Effects of universal vs bedside leukoreductions on the alloimmunization to platelets and the platelet transfusion refractoriness

ARTICLE IN PRESS Transfusion and Apheresis Science ■■ (2014) ■■–■■

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Effects of universal vs bedside leukoreductions on the alloimmunization to platelets and the platelet transfusion refractoriness Yuko Mishima a, Nelson H. Tsuno a,*, Mika Matsuhashi a, Tetsuichi Yoshizato a, Tomohiko Sato a,b, Toshiyuki Ikeda a, Naoko Watanabe-Okochi a,b, Yutaka Nagura a, Shinji Sone a, Mineo Kurokawa b, Hitoshi Okazaki a a b

Department of Transfusion Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Department of Hematology & Oncology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

A R T I C L E

I N F O

Article history: Received 21 June 2014 Received in revised form 9 October 2014 Accepted 1 November 2014 Keywords: Platelet transfusion refractoriness Human leukocyte antigens Human platelet antigens

A B S T R A C T

Background: Multiple platelet exposure induces anti-HLA and/or anti-HPA antibody production, which may cause platelet transfusion refractoriness (PTR). In Japan, the universal pre-storage leukocyte reduction (ULR) was fully implemented since 2006, but prior to ULR, in our institution, leukocyte reduction filters were routinely used at the bedside (bedside leukoreduction, BSLR) for all onco-hematological patients receiving multiple platelet transfusions. Objective: We retrospectively compared patients receiving platelet transfusions in the era of ULR with those of BSLR era. Materials and methods: Patients of the BSLR group (409 cases) and the ULR group (586 cases) were compared in terms of alloimmunization and immunological PTR. The clinicopathological features, including gender, history of pregnancy, number of exposed transfusion donors, periods of transfusion, and prior stem cell transplantation were compared, and the risk factors of alloimmunization were determined. Results: The antibody detection rate was significantly higher in the ULR compared to BSLR group (8.7% vs. 5.4%), as well as the immunological PTR rate (7.3% vs. 3.2%). By the multivariate analysis, female gender and the number of platelet donor exposure, but not universal leukoreduction or transfusion period, were found to be the risk factors strongly associated with alloantibody formation. Conclusion: Although ULR may be superior to BSLR in terms of preventing non-hemolytic transfusion reactions, BSLR was found to be as effective as ULR in terms of preventing platelet alloimmunization and refractoriness. Thus, BSLR should be actively indicated as a realistic alternative in developing countries, before the universal leukoreduction is fully implemented. © 2014 Published by Elsevier Ltd.

1. Introduction Abbreviations: SLR, storage leukocyte reduction; HLA, human leukocyte antigen; HPA, human platelet antigen; MPHA, mixed passive hemagglutination assay. * Corresponding author. Department of Transfusion Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 3815 5411 (Ext. 35166); fax: +81 3 3816 2516. E-mail address: [email protected] (N.H. Tsuno).

Platelet transfusion refractoriness (PTR), as referred to persistent suboptimal platelet count increment after a platelet transfusion, is a knotty issue for thrombocytopenic patients in that subsequent hemorrhagic manifestations are often critical and may be fatal to these patients.

http://dx.doi.org/10.1016/j.transci.2014.11.001 1473-0502/© 2014 Published by Elsevier Ltd.

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In addition, PTR is closely associated with plenty of adverse clinical outcomes such as inferior survival [1], longer duration of hospitalization [2] and higher inpatient hospital costs [2]. Because the use of intensive treatment modalities for cancers, especially those of hematological malignancies, is frequently correlated with transient but profound aplasia of bone marrow, prolonged blood component support, particularly platelets, is needed to avoid lifethreatening bleeding complications. The vast majority (about 70%) of clinical PTR has been attributed to non-immune factors, e.g. fever/sepsis, splenomegaly, hematopoietic progenitor cell transplantation, disseminated intravascular coagulation, graft-versus-host disease, vaso-occlusive diseases, drug-induced thrombocytopenia and hemorrhage [3–6]. The remaining (about 30%) cases are associated with immunization to platelet alloantigens [3]. Transfusioninduced exposures to a wide array of alloantigens expressed on the surface of donor blood cells can make multiple transfused patients vulnerable to be alloimmunized mainly to human leukocyte antigens (HLAs) and to a lesser extent, other antigens including human platelet antigens (HPAs) and ABO antigens [4,7]. Clinically relevant HLA antibodies have been revealed to be mostly immunoglobulin G (IgG) and to be directed against class I epitopes, such as the A and B locus antigens [8], and these antibodies are generated with rates ranging from 7% to 55% [9–11]. Followed by the detection of these antibodies for the first two weeks after exposure, some antibodies can be short-lived, while others can persist for years after the last transfusion [10,12]. HPA alloimmunization appears to be less frequent, with reported rates of up to 2% [13–15], possibly reflecting the lower immunogenicity of HPA antigens compared to HLA. For cases with anti-platelet alloantibodies, transfusions of ABOcompatible and HLA and/or HPA-compatible platelet are indicated, as the best alternative to prevent the condition. In Japan, ABO-compatible platelets are used routinely, and the Japanese Red Cross Blood Center (JRCBC) has a registry system established, in which HLA- and/or HPAcompatible platelets, obtained from registered donors, are provided for transfusion, after the compatibility is confirmed by the cross-match test. The importance of leukocyte reduction in preventing platelet refractoriness (PTR) caused by HLA alloimmunization has been widely accepted in special situations, such as hemato-oncological and transplant patients, which usually require heavy transfusion of platelets [16,17]. The TRAP study, a multicenter randomized controlled trial to reduce alloimmunization against platelets, conducted in the 1990s, has demonstrated the effective preventive value of either leukoreduction against alloantibody-induced refractoriness [18]. Traditionally, leukoreduction has been done through depletion of buffy coats after centrifugation, and subsequently, pre- and post-storage filtrations have been introduced as more effective measures for WBC removal. Because evidences have been accumulated on the usefulness of leukoreduction in preventing febrile nonhemolytic transfusion reactions (FNHTR), transmission of cytomegalovirus (CMV), and PTR, it has been mainly applied for patients at risk of developing such complications [19,20]. Also, considering the potential value in reducing the transmission of pathologic prion protein of variant Creutzfeldt–Jakob disease,

