Further studies to evaluate methods of leucoreduction to prevent alloimmune platelet refractoriness and induce tolerance in a dog platelet transfusion model.

OBJECTIVES: Three leucoreduction filters were evaluated - when used alone or combined with centrifuge leucoreduction (C-LR) - to prevent alloimmune platelet refractoriness in a dog platelet transfusion model. MATERIALS AND METHODS: Donor platelet-rich plasma (PRP) or buffy coat (BC) platelets were either filter leucoreduced (F-LR) or F-LR/C-LR, (51) Cr radiolabelled and transfused. Weekly transfusions were given for up to 8 weeks or until platelet refractoriness. Recipients who accepted treated transfusions were then given non-leucoreduced (non-LR) platelets to determine whether donor-specific tolerance had been induced. RESULTS: Acceptance of F-LR PRP transfusions ranged from 29% to 66%. F-LR/C-LR transfusions prepared from PRP were accepted by 92%, from BC by 63% and from pooled PRP by 75% of recipients (p=NS); overall acceptance rate of F-LR/C-LR transfusions was 83%. Tolerance to subsequent non-LR transfusions occurred in 45% of the F-LR-/C-LR-accepting recipients unrelated to DR-B compatibility between donors and recipients (P = 0·18). CONCLUSION: In a dog platelet transfusion model, acceptance of donor platelets required combining F-LR with C-LR as apparently each process removes different immunizing WBCs.

Clinical and laboratory correlates of platelet alloimmunization and refractoriness in the PLADO trial.

BACKGROUND AND OBJECTIVES: Platelet alloimmunization and refractoriness to platelet transfusion are complications of platelet transfusion therapy. The platelet dose (PLADO) trial, as the largest prospective randomized trial of prophylactic platelet therapy to date, afforded an opportunity to analyse these two issues. MATERIALS AND METHODS: PLADO patient records were examined for evidence of platelet alloimmunization, defined as an increase in HLA Class I panel-reactive antibodies (PRA) to ≥20%, and clinical refractoriness, defined as two consecutive ≤4 h posttransfusion corrected platelet count increments (CCI) of <5000. Multivariate logistic regression, restricted to platelet-transfused subjects who received exclusively either in-process leucoreduction apheresis or whole blood-derived (WBD) leucocyte-reduced platelets, compared the frequency of these outcomes by platelet unit and patient characteristics. RESULTS: Forty of 816 evaluable platelet-transfused patients (5%) became alloimmunized during the trial. Prior pregnancy, chemotherapy only compared to progenitor cell transplant, and low platelet dose - all were associated with significantly higher rates of alloimmunization. Among 35 alloimmunized patients evaluated for refractoriness, 8 (23%) had two consecutive CCI < 5000/µl. Regardless of alloimmunization status, CCIs < 5000/µl were observed following 17% of platelet transfusions. Among 734 patients receiving at least two platelet transfusions, two consecutive CCIs of ≤5000 occurred in 102 (14%). CONCLUSIONS: The incidence of new platelet alloimmunization was low in the PLADO study, but follow-up was at most 30 days. Alloimmunization was present in only 8 of 102 (8%) of observed cases of refractoriness, suggesting that other causes of poor posttransfusion increments are frequent.

Extended storage of autologous apheresis platelets in plasma.

BACKGROUND AND OBJECTIVES: The purpose of our studies was to determine the effects of extended platelet storage on poststorage platelet viability. MATERIALS AND METHODS: Normal subjects were recruited to donate platelets using two different apheresis systems: either the COBE Spectra (n = 58) or the Haemonetics MCS+ (n = 84). Platelet recovery and survival data from the two systems were compared with each other and with in vitro measurements of the stored platelets. RESULTS: There were no significant differences in either platelet recoveries or survivals between the two machines between 1 and 8 days of storage. Combining the data from both machines, platelet recoveries decreased by 2.6% and survivals by 0.3 days/storage day. In vitro assays did not predict either platelet recoveries or survivals during storage for 5-8 days. After 9 days of storage, pHs were unacceptable (≤ 6.1), suggesting that 8 days will be the longest possible storage time. CONCLUSIONS: These data suggest that, if stored platelet bacterial contamination issues are resolved, significant extension of platelet storage times is possible.

Homocystine-induced arteriosclerosis. The role of endothelial cell injury and platelet response in its genesis.

