Platelet Refractoriness Research Articles

Refractoriness to platelet transfusion.

This review discusses the causes of refractoriness to platelet transfusions and presents three options for its management. Platelet refractoriness is a complication of platelet transfusion that affects variable proportions of patients, mostly depending on their diagnosis, previous immunologic stimuli, and type of blood products used for transfusion. A large recent study showed that platelet refractoriness develops in 13% of patients with acute leukemia transfused with traditional blood products and in 3 to 4% of recipients of white cell-reduced blood components. Options to manage platelet refractoriness include platelets from HLA-typed donors, platelet cross-matching, and the antibody specificity prediction method. The selection of the most convenient approach depends on local skills and the available economic and organizational resources. Finally, emerging concepts are presented which could impact the management of platelet refractoriness.

Immune complications associated with chronic transfusion.

Blood transfusion and component therapy are an integral part of treatment for patients with certain life-threatening illnesses. Any benefit derived from transfusion must be weighed against the potential risk for complications. Patients who are chronically transfused present a unique challenge to practitioners. The presentation will focus on alloimmunization to red blood cell antigens, platelet refractoriness, transfusion-associated graft-versus-host disease, and cytomegalovirus infection. Prevention or management of these complications also will be discussed.

Inline-filtration

Background: Routine leukocyte-depletion (LD) of cellular blood products, and even plasma, is currently being implemented in most European countries, as a result of the fear that the variant Creutzfeldt-Jakob-disease (vCJD) might be transmissible by transfusion. However, not only is the scientific evidence supporting such a notion scarce, but the benefits of applying this procedure to all patients also remain unfounded. Methods: A MEDLINE-research for studies dealing with the indications for LD was performed. In addition, the guidelines and recommendations of national and international health authorities were scrutinized. Results: To date,the only proven benefit of LD that can be applied to all patients is the reduction of non-hemolytic febrile transfusion reactions. In addition, LD reduces HLA-immunization and platelet refractoriness in multi-transfused patients. In immunocompromized patients, LD reduces transfusion-transmitted CMV-disease. Furthermore, a minority of 5–10% of transfusion-related-acute-lung-injury cases can be prevented by LD. However, the potential of reducing the immunomodulating effects of transfusion such as postoperative infection, cancer-recurrence-related or overall mortality and of reducing septicemia due to bacterial contamination is still at issue. AIDS patients do not benefit from LD, at least. The suitability of LD for preventing the transmission of vCJD is at best hypothetical. Potential risks of LD like increased leakages have not been taken into account adequately to date. Conclusions: At present, the scientific evidence does not justify the introduction of LD as a routine measure. In times of limited health care resources, this costly procedure might limit access to medical services with proven effectiveness and efficiency. In addition, the loss of 5–10 % of the red cell pool is predicted to lead to more blood supply shortages than previously seen.

Human leucocyte antigen alloimmunization after bone marrow transplantation: an association with chronic myelogenous leukaemia

Platelet refractoriness due to human leucocyte antigen (HLA) alloimmunization is a significant risk to allogeneic bone marrow transplant recipients. To identify factors contributing to this risk, we reviewed the records of 317 consecutive, paediatric, allogeneic bone marrow transplant recipients at a single institution. The 6-year estimated cumulative incidence of platelet refractoriness due to HLA alloimmunization was 2.6% +/- 0.9%. The incidence among patients with chronic myelogenous leukaemia (CML) 12.5% +/- 5.3% was significantly greater than that of other patients (1.1% +/- 0.6%, P < 0.001). Graft rejection (P = 0.003) and the number of platelet transfusions during the first 90 d after bone marrow transplantation (BMT) (P = 0.0025) were also significantly associated with alloimmunization. The association with CML and with graft rejection was not seen among patients alloimmunized before transplantation. Eight patients developed alloimmunization, of whom three had mismatched grafts and four had unrelated grafts. Alloantibody specificities, identified in seven patients, were unrelated to host or graft major histocompatibility complex (MHC). Host recognition of alloantigens in transfused blood products, not graft-host recognition, therefore seems predominantly responsible for the alloimmunization. These results show that paediatric CML patients have a significantly increased risk of platelet refractoriness due to HLA alloimmunization after BMT. Identifying the mechanism for the increased alloimmunization risk may assist in the development of therapies to prevent platelet refractoriness.