universal pre-storage leukoreduction (ULR) of all cellular blood products was started in the UK in 1999. Additionally, considering the unrecognized adverse effects of transfusion associated with contaminated WBC, such as transfusion-related immunomodulation (TRIM), ULR has been implemented also in European countries, Canada and United States after 2000. These movements provided further evidence on the usefulness of ULR for preventing PTR. A study conducted in Canada has also confirmed the significant reduction of both the incidence of alloimmunization and alloimmune refractoriness, by the implementation of universal pre-storage leukocyte reduction, in chronically transfused hematological patients [21]. In Japan, the universal leukoreduction (ULR) was implemented by the JRCBC, starting with platelet concentrates (PC) in 2004, and being completed in 2007, with subsequent covering of red cell concentrates (RCC) and plasma products. Nowadays, PC products are prepared by single-donor apheresis, using high performance apheresis machines with ability to reduce leukocyte counts to levels below 1 × 106 cells per bag, a number theoretically not sufficient for alloimmunization through direct antigenic exposure [22]. Before the dawn of ULR in Japan, in our institution, leukocyte reduction filters were routinely used at the bedside (bedside leukoreduction; BSLR) especially for patients with hematological malignancies and transplantation recipients, mainly due to the inefficient performance of apheresis machines in terms of leukocyte reduction. Taking these facts, instead of the assessment of the real effectiveness of the ULR in preventing alloimmunization and PTR, we aimed to compare the difference between the pre-storage (ULR) and the post-storage (BSLR) leukoreductions in terms of alloimmunization against HLA and HPA. The most important difference between pre-storage LR and post-storage LR is the accumulation of cytokines released by leukocytes during the storage period, as well as of soluble HLA [23], which can be prevented by pre-storage LR, but not by poststorage LR. Soluble HLA has been suggested to be an immunogenic agent, and may be responsible for the antiHLA antibody production [24]. 2. Materials and methods 2.1. Patients A total of 995 patients with hematological diseases receiving platelet transfusions in our institution from January 1999 to December 2010 were retrospectively analyzed. Those transfused in the period between January 1999 and December 2003, i.e., prior to the ULR for platelets by the JRCBC, but with use of bedside filtration, were classified as BSLR group, and those receiving transfusion in the period between January 2006 and December 2010, i.e., after the implementation of ULR, were classified as ULR group. These two groups were compared related to the patients’ clinico-pathological features, including history of pregnancy, number of exposed transfusion donors, and the frequency of anti-platelet antibody detection, and the incidence of PTR. Those cases transfused in the period 2004–2005 were excluded, because the ULR was gradually implemented by the JRCBC, starting from RBC in November 2004, then to platelets in January

