HLA class I depletion by citric acid, and irradiation of apheresis platelets for transfusion of refractory patients

Abstract

Background Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti‐HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA‐matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. Methods and Materials Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice‐cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). Results Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5‐fold compared to untreated PLTs. Acid‐treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady‐state activation, CD62P and CD63 were strongly upregulated on both acid‐treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X‐ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. Conclusion The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion‐associated HLA sensitization and overcome PLT refractoriness.

Proteomic study of apheresis platelets made HLA class I deficient for transfusion of refractory patients

“…Such denaturation of proteins is not detected by proteomics, but was revealed by lack of specific anti‐HLA antibody binding as shown by flow cytometry [3,7]. Furthermore, the β 2 ‐microglobulin expression was down‐regulated 1.9‐fold (Table 1B), which is in line with an earlier study [3] and our flow cytometry results [7] .…”

“…Protocol for acid treatment was done as described by Mirlashari MR et al. [7] and [3]. Briefly: PLTs from apheresis concentrates ( n = 7) (420–500 mL and1‐1.5 × 10 9 PLTs/mL) were split in two and transferred to two Terumo transfer bags (600 mL, Compoflex R6R2021; Terumo BCT, Lakewood, Colorado, USA) 24 h. after processing and centrifuged (1506 × g , 10 min, accel 8, decel 2, temp 22°C), one half of the initial product was acid treated, the other half served as a control.…”

“…For activation analysis, samples from the pellets were mixed with their original, now PLT free plasma. The samples were tested for surface expression of the activation markers CD62P and CD63 before and after TRAP stimulation, the results are recently published [7]. Prior to the proteomic analysis, the pellets were gently mixed to dissolve potential aggregates, and PLTs were resuspended in T‐PAS+ (equal volume as original plasma), to avoid contamination of plasma proteins in the PLT proteomic analysis.…”

“…Acid treatment will reduce the number of intact HLA class I molecules on the PLT membrane, and reactivity to anti‐HLA class I‐specific antibodies will decrease. We have recently optimized the acid treatment method and shown that acid treated apheresis PLTs can be an attractive alternative for transfusion of PLTs to refractory patients [7]. Since X‐ray irradiation had no negative effects on the PLTs [7], acid treatment could even be employed when irradiation of the transfusion product is required, as is the case for many hematologic patients.…”

“…Our recent in vitro results indicated that acid treated apheresis PLTs could be used as an alternative to HLA‐matched PLT transfusions for refractory patients [7]. However, we wished to perform a quantitative proteomic study as well to identify possible detrimental changes from acid treatment on the PLT proteome, before investigating the clinical effect of acid treated PLTs in refractory patients.…”