Cerebral blood flow restoration and reperfusion injury after ultraviolet laser-facilitated middle cerebral artery recanalization in rat thrombotic stroke.

A reversible model of focal thrombotic stroke was developed in the rat and examined for histological evidence of reperfusion injury after clinically relevant times of recanalization. The distal middle cerebral artery of 28 male Sprague-Dawley rats was occluded by 562-nm laser-driven photothrombosis for 0.5, 2, and 3 hours or permanently (each n=7) and was recanalized by 355-nm UV laser irradiation. Occlusive material was examined by transmission electron microscopy. Cortical cerebral blood flow was monitored by laser-Doppler flowmetry. Brain infarcts were examined histologically at 3 days. After occlusion, cortical cerebral blood flow was reduced to 33+/-4% of baseline for all groups and was restored to 82+/-9%, 75+/-3%, and 93+/-7% of baseline for the 0.5-, 2-, and 3-hour groups, respectively, following recanalization after 29+/-8, 38+/-20, and 70+/-33 minutes of UV laser irradiation. The thrombotic occlusion contained compactly aggregated platelets but no fibrin, with length (1.2 to 1.8 mm) proportional to the ischemic period. During recanalization, microchannels containing erythrocytes and scattered leukocytes and bordered by intact disaggregated platelets infiltrated the thrombus. Infarct volumes (mm3) at 3 days were 12+/-3 for the permanent case and 8+/-4, 24+/-3, and 30+/-9 for the 0.5-, 2-, and 3-hour cases, respectively, thus demonstrating reperfusion injury histologically in the latter 2 groups. No hemorrhage was seen. UV laser-facilitated dissolution of a conventionally refractory platelet thrombus provides a novel and effective method for restoring blood flow without hemorrhagic complications during thrombotic stroke. This was the first observation of histologically confirmed reperfusion injury in such a model.

Treatment of Bleeding in Patients with Platelet Disorders: Is There a Place for Recombinant Factor VIIa?

The mechanism of action of recombinant factor VIIa (rFVIIa), i.e. increased thrombin generation on the membrane of activated platelets, as well as the results from in vitro and ex vivo models of thrombocytopenia or inherited thrombocytopathia may support some potential of rFVIIa in thrombocytopenia/thrombocytopathia. rFVIIa was reported as effective to stop or to decrease bleeding in few patients with severe thrombocytopenia resistant to platelet transfusions; however data are still scarce and clinical studies are really needed to define efficacy/safety ratio as well as optimal treatment regimen in this potential indication. Some data in patients with Glanzmann thrombasthenia (GT) may support the use of rFVIIa outside its primary indication in the cases in which there is no real treatment alternative (GT patients with antibodies to GP IIb-IIIa or with platelet refractoriness).

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

Transient adhesion refractoriness of circulating platelets under shear stress: the role of partial activation and microaggregate formation by suboptimal ADP concentration.

