgG1, IgG2, and IgG4 molecules per RBC in Group 1 was about three times that in Group 2, whereas the mean number of IgG3 molecules per RBC was 10 times as high (p < 0.001). It follows that IgG3 was more common in patients of Group 1, but it was also detected in patients of Group 2.

A Quantitative ELISA for Measuring Red Cell-Bound Immunoglobulins

“…The fact that active haemolysis can some times occur at relatively low levels of red cell sensitization is well documented [26] as for example in some cases of Coombs-negative haemolytic anaemia [27,28], All the IgG values found here were above the level required for a positive agglutination test. The results are comparable with those of previous investigators who have studied the degree of IgG sensitization of red cells in autoimmune haemolytic anaemia, the values reported ranging from 230 to about 30,000 molecules per cell [12,17,18,29,30],…”

“…The fact that IgG coating of red blood cells can be dem onstrated in healthy subjects is well known and values similar to the median of 40 molecules per red cell found here have been reported in other investigations using a variety of methods, including ELISA [6,7,17,18], com plement-fixing antibody consumption [19,20] and ra dioimmunoassay techniques [12,21,22]. There appear to have been no previous quantitative data for bound IgA on normal human red cells but its presence in small amounts has been clearly established [6,23,24], albeit at the lower limit of detection [18]; it has also been demonstrated on the erythrocytes of normal dogs [25].…”

“…There appear to have been no previous quantitative data for bound IgA on normal human red cells but its presence in small amounts has been clearly established [6,23,24], albeit at the lower limit of detection [18]; it has also been demonstrated on the erythrocytes of normal dogs [25]. Such surface immu noglobulins are thought to have a role in homeostasis by identifying senescent red cells for removal by mononu clear phagocytes [23,24].…”

Comparison of traditional methods and mitogen-stimulated direct antiglobulin test for detection of anti-red blood cell autoimmunity

“…Eluates, performed in 19 cases, revealed the presence of RBC-autoantibodies in 7 out of 11 DAT-tube-positive cases (3 anti-e, 1 anti-c, 1 anti-nl, and 2 panreactive) and 2 out of 8 microcolumn/solid-phase-positive cases (panreactive). Anemia was present in all groups, but all tests were negative, whereas hemolytic parameters were more frequently abnormal in tube-positive cases (10 out of 14), in half of DAT-tube-negative and other method-positive cases (5 out of 10), and also in half of cases positive only using MS-DAT (8 out of 16, analytical data shown in Table 3). DAT tube was the most specific but least sensitive test (0.87 and 0.43, respectively); other traditional DAT methods showed reduced specificity but increased sensitivity (0.70 and 0.65, respectively), and MS-DAT was the least specific but the most sensitive test (0.59 and 0.88, respectively).…”

“…It is known that DAT tube, which has been considered the gold standard to identify positive and negative cases [2], effectively diagnoses AIHA when at least 500 molecules of autoantibodies are bound to RBCs [13], whereas microcolumn and solid phase require approximately 200-300 molecules per single RBC to yield a positive result [14]. The flow cytometry test is able to detect a lower number of molecules per single RBC [15], but is not routinely performed in most laboratories.…”