The atherogenic mechanism of homocystinemia has been defined by measuring endothelial cell loss and regeneration, platelet consumption, and intimal lesion formation in a primate model. Three groups of baboons were studied: (a) 8 control animals; (b) 15 animals after 3 mo of continuous homocystinemia; and (c) 11 animals after 3 mo of combined homocystinemia and oral treatment with dipyridamole. Experimental homocystinemia caused patchy endothelial desquamation comprising about 10% of the aortic surface despite a 25-fold increase in endothelial cell regeneration. Neither endothelial cell loss nor regeneration was changed significantly by dipyridamole. Homocystine-induced vascular deendothelialization produced a threefold increase in platelet consumption that was interrupted by dipyridamole inhibition of platelet function. All homocystinemic animals developed typical arteriosclerotic or preatherosclerotic intimal lesions composed of proliferating smooth muscle cells averaging 10-15 cell layers surrounded by large amounts of collagen, elastic fibers, glycosaminoglycans, and sometimes lipid. Intimal lesion formation was prevented by dipyridamole therapy. We conclude that homocystine-induced endothelial cell injury resulted in arteriosclerosis through platelet-mediated intimal proliferation of smooth muscle cells that can be prevented by drug-induced platelet dysfunction.

Mechanisms of thrombocytopenia in chronic autoimmune thrombocytopenic purpura. Evidence of both impaired platelet production and increased platelet clearance.

Mechanisms of thrombocytopenia were studied in 38 patients with mild to moderately severe chronic autoimmune thrombocytopenia (AITP). 51Cr and 111In-labeled autologous platelet turnover studies and in vitro analysis of committed megakaryocyte progenitors (CFU-Meg) were used as independent measures of platelet production. Autologous 111In-labeled platelet localization studies were performed to assess platelet clearance. Although there was no increase in the frequency of marrow CFU-Meg, a specific increase in the CFU-Meg [3H]TdR suicide rate was seen which was inversely correlated with the platelet count (P less than 0.001). Platelet turnover studies showed significant numbers of patients had inappropriate thrombopoietic responses to their reduced platelet counts. Platelet-associated antibody levels correlated inversely with platelet turnover suggesting that antiplatelet antibody impairs platelet production. The circulating platelet count was best predicted by an index relating platelet production (i.e., turnover) to the spleen-liver platelet clearance that correlated directly with platelet survival (P less than 0.001). In summary, both depressed platelet production and increased platelet clearance by the liver and spleen contribute to the thrombocytopenia of AITP.

Platelet and fibrinogen kinetics in canine tumors.

Fifty-three dogs with spontaneously occurring tumors were evaluated for abnormalities in the concentration and in vivo survival of platelets and fibrinogen. Thrombocytopenia occurred only in animals with extensive tumor involving spleen or marrow. Platelet survival was shortened in 6 of 15 (40%) dogs with localized tumor [mean 4.4 days +/- 0.3 (S.E.); normal 5.4 days +/- 0.1] and 30 of 35 (80%) dogs with metastatic tumor (mean 3.2 days +/- 0.2). Platelet survival progressively shortened during studies performed in dogs with ongoing disease. Fibrinogen concentration was increased (mean 420 +/- 30 mg/dl) in 44 of 53 (83%) of tumor-bearing dogs (normal 210 +/- 10 mg/dl). Neither history nor extent of disease, including presence of hepatic metastases, appeared to influence fibrinogen concentration significantly. Fibrinogen survival was below the normal range in 3 of 15 (20%) dogs with localized tumor and in 9 of 34 (26%) dogs with metastatic tumors. Thus, platelet consumption appeared to be the most significant hemostatic abnormality in tumor-bearing dogs. This model may be useful in evaluating the efficiency of antithrombotic therapy in preventing tumor-related hemostatic abnormalities.

Platelet refractoriness and alloimmunization.

The two major methods of modifying donor blood products to prevent alloimmunization are leukocyte reduction or ultraviolet B (UVB) irradiation. Two studies have suggested that leukocyte reduction to levels <5 x 10(6) may be required to prevent alloantibody production. Three prospective, randomized transfusion trials demonstrated a statistically significant (P < 0.05) decrease in both platelet refractoriness and lymphocytotoxic antibody production in patients who received leukocyte-reduced blood components as compared to those who received standard unmodified blood products. The results of the Trial to Reduce Alloimmunization to Platelets (TRAP trial) further confirm the potential beneficial effects of leukocyte-reduced and UVB-irradiated blood products in preventing alloimmune platelet refractoriness. Five hundred thirty antibody-negative patients undergoing induction chemotherapy for acute myeloid leukemia were randomly assigned to receive either unmodified platelet concentrates, filtered leukocyte-reduced platelet concentrates, UVB-irradiated platelet concentrates, or filtered leukocyte-reduced platelets obtained by apheresis. Patients who received modified platelet components had statistically significantly lower rates of both alloimmune platelet refractoriness and lymphocytotoxic antibodies than did patients who received unmodified platelet components. There were no differences in any study endpoints among patients who received any of the three modified platelet components. The investigators concluded that leukocyte-reduced and UVB-irradiated platelet components were equally effective in preventing alloimmune-mediated platelet refractoriness; platelets obtained by apheresis provided no additional benefit.

