The NMJI

VOLUME 18, NUMBER 6

NOVEMBER / DECEMBER 2005

Short Report
Platelet alloimmunization in multitransfused patients with haemato-oncological disorders
Meenu Bajpai, babita Kaura, neelam marwaha, savita kumari, R. R. sharma, S. K. agnihotri

Abstract
Background. We studied the incidence of platelet alloimmunization in multitransfused patients with haemato-oncological disorders and determined the factors influencing alloimmunization. We also assessed the effect of alloimmunization on response to platelet transfusion.
Methods. Fifty patients with haemato-oncological disorders who received multiple transfusions were included. The patients were tested for antibodies before they received any transfusion and then after 3–4 weeks of transfusion. Lymphocytotoxicity and platelet immunofluorescence suspension tests were used to detect antiplatelet antibodies. Symptomatic improvement was used to assess the response to platelet transfusions.
Results. Thirty patients were positive by the lympho-cytotoxicity test, giving an incidence of 60% for anti-HLA antibodies. The panel reactivity of the antibodies ranged from 3% to 100%. Nineteen patients were positive by the platelet immunofluorescence suspension test, 16 of whom were also positive by the lymphocytotoxicity test. The overall incidence of antiplatelet antibodies was 66%. The number of transfusions received and the underlying haemato-oncological disorder were not risk factors for the development of antibodies. Patients with a past history of transfusions and those with a positive obstetric history had a significantly higher incidence of antibodies. The response to transfusion therapy was poor in patients with antibodies, as 71.4% of patients with antibodies were non-responsive compared to only 26.6% of antibody-negative patients.
Conclusion. A high percentage of multitransfused patients developed antiplatelet antibodies. Previous sensitization was an important risk factor for the development of antibodies. Patients with high panel reactivity (HLA) showed non-responsiveness to platelet transfusions. Testing for the presence of antiplatelet antibodies and provision of compatible platelets should be important components in the management of patients with platelet transfusion refractoriness.
Natl Med J India 2005;18:134–6

INTRODUCTION
Platelet transfusion is an essential component of supportive therapy in patients with haemato-oncological disorders as many of them present with varying degrees of thrombocytopenia leading to bleeding tendencies. Thrombocytopenia may be either due to the underlying disease or due to the side-effects of therapy. These patients usually require long term platelet transfusion support. However, repeated platelet transfusions may fail to show the desired increment in platelet counts. The cause for this refractoriness to platelet transfusion may be the underlying clinical condition of the patient, e.g. fever, sepsis, drugs, hypersplenism, or it may be immunologically mediated due to the development of alloantibodies.
1 Platelets express a variety of antigens such as human platelet antigens (HPAs), ABO antigens, and human leucocyte antigens (HLAs). The problem of refractoriness due to antibodies to ABO antigens can be overcome by transfusing ABO-compatible platelets. Although alloimmunization to HPAs may occur, they are rarely the sole antibodies responsible for platelet transfusion refractoriness. HLAs are the major antigens implicated in alloimmunization and refractoriness. Data from India on platelet alloimmunization in multitransfused patients are scarce. We studied the incidence of platelet alloimmunization in multitransfused patients with haemato-oncological disorders.

METHODS
Fifty patients with haemato-oncological disorders admitted to the Department of Internal Medicine at the Postgraduate Institute of Medical Education and Research, Chandigarh, who received >4 transfusions of blood or blood components during their hospital stay were included. Two blood samples were taken from each patient. The first sample was collected before the patient received any transfusion during the present admission and the second sample after 3–4 weeks of transfusion. Serum was separated and stored at –20 oC until testing. The indirect platelet suspension immunofluorescence test (PSIFT) and lymphocytotoxicity test (LCT) were used to detect antiplatelet antibodies; PSIFT detects antibodies to both HPAs and HLAs as well as non-specific antibodies attached to platelets. Normal reagent platelets were prepared by pooling platelets from 4 random donors and fixing the platelets with paraformaldehyde after washing in EDTA-PBS. The patients’ sera were incubated with normal pooled platelets followed by incubation with fluorescein isothiocyanate (FITC)-labelled F(ab)2 fragment anti-IgG. The platelets were then examined under ultraviolet illumination for the presence of fluorescence. Sensitized platelets showed strong fluorescence compared to non-sensitized ones.
2 The LCT was used to detect the presence of HLA antibodies and was done as described by Terasaki and McClelland.3 Briefly, the patients’ sera were incubated with a panel of lymphocytes from 30 random donors. Following incubation, rabbit complement was added. Formalin was used to fix–stop the reaction after addition of eosin dye. Lysis of >20% cells in a well was considered positive. Cytotoxicity against even one of the panel lymphocytes was considered positive. The panel reactivity of antibodies (the percentage of panel cells to which the recipient formed cytotoxic antibodies) was calculated.
The clinical response and role of previous sensitization due to pregnancy or transfusion and response to platelet therapy were analysed. The clinical relevance of antiplatelet antibodies was assessed by clinical response to platelet therapy. The patients were considered responsive if there was improvement in their bleeding manifestations. Prevention of bleeding in patients with low platelet counts was also considered a response. Patients who had no improvement in their bleeding manifestations with platelet therapy were labelled as non-responsive. Statistical analysis was done by the chi-square test.

