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.     

Refractoriness to Platelet Transfusion Following Double Valve Replacement in an ITP Patient Who Had Undergone Splenectomy

Abstract Reports of patients with idiopathic thrombocytopenic purpura (ITP) undergoing cardiac surgery are rare, and almost all of the reported cases required platelet transfusion. ITP patients, especially those having a history of splenectomy or a history of heavy bleeding, may have to undergo multiple platelet transfusions. Such transfusions may induce al-loimmunization against the human leukocyte antigen (HLA) and result in refractoriness to subsequent platelet transfusions. We report a case of a 63-year-old female with ITP, with a history of splenectomy and multiple platelet transfusions, who underwent aortic and mitral valve replacement. Although corticosteroid administration, high-dose immunoglobulin therapy, and repeated platelet transfusion led to a temporary increase in platelet count and successful hemostasis, refractoriness to platelet transfusion occurred postoperatively because of the presence of the anti-HLA antibody. In addition, the patient showed complications of pyothorax. Corticosteroids might have exerted an inhibitory influence on the occurrence of pyothorax.     

Platelet alloimmunization after multiple transfusions: a prospective study of 50 patients

Fifty polytransfused patients were prospectively studied to determine the frequency of post-transfusion alloimmunization and its influence on the response to platelet transfusion. Platelet- and HLA-specific antibodies were detected by means of the standard and antiglobulin-augmented lymphocytotoxicity techniques (LCT), the platelet suspension indirect immunofluorescence test (PSIIFT), and monoclonal antibody immobilization of platelet antigens (MAIPA). HLA antibodies were detected in 13 patients (26%) (IgM = 6; IgG = 6; IgM + IgG = 1). The standard LCT was positive in 12 of these 13 patients. Complement-independent HLA antibodies, only detectable in the PSIIFT and the antiglobulin-augmented LCT, were documented in two patients and were associated with poor post-transfusion platelet recovery in the patient who could be evaluated. All the HLA antibodies were detected in the PSIIFT, while only four were detected in the MAIPA. Platelet-specific alloantibodies were found in two patients by means of PSIIFT or MAIPA and may have led to poor post-transfusion platelet recovery in one patient. Platelet autoantibodies were detected in two patients but were not associated with platelet refractoriness. Paraformaldehyde-dependent platelet antibodies were detected in 11 patients but were not associated with platelet refractoriness.     

Genetic and structural characterization of an amino acid dimorphism in glycoprotein Ib alpha involved in platelet transfusion refractoriness.

The platelet-specific alloantigen, Siba, located within the alpha-subunit of the glycoprotein (GP) Ib-IX membrane receptor, has been found to be involved in the pathogenesis of platelet transfusion refractoriness. We have identified the existence of a naturally occurring threonine/methionine dimorphism at position 145 of the GPIb alpha sequence, and determined that the Siba antigen corresponds to the molecule containing methionine145. The diallelic codons can be detected by restriction enzyme analysis of amplified genomic DNA fragments from the GPIb alpha gene. Evaluation of 61 healthy blood donors showed that the allele frequencies are 89% and 11% for the threonine145 and methionine145 codons, respectively. A positive correlation exists between platelet reactivity with the anti-Siba antibody and the presence of a methionine145-encoding allele. Moreover, recombinant expression of two soluble GPIb alpha fragments differing only at residue 145, provided definitive evidence that the human anti-Siba antibody reacts only with the molecule containing methionine145. These results explain the structural basis of the Siba human alloantigen system and define screening methodologies useful in transfusion medicine to match donor and recipient platelets accordingly.     

Audit of Practice in Platelet Refractoriness

Objectives: A significant number of patients become refractory to platelet transfusion and prompt investigation of the cause will encourage appropriate selection of platelet products. Methods: We surveyed haematologists to assess perceived practice concerning platelet refractoriness because of the high cost and limited availability of HLA-compatible platelets. Some 56 of 58 consultant haematologists participated. Results: Clinicians differed on their definition of platelet refractoriness, and non-immune factors were not considered as important as immune causes of platelet refractoriness. A working group, including an invited moderator, was established to produce guidelines on recommended practice for the management of platelet refractoriness. Re-audit after implementation of the guidelines showed that more patients receiving HLA-compatible platelets had been tested for HLA antibodies. There was a mean 50.9% reduction in the use of HLA-compatible platelets. Conclusions: Increased testing for leucocyte and platelet antibodies resulted in reduced demand for and more selective use of HLA-compatible platelets, with no apparent increase in haemorrhagic complications.     