A platelet monoclonal antibody inhibition assay for detection of glycoprotein IIb/IIIa-related platelet alloantibodies.

Post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAT) result from formation of alloantibodies to platelet membrane glycoprotein-associated antigens. The detection and identification of platelet-specific alloantibodies in patient sera is often complicated by the presence of co-existing HLA antibodies and/or more than one platelet specificity in the same serum. We describe a solid phase assay that specifically detects antibodies to platelet membrane associated alloantigens by measuring the ability of patient antisera to inhibit the binding of glycoprotein GPIIb or GPIIIa monoclonal antibodies to intact platelets. When tested in the GPIIIa assay against a panel of random platelet donors, the reactivities of two known PLAI antisera that also contained different HLA antibodies were highly correlated (r = 0.99) and allowed PLA phenotyping of the population. A standard direct binding platelet ELISA, on the other hand, was unable to accurately PLA phenotype the same population. The reactivities of two known Baka antisera (one containing additional anti-PLA2 and the other anti-Brb specificities) were highly correlated (r = 0.95) in the GPIIb assay, and Bak phenotype determination was similarly accomplished for a random platelet panel. Furthermore, a comparison of platelet phenotype results (using the monoclonal inhibition assay) and genotype results (using DNA analysis) for the PLA and Bak systems showed a concordance of 98% for 146 alleles tested. In conclusion, the platelet monoclonal antibody inhibition assay: (1) allows determination of platelet-specific alloantibodies in the presence of contaminating HLA antibodies and/or in sera containing multiple platelet alloantibodies; (2) allows accurate platelet phenotyping for the GPIIIa-associated PLA and GPIIb-associated Bak antigen systems; and (3) may be applicable to the detection of other known or even novel platelet glycoprotein-associated antigens.

Plasma exchange for platelet alloimmunization.

We studied the effectiveness of plasma exchange for reversing antiplatelet alloimmunization in 18 patients undergoing marrow transplantation. Patients received one-to-three daily exchanges. Most exchanges occurred during the pretransplant conditioning phase. Overall, eleven of 18 patients had a beneficial response to plasma exchange, defined as an improvement in post-platelet-transfusion increments. More patients responded who had received two or more exchanges. Patients who had lymphocytotoxic antibodies pretransplant were more likely to benefit from the exchange. Our results show that plasma exchange may improve the posttransfusion platelet increments in alloimmunized patients undergoing marrow transplant.

The treatment of rejection. A trial of acetylsalicylic acid, dipyridamole, and heparin.

Serial studies of platelet and fibrinogen survival were performed in 26 nonimmunosuppressed dogs after allogenic renal transplant operations. Treatment with acetylsalicylic acid, dipyridamole, and heparin failed to improve the selective platelet destruction which occurred in untreated animals, and it did not improve postoperative longevity. There was a high incidence of postoperative wound and intrarenal hemorrhage after heparin treatment. These results are consistent with the hypothesis that platelet destruction is a consequence rather than the cause of acute graft rejection, and it is concluded that antithrombotic therapy is not of practical benefit in preventing acute rejection.

Platelet destruction in autoimmune thrombocytopenic purpura: kinetics and clearance of indium-111-labeled autologous platelets.

Using autologous 111In-labeled platelets, platelet kinetics and the sites of platelet destruction were assessed in 16 normal subjects (13 with and three without spleens), in 17 studies of patients with primary autoimmune thrombocytopenic purpura (AITP), in six studies of patients with secondary AITP, in ten studies of patients with AITP following splenectomy, and in five thrombocytopenic patients with myelodysplastic syndromes. In normal subjects, the spleen accounted for 24 +/- 4% of platelet destruction and the liver for 15 +/- 2%. Untreated patients with primary AITP had increased splenic destruction (40 +/- 14%, p less than 0.001) but not hepatic destruction (13 +/- 5%). Compared with untreated patients, prednisone treated patients did not have significantly different spleen and liver platelet sequestration. Patients with secondary AITP had similar platelet counts, platelet survivals, and increases in splenic destruction of platelets as did patients with primary AITP. In contrast, patients with myelodysplastic syndromes had a normal pattern of platelet destruction. In AITP patients following splenectomy, the five nonresponders all had a marked increase (greater than 45%) in liver destruction compared to five responders (all less than 40%). Among all patients with primary or secondary AITP, there was an inverse relationship between the percent of platelets destroyed in the liver plus spleen and both the platelet count (r = 0.75, p less than 0.001) and the platelet survival (r = 0.86, p less than 0.001). In a stepwise multiple linear regression analysis, total liver plus spleen platelet destruction, the platelet survival and the platelet turnover were all significant independent predictors of the platelet count. Thus platelet destruction is shifted to the spleen in primary and secondary AITP. Failure of splenectomy is associated with a marked elevation in liver destruction. The magnitude of spleen and liver destruction appears to be of considerable importance in the severity of the disease, as reflected in the platelet survival and platelet count.