RESULTS
Patients
Of the 50 patients included, 31 were men and 19 women. The age of the patients ranged from 13 to 75 years with a mean (SD) of 34.9 (17.8) years. Aplastic anaemia (16) and acute leukaemias (23) were the main underlying disorders. The others were chronic myeloid leukaemia (6), non-Hodgkin lymphoma (2) and myelo-fibrosis (1). All patients with chronic myeloid leukaemia were in blast crisis. The number of transfusions of blood and/or blood components received by the patients ranged from 4 to 63 with a mean (SD) of 21.0 (13.7). Eleven women had a history of previous childbirth while one was pregnant at the time of the study. A history of transfusions in the past was present in 12 patients.

Serological profile
Fifty pairs of samples (before and after transfusion) were tested for HLA antibodies by LCT. Forty-seven patients were antibody negative in their initial samples. Of these, 27 became antibody positive in the second sample, suggesting the development of antibodies during follow up. Three patients were antibody positive in both samples suggesting the presence of pre-existing anti-bodies. However, the panel reactivity of antibodies in the initial samples of these 3 patients was low, with reactivity against 6%, 6% and 10% of the panel, respectively. The antibodies in the subsequent samples of these patients showed a higher panel reactivity of 16.6%, 26.7% and 100%, suggesting the appearance of new antibodies. The remaining 20 patients were antibody negative in both samples. The incidence of anti-HLA antibodies by LCT was 60%. The panel reactivity of the antibodies ranged from 3% to 100%; 19 patients showed reactivity with more than 20% of the panel and, of these, 2 samples showed reactivity with 100% of the panel.
   PSIFT was positive in 19 patients. All of them showed antibodies only in their second samples. Of the 19 positive samples, 16 were also positive by LCT. Three samples showed positivity only for PSIFT. The incidence of antibodies using PSIFT was 38%. The overall incidence of antibodies (detected by LCT and/or PSIFT) was 66%.
   Women with a positive obstetric history showed a higher incidence of antibodies (83%) as compared to patients with no such history (60.5%), though this was not statistically significant. The incidence of antibody positivity was higher in patients with a previous history of transfusion (91.7%) as compared to patients with no such history (57.9%), and this difference was statistically significant (p<0.1). There was no correlation between the number of transfusions and antibody formation. The underlying disorder also did not correlate with antibody formation.
Table I. Antibodies and response to platelet therapy*

Antibodies Responsive (%) Non-responsive (%) Total
Present 8 (28.6) 20 (71.4) 28
Absent 11 (73.3) 4 (26.6) 15
Total† 19 (44.1) 24 (55.8) 43
* p<0.05 (presence of antibodies significantly reduces the response to platelet therapy)
† 7 patients did not require platelet transfusions but received red blood cells and fresh frozen plasma.

Clinical response
Seven patients did not require any platelet transfusions (they received transfusions of red blood cells and fresh frozen plasma) during the follow up and hence were not included while assessing the response (Table I). The response to platelet therapy was significantly less in patients with antibodies (p<0.05).
   Seven of the 11 patients with a panel reactivity of <20% were responsive to platelet therapy whereas only 1 of the 19 patients with a panel reactivity of >20% were responsive.

