New Approach to Eliminate HLA Class I Antigens from Platelet Surface without Cell Damage: Acid Treatment at pH 3.0

Abstract

A new method was studied for eliminating HLA class I antigens from the surface of platelets without damaging the cells. Platelets were exposed to an acid solution (pH 3.0) to eliminate the antigenicity of HLA class I antigens. The reduction in antigenicities of HLA class I common antigen and individual HLA class I antigens by acid treatment was marked. Patients' sera which contained multispecific HLA antibodies reacted with PBS-treated platelets, but not with acid-treated platelets. No changes were observed in the antigenicities of glycoprotein Ib or glycoprotein IIb/IIIa. The viability of acid-treated platelets was 83%. Ultrastructural investigations revealed no significant difference between the PBS-treated platelets and acid-treated platelets. The platelet function studies showed that the aggregation of acid-treated platelets induced by various agonists was only slightly reduced compared with PBS-treated platelets. We propose that acid-treated platelets are promising for clinical use in patients refractory to platelet transfusions and may be superior to chloroquine-treated platelets for analysis of the specificity of antiplatelet antibodies.

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“…This method was developed by Kurata et a1. 42 and was similar to that successfully applied in one case by Shanwell et a1. platelet-rich plasma and buffy coat methods4 In our studies, we replaced the plasma employed by Shanwell et al with Plasmalyte-A (Baxter, Deerfield, IL), a crystalloid solution used as a replacement fluid in surgery, which proved to be a suitable platelet storage medium.…”

Acid Treatment of Platelets as a Simple Procedure for Distinguishing Platelet-Specific Antibodies from Anti-HLA Antibodies: Comparison with Chloroquine Treatment

“…Alloantiserum having antibody speci ficity to HLA A24 and B51 was obtained from a patient with chronic renal failure [14],…”

“…Immunofluorescence flow cytometry (FCM) was performed as pre viously described [14], Briefly, fresh platelets were treated with acid, chloroquine or PBS as described above, washed once, and then fixed with paraformaldehyde. After washing three times, the platelets were incubated with test sera for 45 mm at 37°C.…”

“…However, the serologic identification of these platelet-spe cific antibodies is complicated by the presence of anti-HLA antibodies since the frequency of pregnant women who are immunized against HLA antigens has reached 45% [7]. [14].…”

Platelet Antibodies and Diagnosis of Immune-Mediated Thrombocytopenia

“…From their observations that incubation of platelets in acid hypertonic solutions of chloroquine removes HLA-A and HLA-B antigenic material from platelets but not from lymphocytes, Blumberg et al [1] concluded that HLA class I antigens on platelets are adsorbed from plasma rather than that they are integral platelet membrane proteins. HLA antigenicity can also be eluted in acid buffers at pH 3 [2]. 'Elution of HLA antigens' has been explained by Neumüller et al [3] as loss of conformational integrity of the HLA molecule following dissociation of β2-microglobulin and the HLA heavy chain.…”