Exposure of whole blood (WB) to subendothelial extracellular matrix (ECM) under shear stress in the cone and plate(let) analyser (CPA) results in platelet adhesion, followed by release reaction and aggregation of circulating platelets on the adherent platelets. The properties of circulating non-adhered platelets in the CPA was studied by exposure of WB to ECM at a high shear rate (1300/s) for 2 min (1st run), followed by transfer of the suspension to a new ECM-coated well for a second run (2nd run) under similar conditions. The results of the 2nd run demonstrated transient adhesion refractoriness associated with platelet microaggregate formation in the suspension. The adhesion refractoriness was dependent on platelet activation during the 1st run and was prevented by addition of apyrase (ADP scavenger) or ADP receptor inhibitor, suggesting a role for ADP in mediating this response. Furthermore, exposure of WB samples to suboptimal concentrations of ADP (0.4-1 micromol/l) or a thrombin receptor activating peptide (TRAP) (5 micromol/l) for 2 min resulted in a similar transient platelet adhesion refractoriness to ECM under flow conditions. The transient platelet refractoriness and microaggregate formation induced by ADP was associated with a transient reduction in glycoprotein (GP)Ib, increased P-selectin expression and increased fibrinogen binding by circulating platelets. These data suggest a role for platelet agonists at suboptimal concentrations in modulating platelet function and limiting the expansion of the thrombus.

Research opportunities in transfusion medicine.

In recent years, the translation of basic research in transfusion medicine has led to development of novel cellular therapies using well-characterized cell populations isolated from either bone marrow or blood (eg, hematopoietic stem and progenitor cells, T lymphocytes, dendritic cells). Refinements in cell therapies will make possible optimal stem cell engraftment, gene therapy, immunotherapy of cancer and infectious disease, and even solid organ regeneration. Moreover, the immune consequences of transfusion therapy are better appreciated and opportunities are at hand to prevent or blunt unwanted immune responses, such as platelet refractoriness and graft-vs-host disease. Transfusion medicine has become a broad, multidisciplinary field that has evolved beyond issues related to blood procurement and storage. The next series of advances in transfusion medicine will complement the current approaches of donor blood screening and viral/bacterial inactivation steps to ensure a safe and adequate blood supply.

Current issues with blood transfusions in sickle cell disease

With increased recognition of the profound morbidity of sickle cell disease and with growing evidence of the efficacy of transfusion therapy in prevention and treatment of sickle cell complications, most patients now receive intermittent transfusion therapy. The purpose of this report is to review blood component therapy and its risks for sickle cell patients. Packed red cells are the preferred blood component. Leukocyte-reduced units should be standard because of their beneficial effects in reducing alloimmunization, transfusion reactions, platelet refractoriness, and infection transmission. The use of washed, frozen, or irradiated units is limited to specific problems. Sickle trait-positive units function normally, but because of difficulties with calculating hemoglobin S percentages and leukocyte filters, they are not routinely used. Transfusion-acquired infections have shown a marked decrease but still present a major risk. Viral hepatitis transmission is currently low, but at least 10% of adult sickle cell patients are hepatitis C positive, and they often have liver damage. Although bacterial infections are rare, they account for 16% of transfusion-related fatalities. Patients who are iron overloaded are particularly vulnerable to Yersina enterocolitica. Red cell alloimmunization is a serious problem that could potentially affect 50% of transfused patients. However, preventive phenotypic matching for common antigens can minimize alloimmunization; limited matching for at least E, C, and K has become the standard of care. Recently, more patients are being identified who have developed red cell autoantibodies, which can mask alloantibodies and occasionally are hemolytic. Careful laboratory evaluation of all cases is essential. Transfusions also may trigger sickle cell events, including pain crises, stroke, and acute pulmonary deterioration. In part, these are induced by blood viscosity and increased blood pressure. Diuretic therapy and close monitoring of transfusion volume and vital signs can minimize these events. In summary, transfusion therapy carries risks, but the routine use of leukocyte-reduced, phenotypically matched units in conjunction with close monitoring of patients can make transfusion therapy safer.

