Alloimmunization following platelet transfusion: the absence of a dose- response relationship

A major concern about the use of prophylactic platelet transfusions is the development of alloimmunization. To determine whether the rate of alloimmunization is related to the number of platelet transfusions, we measured the development of lymphocytotoxic antibody in the first 2 mo of induction therapy in patients with acute nonlymphocytic leukemia. All patients received prophylactic random donor platelets and packed red blood cells during induction. No patient had lymphocytotoxic antibody present at admission. One hundred and six patients received an average of 9.3 platelet transfusions (range 2-34) containing an average of 61 U (range 9-236). The rate of alloimmunization was 38% overall and correlated with refractoriness to platelet transfusions. Ten of 19 patients receiving less than or equal to 4 transfusions became immunized, compared with 30/87 patients receiving less than 4 transfusions. There was no relationship between the number of platelet transfusions given and the rate of severity of alloimmunization, suggesting prophylactic platelets need not be withheld expressly to prevent alloimmunization.     

Prevention and Management of Platelet Transfusion Refractoriness

Platelet transfusion refractoriness is a major complication of long-term platelet supportive care. Refractoriness may lead to fatal bleeding complications in thrombocytopenic patients. Major factors involved are factors related to the clinical condition of the patient as well as HLA alloimmunisation. Non-alloimmune factors may occur in up to 80% of the patients. However, platelet transfusion outcome is impaired in only 50% of the patients having these conditions. HLA alloimmunisation has been convincingly reduced by the use of leucocytedepleted transfusions. UV-B irradiation of platelet transfusions may be alternatively used to reduce HLA alloimmunisation. Despite these measures, patients with a history of pregnancy or non-leucocyte-depleted transfusions form HLA antibodies in a high proportion (up to 50%). HPA antibodies play a minor but relatively important role in patients with HLA antibodies. ABO antibodies may play a role in refractoriness, which can be abolished by transfusion of ABO-identical platelets. Screening for the presence of HLA and/or HPA antibodies is indicated in case of transfusion failure after ABO-identical or HLA-matched platelets. If no alloantibodies are detected, further analysis to define a role of drugrelated or autoantibodies is required. In case of HLA and/or HPA alloimmunisation associated with refractoriness, matched platelet transfusions are indicated. In case of non-alloimmune factors associated with increased platelet consumption, increasing the transfusion frequency can be considered. Additional investigations are still necessary to define risk factors for secondary HLA alloimmunisation and refractoriness due to non-immune factors to further decrease the incidence of refractoriness.     

Factors influencing the transfusion response to HLA-selected apheresis donor platelets in patients refractory to random platelet concentrates

Immune and nonimmune causes of platelet refractoriness were evaluated in a group of patients receiving HLA-selected single-donor platelet transfusions. During a 1-year observation period, 1 h and 24 h platelet recoveries wre determined after 522 single-donor platelet transfusions given to 43 patients persistently refractory to pooled random-donor platelet transfusions. 72% of patients tested ultimately developed lymphocytotoxic antibodies suggesting they were alloimmunized. When significant lymphocytotoxic antibodies were demonstrable in these patients, HLA well-matched platelet transfusions consistently produced good transfusion responses. In contrast, patients without lymphocytotoxic antibodies had clinical factors that adversely affected transfusion outcome (P less than 0.0001). Fever and splenomegaly markedly reduced 1 h post-transfusion platelet recoveries, while sepsis compromised the 24 h platelet recovery. Overall, the presence of any clinical factor was most likely to reduce 1 h platelet recovery, while donor-recipient HLA incompatibilities correlated best with poor 24 h post-transfusion platelet recovery. A platelet crossmatch test predicted the transfusion response when non-immune clinical factors were absent.     

