Clinical application of a flow cytometric direct antiglobulin test.

BACKGROUND: The clinical application of flow cytometric direct antiglobulin test (FC-DAT) has rarely been evaluated for patients with various diseases including immune and nonimmune hemolytic anemia. STUDY DESIGN AND METHODS: Blood samples from 380 patients with a variety of diseases were studied using the tube direct DAT and FC-DAT. The results of tube DAT and FC-DAT were compared. The predictive values of DAT for hemolysis were evaluated. RESULTS: Of 57 patients with autoimmune hemolytic anemia (AIHA), 6 of the 17 with a negative tube DAT (immunoglobulin G [IgG]) had a positive FC-DAT (IgG) and 23 of the 36 patients with a negative tube DAT (complement 3d [C3d]) had a positive FC-DAT (C3d). In 57 patients with AIHA, the incidence of positive results of FC-DAT (IgG) and tube DAT (IgG) were similar (42 positive vs. 40 positive); but in 323 patients without AIHA, the incidence of positive FC-DATs (IgG) was higher than that of tube DAT (IgG; 47 positive vs. 9 positive). The higher incidence of positive FC-DAT (C3d) than that of tube DAT (C3d) was seen in patients with AIHA (42 positive vs. 21 positive) as well as in patients without AIHA (61 positive vs. 5 positive). Both DAT (IgG) and DAT (C3d) positive has highest positive predictive value for hemolysis, followed by DAT (IgG) alone positive and DAT (C3d) alone positive. CONCLUSIONS: FC-DAT is a complementary test for diagnosing AIHA. There is a synergistic effect of the red blood cell-bound IgG and complement in predicting hemolysis.

Unappreciated HLA antibodies in adult immune thrombocytopenic purpura.

BACKGROUND/PURPOSE: Immune thrombocytopenic purpura (ITP) is an autoimmune disease. Platelet refractoriness is frequently seen in patients with ITP. Platelets express platelet-specific antigens and human leukocyte antigens (HLA). Platelet antibodies to platelet-specific antigens and HLA may be present, but HLA antibodies in patients with ITP have rarely been reported. METHODS: Sera from 44 adult patients with ITP were screened for platelet antibodies by two flow cytometric assays. In method I, platelets from normal donor platelets were used as target cells to screen both platelet-specific antibodies and HLA class I antibodies. In method II, the FlowPRA Class I Screening Test kit was used to screen HLA class I antibodies. Fluorescein isothiocyanate (FITC)-conjugated sheep anti-human IgG Fc was used as the staining reagent in both methods. The negative serum control was from one of the normal males with AB blood group who had never received a transfusion. Sera from a pool of five highly sensitized patients were used as the positive control. RESULTS: Of the 44 sera from patients with ITP, 31 (70.5%) were method I positive, and 28 (63.6%) were method II positive. There was no significant difference between the results of method I and method II (p = 0.439). The distribution of the results of these two tests was: both tests positive in 22 sera, method I positive and method II negative in nine sera, method I negative and method II positive in six sera, and both tests negative in seven sera. The mean platelet counts of patients with positive (41.0 +/- 40.0 x 10(9)/L) and negative (40.4 +/- 26.8 x 10(9)/L) tests by method I did not differ significantly (p = 0.643). The mean platelet counts of patients with (36.7 +/- 31.5 x 10(9)/L) and without (48.1 +/- 43.6 x 10(9)/L) HLA class I antibodies did not differ significantly (p = 0.59). CONCLUSION: HLA class I antibodies are frequently found in ITP. The screening of platelet antibodies including platelet-specific antibodies and unappreciated HLA class I antibodies is warranted in patients with ITP.

Screening for platelet antibodies in adult idiopathic thrombocytopenic purpura: a comparative study using solid phase red cell adherence assay and flow cytometry.

BACKGROUND: Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder caused by antiplatelet autoantibodies. In this study, we compared 2 methods for screening serum platelet antibodies in patients with ITP. METHODS: A total of 44 adult patients were clinically classified with ITP. We used 2 indirect tests to detect human leukocyte antigen antibodies and/or platelet-specific antibodies in their sera. In method I, we used solid phase red cell adherence (SPRCA) assay. In method II, by flow cytometry, platelets from plateletpheresis components were used as target cells, and fluorescein isothiocyanate-conjugated sheep anti-human IgG Fc was used as the staining reagent. Positive results were defined as any test with the percentage of fluorescence exceeding the reference range by 3% or more in method II. Direct tests detecting platelet-associated IgG on platelets of patients with ITP were done by flow cytometry. RESULTS: Serum specimens from 44 adult patients with ITP (28 female, 16 male) were tested. SPRCA assay could only detect platelet antibodies in 22 patients (50%). By method II, 31 serum specimens (70.5%) yielded positive results. There was a difference between the results of the SPRCA test and method II, with a high degree of significance (p < 0.001) by the McNemar test. No significant difference in platelet counts was observed for patients with and without discernible platelet antibodies by SPRCA assay (p = 0.90). The direct test was positive in 12 patients (66.7%) out of 18 ITP patients tested. CONCLUSION: Flow cytometry is more sensitive than SPRCA assay for detecting platelet antibodies. Detection of platelet antibodies is useful in explaining the immune mechanism and platelet transfusion refractoriness in ITP.