Application of the particle gel agglutination assay in the typing of single human leucocyte antigens.

We describe a simple and rapid particle gel agglutination assay (PaGIA) for typing of the human leucocyte antigens (HLA) HLA-A2, HLA-B7 and HLA-B27. Superparamagnetic streptavidin particles were coated with biotinylated monoclonal antibodies (MoAbs) to HLA-A2, HLA-B7 and HLA-B27. Anticoagulated whole blood samples from healthy blood donors (n = 118) with known HLA patterns were incubated with MoAb-coated particles, transferred into a standard ID-gel card, and subsequently centrifuged. Samples were evaluated macroscopically, with antigen-positive samples resulting in a visible agglutination reaction. A clear distinction could be made between all positive and negative samples tested. Fifty-seven samples were found to be positive for HLA-A2 (48%), 26 samples for HLA-B7 (22%) and 5 samples for HLA-B27 (4%).

A rapid gel agglutination test for the determination of fetomaternal haemorrhage.

Determination of fetomaternal haemorrhage (FMH) remains an area of difficulty. In most cases, prophylactic Rh immunoglobulin is usually administered to affected women without testing for foetal red blood cells (RBC). Here, we describe a new particle gel immunoassay (PaGIA) for the determination FMH (FMH-PaGIA). Superparamagnetic particles were coated with monoclonal anti-D and mixed with ethylenediaminetetraacetic acid-anticoagulated blood samples from D-negative pregnant women. The particles were isolated using a magnetic particle concentrator and then placed into the reaction chamber of a gel card. Agglutinated particles on top or dispersed through the gel matrix indicated the presence of D-positive cells. After the test was adapted to detect >or=0.3% D-positive RBC, randomly selected postpartum samples from 208 women were analysed in parallel with the Kleihauer-Betke test (KBT). In addition, all discrepancies were further analysed by flow cytometry. A total of 203 of the 208 postpartum samples were negative in both tests. One sample reacted positive with both assays. Two samples were strongly positive in the new FMH-PaGIA, but negative in the KBT. A serological re-examination revealed that both women were D positive. The KBT gave a false-positive result in two cases because of hereditary persistence of haemoglobin F. The new test is specific, easy to perform and can be done at any time in all laboratories.

A novel antigen-specific capture assay for the detection of platelet antibodies and HPA-1a phenotyping.

BACKGROUND AND OBJECTIVES: The antigen-specific assays currently used for the laboratory investigation of platelet antibodies and antigens are technically complex and cannot be used in most routine laboratories. Here, we describe a simple antigen-specific capture assay (ASCA) for the detection of serum platelet antibodies and for human platelet antigen-1a (HPA-1a) phenotyping. MATERIALS AND METHODS: For the detection of platelet antibodies, platelets from healthy blood donors were incubated with biotinylated monoclonal antibodies to platelet glycoprotein complexes (GP), then solubilized and mixed with superparamagnetic streptavidin particles. Serum samples from patients with autoimmune thrombocytopenia (n = 39), from patients with platelet alloantibodies (6 HPA-1a, 1 HPA-2b, 1 HPA-3a, 6 HPA-5b), and from healthy blood donors (n = 70), were tested. All serum samples from the patients were investigated in parallel by the indirect monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). For HPA-1a phenotyping, superparamagnetic particles were coated with a monoclonal antibody to HPA-1a and mixed with diluted whole blood samples from healthy blood donors (n = 139), who had previously been genotyped for platelet alloantigens. Results The indirect MAIPA detected autoantibodies in 18%, and the direct MAIPA in 50% of patients tested. In contrast, the new ASCA demonstrated positive results in 77% of patients. All tested alloantibodies reacted positive by the ASCA, and all serum samples from healthy blood donors were negative. The results of HPA-1a phenotyping were in concordance with those of genotyping in all cases. CONCLUSION: In our opinion, the ASCA is easy to perform and much more sensitive than the currently available antigen-specific assays for the detection of platelet antibodies.

A simple and practical agglutination assay for human leucocyte antigen-B27 typing.

BACKGROUND AND OBJECTIVES: The human leucocyte antigen (HLA) B27 is the most frequently typed single antigen that is associated with diseases. Here, we describe a simple and rapid particle agglutination assay (PaGIA) for HLA-B27 typing. MATERIALS AND METHODS: Superparamagnetic particles were coated with a monoclonal antibody to HLA-B27 and subsequently used for testing. Anticoagulated whole-blood samples were obtained from healthy blood donors (n = 194) with known HLA patterns and from patients (n = 51) who had been typed positive for HLA-B27 by flow cytometry. RESULTS: The particles agglutinated only after incubation with HLA-27-positive blood samples, using the ID-microtyping system. Positive reactions were clearly distinguishable from negative reactions in all samples tested. Flow cytometric HLA-B27 typing revealed an indeterminate result in one patient. CONCLUSIONS: The new HLA-B27 PaGIA is suitable for rapid typing of HLA-B27. The assay is simple and easy to perform, and can be implemented in any routine laboratory.