Clinical factors influencing the efficacy of pooled platelet transfusions

To determine the relative importance of clinical factors on the efficacy of platelet transfusions, 941 pooled platelet transfusions from HLA-unmatched donors were studied prospectively in 133 patients with bone marrow failure. Multiple linear regression analyses identified the major factors influencing one-hour-corrected increments (CI) as prior splenectomy, bone marrow transplantation, disseminated intravascular coagulation, concurrent intravenous amphotericin B, splenomegaly, and HLA antibody grade. The relative impact of these factors on CI has been quantitated by using a formula developed from these data. A linear relationship was demonstrated between increasing percentage of HLA antibody grade and decreasing CI. A number of other factors were less important in the linear regression model than the aforementioned major factors. These included platelet-specific antibodies, concurrent antibacterial antibiotics, clinical bleeding grade, and temperature. Factors that did not influence CI included the number of prior platelet transfusions, prior granulocyte transfusions, prior red cell transfusions, infection, age, blood group, diagnosis, sex, pretransfusion platelet count, prior pregnancies, and concurrent antineoplastic drugs. This study identified major clinical factors that significantly influenced CI and were major causes of refractoriness to pooled platelet transfusions.     

The impact of platelet transfusion characteristics on posttransfusion platelet increments and clinical bleeding in patients with hypoproliferative thrombocytopenia

Platelet characteristics, such as platelet dose, platelet source (apheresis vs pooled), platelet donor-recipient ABO compatibility, and duration of platelet storage, can affect posttransfusion platelet increments, but it is unclear whether these factors impact platelet transfusion efficacy on clinical bleeding. We performed secondary analyses of platelet transfusions given in the prospective randomized Platelet Dose Study, which included 1272 platelet-transfused hematology-oncology patients who received 6031 prophylactic platelet transfusions. The primary outcome of these analyses was time from first transfusion to first World Health Organization ≥ grade 2 bleeding. Platelet transfusion increments were assessed at 0.25 to 4 hours and 16 to 32 hours after platelet transfusion. There were 778 patients evaluable for analysis of time to bleeding. Adjusted models showed that randomized dose strategy, platelet source, ABO compatibility, and duration of storage did not predict this outcome. Platelet increments were generally higher for transfusions of apheresis platelets, ABO-identical platelets, and platelets stored 3 days versus 4 to 5 days. Thus, although platelet source, ABO compatibility, and duration of storage exert a modest impact on both absolute and corrected posttransfusion platelet increments, they have no measurable impact on prevention of clinical bleeding. This trial was registered at www.clinicaltrials.gov as #NCT00128713.     

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.     

Successful treatment of platelet transfusion refractoriness: the use of platelet transfusions matched for both human leucocyte antigens (HLA) and human platelet alloantigens (HPA) in alloimmunized patients with leukaemia

Six patients, 4 with acute myeloid leukaemia and 2 with a myelodysplasia syndrome who were refractory to random donor platelet transfusions and alloimmunized to human leucocyte antigens (HLA) and human platelet alloantigens (HPA), were treated with HLA-and HPA-matched platelet transfusions. In all the patients refractoriness and alloantibodies to HLA as well as HPA-1b or HPA-5b were detected simultaneously. Sixty-seven transfusions (445 units) of HLA-and HPA-matched platelets were given and responses to them were, in general, satisfactory in all the patients. No major spontaneous bleeding occurred. Four patients underwent bone marrow transplantation despite alloimmunization. The percentages of platelet transfusion days with a platelet nadir below 20×10⁹/l were 88% for the last 3 random donor platelet transfusions and 39% for the first 3 HLA-and HPA-matched platelet transfusions, respectively (p=0.009, Fisher's exact test). Four patients received also HLA-matched platelets, but responses to them were poor. The small number of transfusions with HLA-matched platelets precluded comparisons to either the random donor or HLA-and HPA-matched platelet transfusions. It seems that HLA-and HPA-alloimmunized patients can be successfully supported with HLA-and HPA-matched platelet concentrates.     