Platelet refractoriness – practical approaches and ongoing dilemmas in patient management

Platelet refractoriness can represent a significant clinical problem that complicates the provision of platelet transfusions, is associated with adverse clinical outcomes and increases health care costs. Although it is most frequently due to non‐immune platelet consumption, immunological factors are also often involved. Human leucocyte antigen (HLA) alloimmunization is the most important immune cause. Despite the fact that systematic reviews of the clinical studies evaluating different techniques for selecting HLA compatible platelets have not been powered to demonstrate improved clinical outcomes, platelet refractoriness is currently managed by the provision of HLA‐matched or cross matched platelets. This review will address a practical approach to the diagnosis and management of platelet refractoriness while highlighting on‐going dilemmas and knowledge gaps.     

Relative Importance of Immune and Non-Immune Causes of Platelet Refractoriness

In this prospective study, 26 consecutive patients being treated for haematological malignancies receiving standard (i.e. non-leucocyte-depleted) blood components were observed for the development of refractoriness to platelet transfusions. One hundred and sixteen of the 266 (44%) platelet transfusions failed to produce a satisfactory response. In 102/116 (88%), the poor response was in the presence of non-immune factors known to be associated with platelet refractoriness. Non-immune factors were present alone in 78/116 (67%), and in combination with immune factors in a further 24/116 (21%). Immune factors (HLA and platelet-specific antibodies) were present during 29/116 (25%) of unsuccessful platelet transfusions. Statistical analysis confirmed that platelet refractoriness was significantly associated with the presence of nonimmune factors. The non-immune factors associated with refractoriness were often multiple, most frequently a combination of fever, infection and antibiotic therapy. This study provides evidence that immune mechanisms were not the predominant cause of platelet refractoriness in the patient population studied. It also suggests that measures for the prevention of HLA alloimmunisation, such as leucocyte depletion, may have a limited impact in reducing the incidence of refractoriness to platelet transfusions.     

A pilot trial of complement inhibition using eculizumab to overcome platelet transfusion refractoriness in human leukocyte antigen allo-immunized patients

Heavily transfused patients frequently develop human leukocyte antigen (HLA) allo-immunization resulting in platelet transfusion refractoriness and a high risk for life-threatening thrombocytopenia. Data suggest complement activation leading to the destruction of platelets bound by HLA allo-antibodies may play a pathophysiologic role in platelet refractoriness. Here we conducted a pilot trial to investigate the use of eculizumab, a monoclonal antibody that binds and inhibits C5 complement, to treat platelet transfusion refractoriness in allo-immunized patients with severe thrombocytopenia. A single eculizumab infusion was administered to 10 eligible patients, with four (40%) patients overcoming platelet refractories assessed measuring the corrected platelet count increment (CCI) 10–60 min and 18–24 h post transfusion. Responding patients had a reduction in the requirement for subsequent platelet transfusions and had higher post-transfusion platelet increments for 14 days following eculizumab administration. Remarkably, three of the four responders met CCI criteria for response despite receiving HLA-incompatible platelets. Our results suggest that eculizumab has the ability to overcome platelet transfusion refractoriness in patients with broad HLA allo-immunization. This study establishes proof of principle that complement inhibition can treat platelet transfusion refractoriness, laying the foundation for a large multicentre trial to assess the overall efficacy of this approach (ClinicalTrials.gov, identifier: NCT02298933).     

Bleeding risk and platelet transfusion refractoriness in patients with acute myelogenous leukemia who undergo autologous stem cell transplantation

Therapy for acute myelogenous leukemia can be complicated by alloimmunization to histocompatibility antigens (HLA), with resultant refractoriness to platelet transfusions. Autologous peripheral blood or bone marrow stem cell transplantation (referred here collectively as 'autoBMT') is emerging as a standard consolidative strategy in acute myelogenous leukemia (AML). We had noted life-threatening bleeding associated with platelet transfusion refractoriness following autoBMT; we therefore retrospectively analyzed 39 AML patients for this complication following BMT. All patients received high-dose chemoradiotherapy, followed by infusion of allogeneic sibling donor (n = 12, alloBMT) or autologous (n = 27, autoBMT) stem cells. HLA alloimmunization was assessed if patients were suspected of immune refractoriness to random donor platelet transfusions. Within 100 days of stem cell infusion, one of three alloBMT and six of 12 autoBMT recipients tested were HLA alloimmunized (not statistically significant, NS). Five of six HLA alloimmunized autoBMT patients experienced delayed bleeding, which contributed to their demise while still in remission (P < 0.001). Increased platelet requirements in HLA alloimmunized autoBMT recipients were observed between days 61 and 100 post-BMT, at a median of 211 platelet transfusions vs 0 in non-alloimmunized autoBMT patients (P < 0.01) and 17 in alloBMT patients. Our data suggest that platelet transfusion refractoriness, when associated with HLA alloimmunization, is a risk factor for increased platelet transfusion requirements, delayed bleeding, and poor outcome following autoBMT for AML.     