The immunogenicity of Gov alloantigens is comparable to that of the HPA-5 system

The 175 kDa glycosylphosphatidylinositol (GPI)-linked glycoprotein CD109 carries the bi-allelic platelet system, Gov. To date, detection of Gov antibodies has relied on the immunoprecipitation of radio-labelled CD109. We have modified the monoclonal antibody-specific immobilization of platelet antigen assay for the detection of Gov antibodies. Screening of 605 archived patient samples referred for neonatal alloimmune thrombocytopenia (NAITP, n = 305); post-transfusion purpura (PTP, n = 60); and platelet refractoriness (PR, n = 240) revealed 14 Gov antibodies (10 anti-Gova, 4 anti-Govb) which equals the number of HPA-5 antibodies detected in the 605 samples. There is no obvious linkage between the HLA class II type and the ability to mount an immune response. Phenotyping of 113 donors by MAIPA showed frequencies of 19·5%, 40·7% and 39·8% for ‘aa, ab and bb’, respectively, with ‘a’ and ‘b’ allele frequencies of 0·40 and 0·60. Cloning of the CD109 gene and determination of the molecular basis of the Gov antigens has become relevant now that it has been established that Gov alloantibodies are more frequent than previously thought.

Glanzmann thrombasthenia and isoimmunization

Glanzmann thrombasthenia is a rare autosomal recessive inherited bleeding disorder characterized by the inability of the platelets to aggregate. The abnormality results from a quantitative or qualitative defect in the platelet membrane glycoprotein (GP) IIb-IIIa. A potential consequence of platelet membrane GP deficiency is isoantibody (isoAb) production following platelet transfusion or during pregnancy, when the patient has contact with normal platelets. IsoAb have been reported in Glanzmann's thrombasthenia patients lacking GPIIb-IIIa (GTI), which recognize distinct epitopes on the GPIIb-IIIa complex. These antibodies (Ab) are of clinical significance as they can lead to platelet refractoriness by destruction of the transfused platelets and thus to therapeutical difficulties. The development of antigen specific assays (as the MAIPA technique) has been extremely useful, allowing the binding of the antibody to its epitope in a conformational state. One of the major disadvantages of this technique is the existence of false negative results due to a competition between the human Ab and monoclonal antibodies (MoAbs) for the same or closely related epitopes. Negative or discordant results for identification or characterization of Ab are a matter of concern during investigation for isoimmunization. In an attempt to demonstrate the importance of the choice of MoAbs for accurate biological diagnosis and to show the serological heterogeneity among isoAb, we screened serum from GTI patients by the MAIPA technique with a panel of MoAbs. These studies were particularly informative as we found striking differences in the binding of the human Ab to their target antigen depending on the MoAbs present in the reaction. Furthermore the choice of MoAbs can be crucial for detecting a specific Ab in case of low reactivity or low titre. Further investigations are needed to clarify whether this heterogeneity plays a role in clinical conditions.

Detection of Gov system antibodies by MAIPA reveals an immunogenicity similar to the HPA-5 alloantigens.

The glycosylphosphatidylinositol-linked platelet protein CD109 carries the biallelic alloantigen system Gov. There is limited information on the incidence of Gov alloantibodies in neonatal alloimmune thrombocytopenia (NAITP), post-transfusion purpura (PTP) and platelet refractoriness. We adapted the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay to the detection of Gov antibodies and determined their incidence in 605 archived samples (112 with HPA antibodies) referred for the aforementioned conditions. Here, we show that CD109 expression was reduced upon platelet storage in saline or by cryopreservation, but was stable when stored as whole blood or therapeutic platelet concentrate. Fourteen of the 605 samples contained Gov alloantibodies (anti-Gova, n = 10; anti-Govb, n = 4), with the majority in platelet refractoriness (n = 9) and, of the remaining five, four in NAITP and one in PTP. In seven cases, no other HPA antibodies were detected, three being NAITP cases. The incidence of Gov antibodies was significantly lower than HPA-1 system antibodies (n = 87), but equalled the number of HPA-5 system antibodies (n = 14) and outnumbered HPA-2 and -3 system antibodies (10 altogether).