Serial measurement of lymphocytotoxic antibody and response to nonmatched platelet transfusions in alloimmunized patients

Serial evaluations of lymphocytotoxic antibody (LCTAb) and responsiveness to random donor platelet transfusion were reviewed in 234 patients who had developed LCTAb at some time during their treatment course. Seventy (30%) of these patients had significant falls in antibody levels. In 44 patients these declines occurred after further antigenic exposure was reduced either because no transfusions were administered or only histocompatible platelets were transfused. Forty patients with declines in LCTAb levels who were previously refractory to platelet transfusion were rechallenged with random donor platelets. Thirty-four of 35 clinically evaluable patients had good responses to these unmatched transfusions for 2 weeks to 36 months, and in 21 patients antibody did not return despite repeated transfusions. Thus, serial LCTAb measurements are helpful in the management of alloimmunized patients. Many patients will have decreases or a loss of LCTAb, either permanently or transiently, and can be successfully supported with more easily available unmatched random donor platelet transfusions.     

Optimizing platelet transfusion therapy

Platelet transfusions are widely used. Prophylactic transfusions are employed in severely thrombocytopenic patients without evidence of bleeding, but no randomized trial data prove the safety or efficacy of this approach. Randomized trials have demonstrated the equivalence of transfusion triggers of 10,000 and 20,000/microl for prophylactic transfusions. The former threshold is potentially safer for the patient, conservative of donor resources and leads to lower costs, with perhaps a slightly greater risk of minor hemorrhage. Randomized trials have demonstrated the equivalence of pheresis or whole blood-derived platelet transfusions. The former present a lower risk for infectious agents, and the latter are less expensive and a more efficient use of limited donor resources. Randomized trials prove that leukoreduced and ABO identical platelet transfusions reduce the risks of HLA alloimmunization and platelet transfusion refractoriness (both leukoreduction and ABO matching), transfusion reactions (leukoreduction) and CMV transmission (leukoreduction). Leukoreduction and ABO matching of platelet transfusions also have been associated in preliminary observational studies with reduced morbidity and mortality in surgical patients and reduced infections in patients with leukemia. These results require further investigation. Future challenges include (1) determining the best approach to bacterial contamination of platelets, whether by detection methods or pathogen inactivation and (2) determining the threshold for prophylactic platelet transfusions in thrombocytopenic patients undergoing surgery or invasive procedures.     

Relationship of HLA and platelet-reactive antibodies in alloimmunized patients refractory to platelet therapy

Platelet crossmatching assays have been used to predict the outcome of platelet transfusions in alloimmunized patients by detecting antibodies against platelets. The transfusion failure of HLA-matched platelets predicted by platelet crossmatching may be related to HLA antibodies undetected by lymphocytotoxicity but detected by platelet immunoglobulin-binding assays or platelet-specific antibodies (both antibodies defined here as platelet-reactive antibodies). To differentiate platelet-reactive antibodies from lymphocytotoxic HLA antibodies, we used HLA characterized lymphocytes in parallel with platelets from individuals to form separate frozen panels. Sera from 10 allosensitized patients were studied in the lymphocyte panel by lymphocytotoxicity and in the platelet panel by enzyme-linked immunoassay (ELISA). By comparing pattern and percent wells reacting in each panel, lymphocytotoxic HLA antibodies and antibodies reactive with platelets in ELISA were detected separately. In all 10 allosensitized patients, platelet-associated antibodies were present and 7 had additional lymphocytotoxic HLA antibodies. Using this double parallel panel technique, we found platelet-reactive antibodies important in platelet alloimmunization, unrecognized by lymphocytotoxicity. These data indicate platelet-crossmatching be solely used in the selection of platelets for allosensitized patients.     

Refractoriness to random donor platelet transfusions in patients with aplastic anaemia: a multivariate analysis of data from 264 cases

Frequent platelet support is an essential part of the management of patients with severe aplastic anaemia and platelet transfusions from random donors are usually given as initial therapy. To evaluate those parameters that might correlate with the development of refractoriness to platelets from random donors, we performed a retrospective multivariate analysis in 264 patients with severe aplastic anaemia who presented for allogeneic bone marrow transplantation. Two hundred and ten (79.5%) of these patients had received multiple platelet and red cell transfusions, and 71 (34%) were refractory to random donor platelets. The strongest factor correlating with refractoriness was the presence of lymphocytotoxic antibodies, followed by the number of platelet units previously transfused. However, the latter variable attained significance only when the number of platelet units transfused exceeded 40. When given HLA-compatible platelet transfusions, only five (7%) of the refractory patients did not show a reasonable post-transfusion platelet increment. Measures which would delay or prevent platelet alloimmunization might include a policy of therapeutic rather than prophylactic platelet transfusions, and referring patients early in the course of their disease for marrow grafting if a suitable donor is available.     