Engraftment and transfusion requirements after allogeneic marrow transplantation for patients with acute non-lymphocytic leukemia in first complete remission.

This retrospective study analysed factors affecting engraftment and transfusion requirements of platelets and red blood cells in 303 patients transplanted for acute non-lymphocytic leukemia in first remission from HLA-identical or one-antigen mismatched donors. Multivariant analysis showed that the most important factors affecting the speed of engraftment were drugs used for graft-versus-host disease (GVHD) prophylaxis, the development of acute GVHD and HLA matching. Factors affecting only granulocyte recovery included patient age and sex. The radiation regimen used for preparing patients affected the time to platelet independence. Patients transplanted in laminar airflow rooms took longer to achieve red cell independence and required more units of red cells and platelets than patients transplanted in regular rooms. In addition, ABO incompatibility affected red cell transfusion requirements while GVHD prophylaxis and acute GVHD influenced both red blood cells and platelet support.

A pilot study of continuous infusion heparin for the prevention of hepatic veno-occlusive disease after bone marrow transplantation.

Twenty-eight patients undergoing marrow transplantation participated in a pilot study to determine the safety of continuous infusion heparin for the prevention of veno-occlusive disease (VOD) of the liver. Four doses of continuous infusion heparin were administered, ranging from a dose prolonging the partial thromboplastin time (PTT) to 1.5-2.0 times the patients' baseline value, to a dose prolonging the PTT to less than 1.2 times the patients' baseline value. Seven patients (25%) received a full course of heparin, beginning from the day the preparative therapy started through day 14 post-transplant (range 20-26 days on heparin). In 21 patients infusions were ended before day 14 post-transplant, a median of 16 days on heparin (range 1-26 days). Of these, 14 patients were withdrawn from heparin because of bleeding and seven were withdrawn because of anticipated bleeding. Bleeding was observed in 27 patients and was minor in 25. Two patients developed major bleeding in the gastrointestinal tract which was not fatal. Minor bleeding was observed in 27 of 28 case control patients who did not receive heparin. The sites of bleeding were similar in control and heparin treated patients. VOD developed in 20 patients (71%) and was sever or fatal in four (14%). The prevalence of VOD was not influenced by the dosage of heparin or the duration of its administration. We conclude that low dose heparin resulting in marginal prolongation of the PTT may be infused into patients undergoing marrow transplantation with a low risk of serious bleeding. Further studies are needed to evaluate its efficacy.

Transfusion and bone marrow transplantation.

Bone marrow transplantation has prolonged the lives of a significant percentage of patients with a variety of both malignant and nonmalignant disorders. However, the impact of this treatment on a transfusion service is substantial. Large numbers of often specialized blood products are required to support these patients, and the logistics of accomplishing this taxes the ingenuity and resources of even large regional blood programs.

Platelet transfusion therapy.

This article reviews appropriate platelet support of those patients who are chronically thrombocytopenic because of decreased platelet production, including those receiving chemotherapy or radiation treatments and those undergoing bone marrow transplants. The platelet products available for transfusion and the indications for platelet transfusions are discussed. Expected responses to platelet transfusions are reviewed as well as the prevention of platelet alloimmunization. Finally, a discussion of the mechanisms and management of platelet refractoriness is included.

Increasing participation of blood donors in a bone-marrow registry.

In an experiment to increase recruitment of unrelated bone-marrow donors, Ss were selected from a list of people who had donated blood within the past 24 months. They were randomly assigned to 3 groups. Members of the experimental group, 2 months before receiving a mailed brochure about a bone-marrow registry, were complimented on being blood donors and asked to complete a self-descriptive questionnaire. One control group received only the mailed brochure, and the other did not receive any mailing. The experimental group joined the registry at over 2 times the control-group rates. These results appear to be attributable to an attitude change associated with being recognized as a special group that contributed to the community's welfare.