Platelet transfusion reaction associated with interdonor HLA incompatibility

An HLA-compatible platelet transfusion was followed by chills, fever, and severe respiratory distress in a multitransfused patient with chronic lymphocytic leukemia. During the previous 7 days the patient had received blood products without incident, including 8 units of red blood cells (RBC), 24 units of pooled random donor platelet concentrates, and five HLA-compatible platelet pheresis products. The patient had no demonstrable RBC, HLA lymphocytotoxic, platelet or granulocyte antibodies. The platelet donor, a multiparous female, had no granulocyte or RBC antibodies but had lymphocytotoxic antibodies against HLA-A2 CREG (cross-reacting group A2, A28, A23, A24) which reacted not with lymphocytes of the patient but with lymphocytes of the donor whose RBC were transfused 24 h prior to the platelet transfusion reaction and whose HLA type is A23, A24; B44, B57. No RBC donors had HLA lymphocytotoxic, granulocyte, or platelet antibodies against the platelet donor. The patient received three subsequent platelet transfusions from the same donor after removal of the antibody-laden plasma with no adverse reaction. These data suggest an interdonor reaction caused by the presence of cells from the RBC donor received by the patient 24 h prior to the transfusion of donor lymphocytotoxic antibody to HLA-A2 CREG antigens.     

Prevention of Primary HLA Class I Allo-Immunization with Leucocyte-Poor Blood Components Produced without the Use of Platelet Filters

In a prospective study the incidence of allo-immunization and platelet refractoriness was investigated using a consequently leucocyte-poor blood product regime. Twenty-five previously non-transfused patients with acute leukaemia (11 men, 7 women) or autologous bone marrow transplantation for Hodgkin's or non-Hodgkin's lymphoma (2 men, 5 women) received at least 80 donor units of filtered red cells (filtration within 24 h after donation, leucocyte content 8.5 +/- 3.9 x 10(6)/U) and/or of platelet concentrate (produced by the buffy coat method, leucocyte content: 7.8 +/- 4.2 x 10(6)/U). A 1-hour recovery of 20% in three consecutive transfusions, in the absence of clinical factors known to impair increment, was defined as platelet refractoriness. HLA class I antibody screening with a panel of 60 cells was performed before the first transfusion and after 80 U of blood components. Of 25 patients who entered this study, 6 patients developed platelet refractoriness after a mean of 38 units of blood components (range 26-45 U); all 6 were female with a history of multiple pregnancies. In 19 patients regarded as non-refractory, no HLA antibodies were demonstrated (13 men, 6 women). This study, though limited in size, suggests that the use of blood products containing less than 1 x 10(7) leucocytes/donor unit prevents primary HLA class I immunization and platelet refractoriness.     

Use of leukocyte-depleted platelet concentrates for the prevention of refractoriness and primary HLA alloimmunization: a prospective, randomized trial

Compared with conventional transfusion regimes a strong reduction in HLA alloimmunization and refractoriness to platelet transfusions is obtained when both red blood cell concentrates (RBCs) and platelet concentrates (PCs) are depleted of leukocytes by filtration. Because most of the leukocyte contamination is introduced by transfusion of RBCs, filtration of RBCs appears rational, but uncertainty exists regarding the degree of leukocyte-depletion of PCs needed for the prevention of HLA alloimmunization and refractoriness. We conducted a prospective trial and randomized patients with acute leukemia to receive leukocyte-depleted PCs prepared either by centrifugation (mean leukocyte count 35 x 10(6)/PC of 6 U) or by filtration (mean leukocyte count less than 5 x 10(6)/PC of 6 U). Both groups received RBCs that were filtered after prior removal of the buffy coat. Clinical refractoriness occurred in 46% (12 of 26) of the evaluable patients that were transfused with centrifuged PCs and only in 11% (3 of 27) in the filtered group (P less than .005). De novo anti-HLA antibodies were detected in 42% (11 of 26) patients in the centrifuged group and only in 7% (2 of 27) of the patients receiving filtered PCs (P less than .004). In 8 of 11 alloimmunized patients in the centrifuged group antibodies were detected in the first 4 weeks of transfusion therapy while none of the patients in the filtered group became immunized against HLA antigens during that period. We conclude that for the prevention of HLA alloimmunization and refractoriness to platelet transfusions from random donors, both RBCs and PCs have to be leukocyte-depleted by filtration.     