HLA‐C Expression on Platelets: Studies with an HLA‐Cw1‐Specific Human Monoclonal Antibody

Abstract Background and Objectives: The expression of HLA‐C on the surface of platelets is rarely studied due to the lack of proper alloantisera. We addressed this question using an IgM human monoclonal antibody directed against HLA‐Cw1 (VP6G3). Material and Methods: Both flow cytometry and complement dependent cytotoxicity studies were used in the current analysis. Results: The expression of the HLA‐Cw1 antigen on platelets is lower than on peripheral blood lymphocytes as shown by flow cytometry. Variation in expression levels between individuals is also observed. Using this antibody in a complementdependent cytotoxicity assay, we did not observe lysis using platelets as targets, whereas peripheral blood lymphocytes of the same blood donors were adequately lysed. Conclusions: These results confirm that platelets indeed express HLA‐C. Furthermore, the results support the insignificant role of HLA‐C in immunological platelet refractoriness.

HLA-C Expression on Platelets: Studies with an HLA-Cw1-Specific Human Monoclonal Antibody

Background and Objectives: The expression of HLA-C on the surface of platelets is rarely studied due to the lack of proper alloantisera. We addressed this question using an IgM human monoclonal antibody directed against HLA-Cw1 (VP6G3). Material and Methods: Both flow cytometry and complement dependent cytotoxicity studies were used in the current analysis. Results: The expression of the HLA-Cw1 antigen on platelets is lower than on peripheral blood lymphocytes as shown by flow cytometry. Variation in expression levels between individuals is also observed. Using this antibody in a complement-dependent cytotoxicity assay, we did not observe lysis using platelets as targets, whereas peripheral blood lymphocytes of the same blood donors were adequately lysed. Conclusions: These results confirm that platelets indeed express HLA-C. Furthermore, the results support the insignificant role of HLA-C in immunological platelet refractoriness.

Leukocyte-reduced blood transfusions: perioperative indications, adverse effects, and cost analysis.

Leukocyte-reduced blood transfusions: perioperative indications, adverse effects, and cost analysis.

The evaluation and management of platelet refractoriness and alloimmunization

T HE ARREST of hemorrhage associated with an increase in the patient's platelet count after blood transfusion was first reported by Duke in 1910.' It was not until the 1960s that clinical studies showed that therapeutic platelet transfusions decreased fatal hemorrhagic complications in thrombocytopenic patients2 and that prophylactic platelet transfusions reduced episodes of bleeding.3 More recent reports have suggested that a threshold of I O,OOO/~ for prophylactic platelet transfusion is equivalent to 20,OOO/~ in the absence of additional complicating clinical factors such as platelet dysfunction, associated coagulopathy, or primary defects of vascular integrity.4.5 The hemostatic benefit of platelet transfusion is presumably mediated through an increase in the posttransfusion platelet count above a target level, and clai!s that platelet transfusion may be beneficial even in the absence of such an increase are not commonly accepted.

Evaluation of Cobe Trima for the collection of blood components with particular reference to the in vitro characteristics of the red cell and platelet concentrates and the clinical responses to transfusion

This study evaluated Cobe Trima for donor and operational acceptability, the quality and storage stability of the blood components collected, and the clinical responses to transfusion. The study was carried out in 2 phases; phase 1 assessed the efficiency of red cells and platelet collection, and the characteristics of the components collected before and after storage. Phase 2 was an evaluation of operational issues and the in vitro characteristics of the red cells and platelet concentrates at the time of transfusion in respect to their cellular content, and leucocyte (interleukin IL-6 and IL-8) and platelet-derived (Rantes) cytokine levels. Cytokine levels were also measured in the donors before and after the collection procedure and in patients both before and after transfusion. The clinical responses to a small number of transfusions were assessed. The Cobe Trima was found to be straightforward to use by the operators, although additional operator training was required to manage occasional uncertainty with alarm messages. It was acceptable to the donors except for the occurrence of citrate reactions in 3/6 donors in phase 1; this problem persisted in phase 2 (6/15 donors), and needs to be addressed in the future. All blood components met UK product specifications apart from 2 platelet concentrates, 2 red cell concentrates, and one unit of FFP; the red cell and platelet concentrates had good storage characteristics. The 2 procedures, which resulted in low platelet yields, were due to occlusion of the plasma line; the method for installation of the harness has been subsequently modified to prevent this. 2 red cell concentrates showed haemolysis; the reason for this was not established. The Factor VIII level was satisfactory in plasma and the cellular content was low. The responses to 12 platelet transfusions were expected as in a group of haematology patients, and no immediate adverse effects were reported with any of the transfusions. Leucocyte-associated (IL-8 and IL-6) and platelet-associated (Rantes) cytokine levels were not elevated in donor samples taken before or after the collection procedure, or in the red cell and platelet concentrates at the time of issue. Pre- and post-transfusion IL-8 levels were raised in one patient with non-immune platelet refractoriness, and normal in 2 patients with excellent or almost satisfactory responses to platelet transfusions raising the question as whether IL-8 could be used as a laboratory marker for non-immune platelet refractoriness due to infection.

