Evaluation of Platelet Alloimmunization by Filtration Enzyme-Linked Immunosorbent Assay

Abstract

The current methods for detecting antiplatelet antibodies are mostly manual and labor-intensive. A convenient and rapid detection method is required for effectively detecting alloimmunization during platelet transfusion. In our study, to detect antiplatelet antibodies, positive and negative sera of random-donor antiplatelet antibodies were collected after completing a routine solid-phase red cell adherence test (SPRCA). Platelet concentrates from our random volunteer donors were also prepared using the ZZAP method and then used in a faster, significantly less labor-intensive process, a filtration enzyme-linked immunosorbent assay (fELISA), for detecting antibodies against platelet surface antigens. All fELISA chromogen intensities were processed using ImageJ software. By dividing the final chromogen intensity of each test serum with the background chromogen intensity of whole platelets, the reactivity ratios of fELISA can be used to differentiate positive SPRCA sera from negative sera. A sensitivity of 93.9% and a specificity of 93.3% were obtained for 50 μL of sera using fELISA. The area under the ROC curve reached 0.96 when comparing fELISA with the SPRCA test. We have successfully developed a rapid fELISA method for detecting antiplatelet antibodies.

Keywords: antiplatelet antibody; fELISA; solid-phase red cell adherence test (SPRCA).

Figure 1

Reversible background filtration enzyme-linked immunosorbent assay (fELISA) reactivity of original platelets and ZZAP-treated platelets. Background chromogenic reactivity of original platelets (mock control) reduced after being treated with ZZAP once (a, a′) and twice (b, b′) in duplicate fELISA tests without adding serum of the first antibody. Mock control platelets reacting with self serum and processing throughout the entirety of fELISA were used as the negative control. After preincubation with self serum, ZZAP-treated platelets (A, A′ and B, B′) regained almost the same chromogen backgrounds as the mock and negative controls. All digital images were processed with according to intensity of darkness, quantified using ImageJ software.

Figure 2

Similar lower filtration enzyme-linked immunosorbent assay (fELISA) reactivity ratios from solid-phase red cell adherence test (SPRCA)-negative sera. Both self serum (negative control) and 8 SPRCA (0/8)-negative sera (nos. 1–8) were individually tested for their reactivity with the ZZAP-treated pool platelets. All digital images were processed according to intensity of darkness, quantified using ImageJ software.

Figure 3

Filtration enzyme-linked immunosorbent assay (fELISA) reactivities’ congruity with the solid-phase red cell adherence (SPRCA) test results. Pooled platelets of eight different platelet concentrates were stripped via ZZAP treatment and then applied as antigens of fELISA. From a total of 24 SPRCA samples, 8 samples were tested each time ((A) first test; (B) second test; (C) third test) for their fELISA reactivity. Numbers in the brackets indicate the SPRCA-positive ratio of samples to eight random platelets. All digital images were processed according to intensity of darkness, quantified using ImageJ software. The negative control membrane’s reactivity was used as the denominator for calculating the reactivity ratio of each test serum.

Figure 3

Filtration enzyme-linked immunosorbent assay (fELISA) reactivities’ congruity with the solid-phase red cell adherence (SPRCA) test results. Pooled platelets of eight different platelet concentrates were stripped via ZZAP treatment and then applied as antigens of fELISA. From a total of 24 SPRCA samples, 8 samples were tested each time ((A) first test; (B) second test; (C) third test) for their fELISA reactivity. Numbers in the brackets indicate the SPRCA-positive ratio of samples to eight random platelets. All digital images were processed according to intensity of darkness, quantified using ImageJ software. The negative control membrane’s reactivity was used as the denominator for calculating the reactivity ratio of each test serum.

Figure 4

Predictions of the solid-phase red cell adherence test (SPRCA) results using the receiver operating characteristic (ROC) curve of the ratio of filtration enzyme-linked immunosorbent assay (fELISA) reactivity. The area under the ROC curve (AUC) of the ROC and statistics are indicated; p ≤ 0.001 is considered statistically significant. We applied (A) 5 uL serum and (B) 50 uL serum to fELISA in these instances.