Evaluation of an enzyme‐linked immunosorbent assay kit (GTI PakPlus®) for the detection of antibodies against human platelet antigens

Abstract

Twenty-six serum samples from 24 patients were investigated for the presence of platelet-specific antibodies in a partly retrospective (n = 15) and partly prospective (n = 9) study. The sera contained either alloantibodies to human platelet antigens (HPA) (n = 23) or were from clinically suspected cases of fetomaternal alloimmune thrombocytopenia (FMAITP) in which platelet-specific antibodies had not been detected (n = 3). Three techniques were used to detect platelet antibodies: the platelet immunofluorescence test, the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and a commercially available enzyme-linked immunosorbent assay--GTI PakPlus (GTI kit). Two alkaline phosphatase-conjugated antiglobulin reagents provided by the manufacturer were used in the GTI kit: an antihuman IgG/IgA/IgM (IgGAM) conjugate and an antihuman IgG conjugate. The GTI kit with the anti-IgGAM conjugate failed to detect eight antibody specificities in seven sera (anti-HPA-1a [n = 3], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1] and anti-HPA-5b [n = 3]). Greater signal-to-background ratios were achieved in the GTI kit with the anti-IgG conjugate but five antibody specificities (anti-HPA-1a [n = 1], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1], anti-HPA-5b [n = 2]) remained undetectable. All the sera were detected by MAIPA assay and, furthermore, the MAIPA assay achieved the greatest signal-to-background ratio in the majority of sera tested. These findings re-emphasize the value of the MAIPA assay in reference laboratories and illustrate that the GTI kit may either fail to detect or incorrectly identify clinically significant HPA antibodies.

External quality assessment of platelet serology and human platelet antigen genotyping: a 10‐year review

“…Four of eight ILs used an SPhE and not a MAIPA assay, which resulted in more false‐positive results and a slightly lower sensitivity for the detection and identification of HPA antibodies, which is in concordance with earlier studies 17,18 . Most unexpected positive reactions in the SPhE were not HPA‐specific but occurred with GPIa/IIa or more than one GP.…”

Antibodies to human platelet antigens form a significant proportion of platelet antibodies detected in Indian patients with refractoriness to platelet transfusions

“…This fact is supported by findings in a study conducted on 26 serum samples from 24 patients who contained antibodies to HPAs. All antibody specificities against HPA antigens were detected by the MAIPA; however, the GTI PakPlus (GTI kit) failed to detect or incorrectly identified clinically significant anti‐HPA antibodies in some sera (Lucas et al, ). In this study, we have described the presence of anti‐HLA antibodies on the Luminex bead‐based assay in a small subset of 10 patient samples in whom antibodies were not detected.…”

Analysis of platelet‐reactive alloantibodies and evaluation of cross‐match‐compatible platelets for the management of patients with transfusion refractoriness

“…However, of the 45 patients with anti‐HPA antibodies detected using ELISA, only 2 patients had platelet‐specific alloantibodies (anti‐HPA‐3a and anti‐HPA‐5b), while another 2 patients were detected as being anti‐GPIIb/IIIa with unknown allospecificities when characterised by MAIPA. Comparing these two methods, MAIPA and ELISA (commercially platelet antibody kit), MAIPA is generally accepted as the ‘golden standard’ in detecting HPA antibodies but needs a high degree of technical expertise, while ELISA is widely used in many laboratories because it is a simple and rapid test but may provide false‐positive results (Lucas & Rogers, ). In our study, we used EDTA‐containing buffers as a storage medium for the extracted platelets, washing platelets and preparing platelet suspensions.…”