Primary refractoriness to platelet transfusion caused by Nak(a) antibody alone

BACKGROUND AND OBJECTIVES: Anti-Nak(a), a platelet-specific antibody, occasionally causes platelet-transfusion refractoriness (PTR) together with human leucocyte antigen (HLA) antibodies. Anti-Nak(a) usually appears after frequent platelet transfusions or pregnancy. We report the first case of PTR caused by anti-Nak(a) alone. MATERIALS AND METHODS: A 19-year-old male patient with testicular tumour showed PTR when receiving his first transfusion of platelets. Screening for platelet antigens and platelet antibodies revealed that he had type I CD36 (Nak(a)) deficiency and that anti-Nak(a), but not anti-HLA, was present before he received his first transfusion. RESULTS: The transfusion of Nak(a)-negative, but HLA non-selected, platelets was effective in raising the platelet count. CONCLUSION: Clinically significant Nak(a) antibody was present as naturally occurring antibody in a platelet glycoprotein IV (CD36)-negative non-transfused male patient.     

Clinical Aspects and Typing of Platelet Alloantigens

Platelet alloantigens can induce the formation of corresponding alloantibodies when exposed to phenotypically negative individuals. These antibodies are responsible for fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, passive alloimmune thrombocytopenia and transplantation- associated thrombocytopenia and may contribute to platelet transfusion refractoriness together with HLA antibodies. Besides antibody detection laboratory diagnosis of the clinical syndromes requires alloantigen typing. Furthermore, typed platelet donors are a prerequisite for effective platelet transfusion therapy. Different techniques for phenotyping are well established and easy to perform but they rely on the availability of antisera. Since the molecular genetic background of the clinically most relevant alloantigens has been elucidated during the last years various genotyping methods have been applied to the platelet membrane polymorphisms and thus facilitated widespread platelet alloantigen typing. Generation of antibodies from phage display libraries and of lymphoblastoid cell lines from donors with all genetic variants will allow further developments.     

Genotype Frequencies of the Human Platelet Antigen,Ca/Tu,in Japanese,Determined by a PCR-RFLP Method

Recently, the polymorphism of a new human platelet antigen, Ca/Tu, was shown to be derived from a G-A nucleotide substitution at base 1564 of GPIIIa cDNA, which leads to a single amino acid difference, Arg/Gln at amino acid 489 of GPIIIa. We developed a PCR-RFLP method to determine the genotypes of Ca/Tu and their frequencies in a Japanese population. Fifteen Ca/Tu^a donors comprising 1 Ca/Tu(a/a) homozygous donor and 14 Ca/Tu(a/b) heterozygous donors were found among the 314 random donors analyzed. The frequencies of Ca/Tu genes were 0.025 (Ca/Tu^a) and 0.975 (Ca/Tu^b). The present study showed that the frequency of Ca/Tu^a individuals in the Japanese (15/314) was approximately 7-fold higher than in the Finnish population (1/150) previously reported by Kekomäki et al. Therefore, attention must be given to the involvement of the Ca/Tu alloantigen in neonatal alloimmune thrombocytopenia and the refractoriness of platelet transfusion.     

Clinical and laboratory factors associated with platelet transfusion refractoriness: a case–control study

In recent years clinical factors have largely surpassed alloimmunization as the predominant cause of platelet refractoriness. This makes it necessary to properly identify and weigh the non-immune factors that have a major impact on refractoriness. A case–control study is suitable for such an analysis, and to our knowledge has not previously been performed to assess this issue.
Fifty-two refractory patients were compared with 52 control patients who were transfused at the same time. Only one transfusion event was analysed per patient. Clinical and laboratory data were recorded at the time of selected transfusion, and their association with refractoriness was investigated by the contingency table method and the Cox stepwise logistic regression.
There were 16 (31%) patients with HLA antibodies in the index group and only one in the control group. The corrected count increment in the group of patients refractory due to HLA antibodies was significantly lower than that in non-alloimmunized refractory patients [median (range): 48.5 (−3560, 4614) and 4058 (−4417, 6886), respectively; U = 493, P  < 0.0001]. In the multivariate analysis, factors associated with refractoriness were the presence of HLA antibodies (odds ratio (OR) 50.7; 95% CI 5.5–463); fever (odds ratio 7.2; 95% CI 2.5–21) and BMT because of chronic myeloid leukaemia (odds ratio 7.3; 95% CI 1.8–30). The latter two were the only factors that remained independently associated with refractoriness after excluding alloimmunized patients and their controls.
We conclude that HLA antibodies are strongly associated with platelet transfusion refractoriness, but account for less than a third of these patients. Fever and BMT because of chronic myeloid leukaemia were the only non-immune factors independently associated with refractoriness.     

