Transfusion of Cryopreserved Autologous Platelets to Overcome HLA Alloimmunization in Patients with Hematological Malignancies,

Abstract 3375

Alloimmunization to platelet antigens occurs in up to 25% of mainly female patients with hematologic disorders receiving intensive chemotherapies. It leads to platelet transfusion refractoriness, increased bleeding risk and often compromises further treatment. Once alloimmunization is established, only HLA-matched platelets improve platelet count increments. However, in patients with rare HLA phenotypes, the identification of HLA matched platelets may be difficult. In this situation, transfusion of autologous platelets is a clinical option, but cryopreservation of platelets has been notoriously difficult. Here, we present our clinical experience with transfusion of cryopreserved autologous platelets in patients with hematological malignancies. The platelets were collected after recovery from chemotherapy. Per patient, 2.5 (1–5) platelet aphereses were performed collecting 298 (190–598) ml per apheresis resulting in 6 (4–8) concentrates. All platelet concentrates were irradiated with 50 Gy, cryopreserved with 10% DMSO and RPMI-1640 without Phenol Red as freezing medium. Platelets were frozen by computer-controlled rate freezing and afterwards kept in liquid nitrogen. For transfusion, platelet concentrates were thawed at bedside in a warming bath (37°C) and transfused immediately without removal of DMSO. Nine patients received 36 cryopreserved autologous platelet transfusions. All patients were female and received intensive chemotherapies for acute leukemias (8) or NHL (1). The median age at diagnosis was 50 (range 30–69) years and the patients had 2 (1–4) prior pregnancies. Alloimmunization occurred 25 (15–458) days after diagnosis with a panel reactivity of 73 (14–93)%. The platelets were transfused 26 (9–133) days after cryopreservation. The platelet count before transfusion was 7 (2–16) G/L and the platelets were transfused prophylactically (32) or therapeutically (4). The median number of platelets per concentrate was 1.7 (0.8–2.9) ×10¹¹. The corrected count increment after 1 hour (1h-CCI) was 5.2 (−1.8–20.2) and after 18–24 hours 4.7 (−5.8–25.2). There was no DMSO toxicity after infusion. The phenotype and function of the cryopreserved platelets were further analyzed in preliminary in vitro experiments by flow-cytometry. As compared to fresh platelets, cryopreservation resulted in a reduction of the size and cytoplasmic complexity. The surface glycoproteins (gp) IIa and IIIb remained unchanged while the expression of the gp Ib was reduced to 54 (47–61)%. The cryopreserved platelets were partially activated, as demonstrated by the expression of negatively charged surface phospholipids (median 73%, normal <5%) and P-selectin (33%, normal <5%). However, the fibrinogen receptor was not activated, as demonstrated by PAC-1 binding (4%, normal <5%). Stimulation with strong platelet agonists (thrombin 0.5 U/ml, convulxin 50 ng/ml) induced full activation of about 1/3 of the cryopreserved platelets, as demonstrated by PAC-1 positivity (increased from 4% to 35%), and further degranulation of the alpha granula (increased from 33% to 55%) and expression of negatively charged phospholipids (increased from 73 to 99%). In conclusion, transfusion of cryopreserved autologous platelets is a feasible alternative to HLA-matched platelets offering an important clinical option for patients with HLA alloimmunization and rare HLA phenotypes. The experimental data demonstrate that the platelets are partially stimulated by the cryopreservation, but can be further activated in vitro by strong agonists, suggesting their functionality.