Alloimmunization after Leukocyte-Depleted Multiple Random Donor Platelet Transfusions

In a prospective study we investigated the development and the course of alloimmunization after leukocyte-depleted red cell and multiple random donor platelet transfusions in 335 patients. Of these 335 patients, who had a negative antibody screening on admission and a negative transfusion history, 69 (21%) developed either transient (n = 18) or permanent (n = 51) lymphocytotoxic antibodies, but only 31 patients (9%; 95% confidence limits 6-12%) developed multispecific alloantibodies necessitating HLA-matched platelet transfusions. There was no difference with regard to the development of antibodies and platelet refractoriness between leukemia patients receiving cytostatic treatment and patients with aplastic anemia receiving prednisone and antithymocyte globulin. Females with previous pregnancies developed platelet refractoriness with an increased incidence (Chi 2 13.38; p less than 0.001) compared to females without previous pregnancies, males, and children.     

High-dose intravenous gamma globulin improves responses to single-donor platelets in patients refractory to platelet transfusion

Ten patients, with bone marrow failure or malignant disorders, became refractory to platelet transfusions using random, as well as partial or fully HLA-matched, single-donor platelets. To determine its effect on platelet refractoriness, intravenous gamma globulin (IV IgG) was administered at 400 or 800 mg/kg/d for five days, and postinfusion platelet responses were monitored. Platelet transfusion responses following intravenous gamma globulin (IV IgG) were graded as follows: Excellent, 48-hour posttransfusion count greater than 50,000/microL; good, 48-hour count greater than 20,000 but less than 50,000/microL; Fair, increased increment, 48-hour count less than 20,000; and failed, no increased increment. Six of ten patients (60%) had improved responses to selected single-donor platelets (two were excellent, three were good, and one was fair). The time to achieve a platelet transfusion count greater than 25,000/microL ranged from one to nine days of IgG therapy. One individual had sustained benefit (greater than 1 year); the remaining responses persisted for 6 to 8 weeks. These results suggest that IV IgG may be useful in the management of platelet refractoriness, especially in patients receiving single-donor platelets.     

Current practice and future directions for optimization of platelet transfusions in patients with severe therapy-induced cytopenia

Platelet transfusions are mainly used for patients with thrombocytopenia due to bone marrow failure, especially cancer patients developing severe chemotherapy-induced thrombocytopenia (e.g. patients with acute leukemia or other hematologic malignancies). A prophylactic transfusion strategy is now generally accepted in developed countries. Some clinical data, however, support the use of a therapeutic transfusion strategy at least for certain subsets of these patients. Several methodological approaches can then be used to evaluate the outcome of platelet transfusions, including peripheral blood platelet increments and bleeding assessments. Several factors will influence the efficiency of platelet transfusions; fever and ongoing hemorrhage are among the most important patient-dependent factors, but the number and quality of the transfused platelets are also important. The quality of transfused platelets can be evaluated by analyzing platelet activation, metabolism or senescence/apoptosis. Only evaluation of metabolism is included in international guidelines, but high-throughput methods for evaluation of activation and senescence/apoptosis are available and should be incorporated into routine clinical practice if future studies demonstrate that they reflect clinically relevant platelet characteristics. Finally, platelet transfusions have additional biological effects that may cause immunomodulation or altered angioregulation; at present it is not known whether these effects will influence the long-time prognosis of cancer patients. Thus, several questions with regard to the optimal use of platelet transfusions in cancer patients still need to be answered.     