DISCUSSION
Platelet transfusion therapy is life-saving for patients with haemato-oncological disorders and bleeding due to thrombocytopenia. Prior to the advent of platelet transfusion therapy, haemorrhage contributed to the death of up to 53% of patients with leukaemia.4 However, platelet transfusion has its own problems. One of them is alloimmunization to HPA and HLA antigens. Alloimmunization depends upon a number of factors such as the type of platelet products transfused, the number of transfusions received and the immune status of the patient. At our centre, non-leucoreduced random donor platelets pooled from 5–6 donors are the mainstay of platelet transfusion therapy, although single-donor apheresis platelets may occasionally be transfused. The former increases the risk for alloimmunization due to greater donor exposure.
   The overall incidence of platelet alloantibodies (both HLA and HPA) in our study was 66%. HLA antibodies are the major antibodies involved in platelet refractoriness in multitransfused patients requiring continued platelet support. The incidence of HLA antibodies was 60% in our study. Three reviews on HLA alloimmunization have mentioned incidences ranging from 25% to 95%, 30% to 100% and 30% to 70% in patients receiving non-leucoreduced blood components.5–7 Various workers have used different criteria and cut-off values to define HLA alloimmun-ization. Currently there are no standard criteria to define alloimmunization. In our study, sera causing cell death of >20% against any panel cell was considered a positive result, whereas in another study >10% cell death against any panel cell was considered positive. Other authors have considered positive results when the panel reactivity exceeded 5% or 20% of the total panel (lymphocyte killing per panel cell not mentioned).8–10 Various studies have reported alloimmunization rates of 34%–60%.9–14
   Kiefel et al.10 in a study on 252 multitransfused patients with haemato-oncological disorders reported an incidence of 42.9% of HLA alloimmunization using the LCT and monoclonal antibody immobilization of platelet antigen (MAIPA). However, they used a cut-off value of >20% panel reactivity while defining HLA alloimmunization. A Spanish study using MAIPA reported a relatively lower incidence (17%) of HLA antibodies in multitransfused patients with haemato-oncological disorders and they attributed it to racial differences.15 Transfusion of leucoreduced blood components leads to a much lower incidence of development of HLA antibodies. A multicentric prospective study by ‘The Trial to Reduce Alloimmunization to Platelets Study Group’ (TRAP) reported an incidence of lymphocytotoxic antibodies ranging from 17% to 21% in patients receiving leucoreduced or leucocyte inactivated blood components as compared to 45% in patients receiving non-leucoreduced blood components.16
   In our study, antibody positivity was detected in 19 patients using PSIFT. Of these, 16 were also positive by the LCT. The antibody positivity in these cases could be due to HLA and/or HPA antibodies. Three samples showed positivity only in PSIFT. Anti-bodies in these three cases could be HPA antibodies, non-complement activating HLA antibodies, auto-antibodies, or rarely due to HLA antibodies against an antigen that was represented in the platelet pool but was missed in the panel of lymphocytes used.
   Previous sensitization due to transfusions was a risk factor for the development of platelet antibodies (p<0.1). Kurz et al.11 also found that patients with a history of previous transfusion had a higher rate of alloimmunization (p<0.013). In our study there was no definite association between the number of transfusions received during the same admission or the underlying diagnosis with development of antibodies. This could be due to the small sample size or variable donor–patient heterogeneity.
   Holohan et al.17 showed a higher frequency of HLA alloimmunization (80%–90%) in patients with aplastic anaemia compared to those with haematological malignancies (40%–60%). Lee and Schiffer18 found that patients with acute myeloid leukaemia were more likely to develop HLA alloimmunization (44%) than patients with acute lymphoblastic leukaemia (18%). These differences were attributed to varying degrees of immuno-suppression and altered immune status resulting from the disease process or from immunosuppressive therapy.19
   A previous study from India20 used PSIFT to determine the incidence of platelet antibodies in 29 multitransfused patients with haemato-oncological disorders, of whom 2 were antibody positive. These 2 were parous females with acute myeloid leukaemia who had received multiple blood transfusions. The low prevalence of antibodies was probably due to the use of only PSIFT for detecting antibodies.
   Platelet refractoriness due to HLA alloimmunization may be overcome by providing HLA-compatible platelets in patients with low panel reactivity, but the problem becomes intractable in patients with high panel reactivity as compatible platelets are difficult to find. In such patients, alternative strategies such as the use of intravenous immunoglobin, treatment with cyclosporin, immunoadsorption using staphylococcal protein A columns and plasmapheresis may be used. In our study, 19 of 30 alloimmunized patients had >20% panel reactivity and all except 1 of these patients were refractory to random-donor platelets. However, 7 of 11 patients with <20% panel reactivity were responsive to random platelet transfusions. Therefore, the panel-reactive antibody level is an important predictor of response to platelet transfusion therapy.6 HPA antibodies are rarely the sole cause of refractoriness to platelet transfusion, but should be sought and HPA-compatible platelets transfused if the patient is not responding to HLA-compatible platelets.
   Major advances have occurred in the treatment of patients with haemato-oncological disorders. Platelet transfusion is a vital support for these patients. However, keeping in view the risks of alloimmunization, leucoreduced components and single-donor rather than random-donor platelets should be transfused. In view of the advances in the treatment of patients with haemato-oncological disorders, transfusion support services in India need to provide leucoreduced blood components to reduce the incidence of HLA alloimmunization. As of now, non-leucoreduced components are being used in most centres due to the cost of leucoreduction. Facilities for the testing of HLA and HPA antibodies and provision of compatible platelets are required urgently at centres treating patients with haemato-oncological disorders.

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Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India
MEENU BAJPAI, NEELAM MARWAHA, R. R. SHARMA,
S. K. AGNIHOTRI Department of Transfusion Medicine
BABITA KAURA Department of Immunopathology
SAVITA KUMARI Department of Internal Medicine
Correspondence to NEELAM MARWAHA; neelam2918@yahoo.com


 

 

 

 



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