Platelet transfusion refractoriness

The platelet, a fascinating anucleate cell, is critically important for haemostasis. Platelet transfusions have greatly reduced the incidence of major haemorrhagic complications associated with the management of haematological and oncological disorders. However, some patients fail to receive the full benefit of platelet transfusions because they do not achieve the appropriate platelet count increment following transfusion. This review will discuss the aetiology, diagnosis, and management of refractoriness to platelet transfusion, a complicated problem for both the treating physicians and the transfusion services supporting these patients. Although advances have been made in the diagnosis and treatment of immune-mediated platelet refractoriness, which is usually caused by anti-human leucocyte antigen antibodies, non-immune causes, such as sepsis, remain problematic.     

Human leukocyte antigen (HLA) class I antibodies and transfusion support in paediatric HLA-matched haematopoietic cell transplant for sickle cell disease

The relevance of donor-specific human leukocyte antigen (HLA) antibodies in HLA-mismatched haematopoietic cell transplant (HCT) is known, but the importance of HLA antibodies in HLA-matched HCT is unclear. We hypothesized that HLA antibodies detected before HCT would cause platelet transfusion refractoriness during HCT and investigated this in a multi-centre study. Pre-HCT samples from 45 paediatric patients with sickle cell disease (SCD) undergoing HLA-matched HCT were tested for HLA class I antibodies. The number of platelet transfusions received before day +45 was compared between those with and without antibodies. Thirteen of 45 (29%) patients had a positive HLA class I antibody screen, and these patients received significantly more platelet transfusions than patients without antibodies (median 19 vs. 7·5, P = 0·028). This platelet transfusion association remained significant when controlling for conditioning regimen. Among alloimmunized patients, there was no association between the panel-reactive antibody and the number of platelet transfusions. Patients with HLA class I antibodies also had a higher incidence of acute graft-versus-host disease (GVHD): 6/13 (46%) vs. 3/32 (9%), P = 0·011. Pre-HCT HLA class I alloimmunization is associated with increased platelet transfusion support and acute GVHD in paediatric HLA-matched HCT for SCD. Further studies are needed to investigate the pathobiology of this association.     

Refractoriness to platelet transfusion in acute myeloid leukemia correlated with the optical density of anti-platelet factor 4/heparin antibodies

A small number of reports have described cases of heparin-induced thrombocytopenia complicating hematological disorders with impaired platelet production. We describe the case of a 66-year-old woman with acute myeloid leukemia who exhibited unexplained refractoriness to platelet transfusion, while receiving heparin flushes, and was found to have anti-platelet factor 4 (PF4)/heparin antibodies with high optical density (OD) values (>2 units) detected by an enzyme-linked immunosorbent assay. After cessation of heparin flushes, her refractoriness to platelet transfusion resolved. We retrospectively confirmed that the OD values for anti-PF4/heparin antibodies declined gradually; refractoriness to platelet transfusion resolved when the OD values fell below 1.0 units. Given the absence of any other evident explanation for this phenomenon, and the correlation between the OD values for anti-PF4/heparin antibodies and the efficacy of platelet transfusions, we conclude that the patient’s refractoriness to platelet transfusion was most likely caused by anti-PF4/heparin antibodies that had platelet-activating properties.     

Platelet transfusions irradiated with ultraviolet-B light may have a role in reducing recipient alloimmunization

The recipients of multiple platelet transfusions frequently develop alloantibodies directed against the human leucocyte antigens (HLA) present on both leucocytes and platelets. Such alloimmunization (AI) may result in refractoriness to further platelet transfusions. Contaminating leucocytes bearing Class II HLA and present in platelet concentrates (PC) are responsible for the formation of HLA antibodies and their removal by filtration reduces the rate of recipient AI. Ultraviolet irradiation (UVR) of PC at an appropriate dose inactivates the contaminating mononuclear leucocytes so that responses in vitro to mitogens and alloantigens are abrogated. It seems likely that UV-irradiation of donor dendritic cells (DC) is important in preventing in vitro responses to alloantigens and in vivo allosensitization. At the same time, satisfactory platelet function and structure is retained when measured by in vitro tests. In vivo assessments of platelet recovery and survival in healthy subjects and the ability to correct the bleeding time in thrombocytopenic patients are comparable to non-irradiated PC. Prospective studies are now in progress to determine if UVR will reduce recipient AI to HLA in multiply-transfused patients with leukaemia and lymphoma.