A flow cytometric platelet immunofluorescence crossmatch for predicting successful HLA matched platelet transfusions

Platelet crossmatching may provide a useful way of selecting donors for effective platelet transfusions in patients refractory to random donor platelet concentrates due to alloimmunization. We assessed the predictive value of a flow cytometric platelet immunofluorescence crossmatch test for the outcome of HLA matched platelet transfusions in a group of alloimmunized patients. Platelet immunofluorescence (PIFT) crossmatches were performed for 104 HLA-matched platelet transfusions administered to 30 patients. A negative PIFT crossmatch correctly predicted a successful platelet transfusion (1 h post-transfusion platelet recovery >20%) in 56/75 (75%) cases. We also considered non-immunological factors that, in combination with alloimmunization, might have contributed to an unsuccessful transfusion result, i.e. fever, septicaemia, splenomegaly, disseminated intravascular coagulation and bleeding. The predictive value of a negative PIFT crossmatch was better when these non-immunological factors were absent [48/59 (81%) correct predictions] than when these factors were present [8/16 (50%) correct predictions] (P=0.01; chi-square test). The effect of ABO incompatibility between donor and recipient on the predictive value of the PIFT crossmatch was also analysed. Positive PIFT crossmatches occurred more frequently in ABO incompatible donor–recipient combinations [in 18/28 (64%) cases] than in ABO-compatible donor–recipient combinations [in 11/76 cases (14%)] (P<0.001, chi-square test). Successful platelet transfusions were observed on 53/76 (70%) occasions in ABO compatible transfusions as compared to 16/28 (57%) in ABO incompatible transfusions. This difference was not statistically significant (P=0.23; chi-square test). Consequently, a negative PIFT crossmatch appeared to be non-predictive for the transfusion outcome in cases of ABO incompatibility between donor and recipient. We conclude that the PIFT crossmatch for platelet donor selection in addition to matching for HLA antigens, is predictive for the outcome of ABO compatible transfusions in alloimmunized recipients and prediction levels are increased when non-immunological causes for platelet refractoriness are absent.     

The ineffectiveness of random donor platelet transfusion in splenectomized, alloimmunized recipients

The effect of splenectomy on the response to random donor platelet transfusion in 15 multitransfused thrombocytopenic patients is presented. Eight patients responded poorly, with low corrected platelet count increments at 1 and 24 hours posttransfusion. These eight patients were clinically alloimmunized and had lymphocytotoxic antibody ( LCTAb ) in their sera. They responded well to closely HLA-matched transfusions. In contrast, seven splenectomized patients responded well to random donor platelets. Five of these patients had no LCTAb and no other evidence of immunization. Two patients who responded well to random donor platelets had "weak" LCTAb , and one responded to platelets presplenectomy in the presence of this antibody. Splenectomy does not improve the response to random donor platelets in alloimmunized recipients.     

The rational use of platelet transfusions in children

Platelet transfusions are undoubtedly effective in securing hemostasis in bleeding children with absent or nonfunctioning platelets. They are, however, abused in some circumstances and are not without risk. The use of platelet transfusions to prevent rather than to treat bleeding in children with malignant disease has increased several times over the last two decades. When joining in this widespread practice, physicians should be aware that there is a relatively unimpressive evidence base supporting it and also that for patients with uncomplicated myelo-suppression the most persuasive studies suggest that a threshold platelet count of 10 x 10(9)/L is no less effective than the more customary 20 x 10(9)/L is. Still lower thresholds await evaluation. For children with nonmalignant conditions the use of platelet transfusions should be carefully evaluated on a case-by-case basis, but they should normally be avoided in the absence of clinically important bleeding. Neonates with thrombocytopenia, particularly those with immune disease due to a maternal alloantibody, are considered an exception to this generalization. The serious hazards of platelet transfusions include alloimmunization and the induction of refractoriness, graft-versus-host (GVH) disease, and the transmission of infection, all of which can be life threatening. Less risky alternative therapeutic approaches may become more widely available in the future, including recombinant thrombopoietin and lyophilized heat-treated platelet membrane preparations.     