Pesquisa de anticorpos antiplaquetários. As primeiras cem amostras estudadas.

Two types of platelet alloantigens are considered: type I, which refers to platelet alloantigens shared by platelets and other cells; and type II, which comprises the platelet-specific alloantigens. The Human Platelet Alloantigens (HPA) System consists of five diallelic alloantigens and eleven low frequency antigens. According to serological antigen capture assays, immunochemical methods and studies of Molecular Genetics, the glycoproteins (GP) of platelet membrane that are most frequently associated with antibodies are GPIIb-IIIa, GPIb-IX and GPIa-IIa. It is possible to detect platelet antibodies using different laboratory methods. Our experience was restricted to a solid phase system. In this method we used the indirect technique for the screening of alloantibodies and the direct technique for the detection of autoantibodies. The procedure with chloroquine solution was performed in order to differentiate between the presence of HLA and non-HLA platelet specific antibodies. Our comments are based on results obtained in a hundred samples tested throughout a period of eleven months and we briefly review the clinical features related to the diagnosis of autoimmune and alloimmune thrombocytopenia, drug-induced thrombocytopenia and other different factors of platelet destruction. The concepts of transfusional practice applied to emergency situations, platelet refractoriness strategy and the comprehensive management of patients are approached in this work.

Effects of Endotoxemia on Thrombopoiesis in Men

Febrile conditions are often associated with increased platelet turnover and refractoriness to platelet transfusions, although several pyrogenic cytokines enhance thrombopoiesis. This study aimed to characterize the effects of experimental human endotoxemia on platelet turnover and thrombopoiesis. Endotoxin (4 ng/kg) was infused into 30 healthy men to study the regulation of thrombopoiesis in systemic human inflammation. Platelet counts, plasma thrombopoietin (TPO) and glycocalicin levels, and reticulated platelets (RP) were measured to evaluate the effect of acute endotoxemia on thrombopoiesis. Ten subjects received pretreatment with 1000 mg aspirin po. to evaluate possible effects of aspirin on platelet turnover, and ten subjects received paracetamol to control for effects of antipyresis. Platelet counts dropped by about 15% (p <0.001) one hour after LPS infusion, began to recover at 24 h, and exceeded baseline values by 81% (CI: 5-12; p <0.001 ) at 7 days after LPS iv. Reticulated platelet counts increased from 1.62% (CI: 1.24-2.0) to a maximum of 2.39% (CI: 1.81-2.98; p = 0.003) at 6 h. TPO levels increased from baseline values of 10 A.U/ml (CI: 8.8-11.2) to 15.5 A.U/ml (CI: 13.6-17.3) at 24 h (p <0.001), whereas plasma glycocalicin was not changed (p >0.05). The number of circulating platelet-neutrophil aggregates increased more than 100% at 6 h (p <0.001). Neither aspirin nor paracetamol affected changes in any of the parameters measured. Low grade endotoxemia induces a rapid fall of platelet counts, which is followed by an early increase in reticulated platelets and TPO levels but not of glycocalicin levels. Finally peripheral platelet counts increase several days after LPS infusion.