Independent adjudicator assessments of platelet refractoriness and rFVIIa efficacy in bleeding episodes and surgeries from the multinational Glanzmann's thrombasthenia registry

Glanzmann's thrombasthenia (GT) is a rare congenital bleeding disorder associated with decreased platelet aggregation due to qualitative/quantitative deficiencies of the fibrinogen receptor. Severe bleeding episodes and perioperative bleeding are typically managed with platelet transfusions, although patients can develop anti‐platelet antibodies or experience clinical refractoriness. The GT Registry (GTR) was established to collect efficacy/safety data on hemostatic treatments for GT, including recombinant factor VIIa (rFVIIa). At the request of the United States Food and Drug Administration, three hematology experts evaluated platelet refractoriness, antibody status, and rFVIIa efficacy data on a case‐by‐case basis to support a potential indication for rFVIIa in GT. Adjudication included 195 patients with 810 events (619 severe bleeding episodes, 192 surgeries), and a consensus algorithm was developed to describe adjudicators' coding of refractoriness and antibody status based on treatment patterns over time. Most rFVIIa‐treated events were in patients without refractoriness or antibodies. Adjudicators rated most rFVIIa‐treated bleeding episodes as successful (251/266, 94.4%; rFVIIa only, 101/109, 92.7%; rFVIIa ± platelets ± other agents, 150/157, 95.5%); efficacy was consistent in patients with platelet refractoriness ± antibodies (75/79, 94.9%), antibodies only (10/10, 100.0%), and neither/unknown (166/177, 93.8%). Adjudicators also rated most rFVIIa‐treated surgeries as successful (159/160, 99.4%; rFVIIa only, 65/66, 98.5%; rFVIIa ± platelets ± other agents, 94/94, 100.0%); efficacy was consistent in patients with platelet refractoriness ± antibodies (69/70, 98.6%), antibodies only (24/24, 100.0%), and neither/unknown (66/66, 100.0%). Unblinding the adjudicators to investigator efficacy ratings changed few assessments. Doses of rFVIIa were narrowly distributed, regardless of other hemostatic agents used.     

Effective prophylaxis against platelet refractoriness in multitransfused patients by use of leukocyte-free blood components

Development of permanent platelet refractoriness is a major problem in multitransfused patients with diseases such as leukemia, aplastic anemia, or pediatric solid tumors. We tried to prevent alloimmunization in these patients by systematic use of leukocyte-free blood components with less than one million of contaminating leukocytes per unit of platelets or red cells. Our study group comprised 26 patients with a minimum of 10 platelet transfusions per patient. These patients were compared with a historical reference group of 21 patients who had received standard blood products. In the leukocyte-free group none developed platelet refractoriness, in contrast to the reference group where 11 of the 21 patients became refractory to random platelets. The median corrected platelet increment for random pooled platelets was significantly higher in the leukocyte-free group compared with the reference group. The increasing number of transfusions did not correlate with the development of platelet refractoriness; instead we propose that the lower limit of antigenic exposure is important. We conclude that systematic use of leukocyte-free blood components effectively prevents development of platelet refractoriness and contributes to optimal supportive care of children with cancer.     

Rituximab,plasma exchange and intravenous immunoglobulins as a new treatment strategy for severe HLA alloimmune platelet refractoriness

Platelet refractoriness (PR) due to HLA alloimmunization is a common and serious complication of patients receiving long-term packed red blood cell and platelet transfusions. Although most alloimmunized patients will respond to HLA-matched platelets, 20–50% of patients will remain refractory even to matched platelets. Several measures have been reported to overcome this complication, such as intravenous immunoglobulins (IVIG), plasma exchange (PE), protein A column therapy, or rituximab. We report a woman with acute myeloid leukemia secondary to myelodysplastic syndrome who was diagnosed with PR because of HLA alloimmunization. Due to difficulties in finding HLA-compatible platelet donors by cross-reactive groups in our panel of HLA-typed platelet donors, the patient received treatment with rituximab, PEs and IVIG. With this treatment strategy, the presence of HLA antibodies decreased from a panel-reactive antibody (PRA) of 89–0%. This allowed the performance of hematopoietic progenitor cell transplantation with random donor platelets. Rituximab, PE, and IVIG may be an option to overcome severe PR due to poly-specific HLA alloimmunization.