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2006, and finally to plasma products of 5 units in March 2006, and to plasma products of 1 and 2 units in August 2007. In this study, we defined as immunological PTR those cases in which anti-platelet antibodies were detected and the satisfactory platelet count increment could not be achieved by platelet transfusion. On the other hand, those cases with satisfactory platelet count increment in spite of the presence of anti-platelet antibody were defined as nonimmunological PTR, and those without any detected antiplatelet antibodies as non-PTR. All patients with immunological PTR required the transfusion of HLA/HPAcompatible platelets. To determine the factors that may have affected the production of anti-platelet antibodies, patients were divided into anti-platelet antibody positive group including immunological PTR cases and negative group including non-immunological PTR cases and non-PTR cases, and the clinico-pathological features, such as patients’ age, gender, history of pregnancy, number of exposed transfusion donors, prior autologous or allogeneic transplantation, and the method of leukocyte reduction (ULR or BSLR), were retrospectively analyzed. This study was approved by the Research Ethics Committee of The University of Tokyo. 2.2. Blood products Before the implementation of the ULR by the JRCBC, PC products were collected by the apheresis method (Trima (Terumo BCT, Lakewood, CO, USA)) or Component Collection System (CCS (Haemonetics, Braintree, MA, USA), or TERUSYS (Terumo, Tokyo, Japan)), without leukoreduction filters, thus, considered non-leukoreduced. Therefore, in our service, bedside leukoreduction with filters was applied to all PC products transfused to the patients enrolled in this study. The following filters were used in the study period: Sepacell PLX-5, -10, RZ200 (Asahi-KASEI, Tokyo, Japan) and Imugard III PL (Terumo, Tokyo, Japan). After the implementation of the ULR for platelets, PC products are routinely collected by apheresis only (Trima, 27.7% of total supplied PC products in Japan), or apheresis using leukoreduction filters (CCS or TERUSYS, 72.3%) from a single donor, thus, bedside leukoreduction by filters was considered no longer necessary. The Trima system is the only one that effectively reduces leukocyte counts to values below 1 × 106 cells per bag, without the need of leukocyte filtration (information provided by the JRCBC). All PC products supplied in the study period, including HLA- and/or HPAmatched ones, were used within 72 hours after donation according to the JRCBC’s recommendations in the period 1999–2007, and were used within 96 hours after donation in the period 2007–2010. 2.3. Mixed passive hemagglutination assay (MPHA) In brief, the antigen-coated plate was rinsed with a washing solution consisting of saline solution containing 0.05% Tween-20, and then the test serum diluted in dilution solution was added 25 μL/well and incubated for 2 hours at room temperature, in a humidified chamber. After washing with the washing solution for 4 times and washing

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more 4 times with new one, the indicator cells, consisting of anti-human IgG-conjugated sheep erythrocytes, were added 25 μL/well, and allowed to react for overnight at room temperature, in a humidified atmosphere. The results are judged by the naked eye, in comparison with the positive and negative control wells. Additionally, the same assay was performed in MPHA plates previously treated with a chloroquine solution for 2 hours at room temperature to remove HLA reactivity. For the determination of the specificity of the anti-platelet antibodies, the MPHA assay using panel kit (Beckman Coulter, Tokyo, Japan) for platelets with known specificities was performed. MPHA is the standard method for the detection of anti-platelet antibodies in Japan, and the kits are commercially available only in Japan. These kits include platelets with HLA and HPA antigens suitable for the Japanese population. They are commercially available since the early 1990s, and the quality of the assay has not changed since then. 2.4. ELISA assay For screening and detection of anti-HLA and anti-HPA alloantibodies, the ELISA assay-based PakPlus platelet antibody screening kits (Immucor GTI Diagnostics, Norcross, GA, USA) were used according to the manufacturer’s instructions. In brief, microtiter well plates coated with platelet glycoproteins or HLA Class I antigens were incubated with test serum. After incubation, the wells were washed to remove unbound proteins, and antibodies bound to the microtiter wells were detected using an alkalinephosphatase-conjugated anti-human IgG and the appropriate substrate. Results were considered positive when the ratio of the mean OD of the test sample to that of normal control sera was higher than 2.0. Positive control samples gave an OD > 2.0 for the monospecific anti-IgG reagent. 2.5. Calculation of the corrected count increment (CCI) The medical records were reviewed and the corrected count increment (CCI) was calculated for each platelet transfusion. The platelet count prior to and 16–24 hours after the transfusion was used for the calculation, after correction for the patient’s body surface area (BSA) and the number of platelets transfused, using the following formula: CCI (×109/ L) = (post-transfusion platelet count – pre-transfusion platelet count × 109/L) × BSA (m2)/platelets transfused × 1011. CCI higher than 4.5 was considered effective, and those lower than 4.5, ineffective (or PTR). 2.6. Transfusion effectiveness according to the platelet antibody specificity The patients with anti-platelet antibodies were analyzed. The platelet transfusion effectiveness was evaluated according to the CCI, as described above, excluding those cases suspected of PTR of non-immunological causes, such as DIC, fever, and splenomegaly. For the analysis of the effectiveness of HLA/HPA-matched platelets, the PTR cases were further divided, according to the type of anti-platelet antibody, into those with anti-HLA antibody only, anti-HPA antibody only or both. The ABO blood group

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incompatibility was not evaluated, because previous reports have shown no significant difference of transfusion effectiveness between ABO compatible and incompatible platelets [25], and in Japan, ABO-compatible platelets are used whenever possible. 2.7. The number of platelet/RBC transfusion donors and the characteristics of the patients The number of platelet/RBC donors and units to which the patients were exposed (exposed donors) was analyzed. For both anti-platelet antibody producing and not producing cases, the total number of exposed platelet/ RBC donors and units during the whole period of the treatment was calculated. When HLA and/or HPA matched PCs were used, they were excluded from the analysis. Also, the clinico-pathological features of the patients were analyzed, since it is well known that female donors, especially those with history of pregnancy (multipara), have a higher risk of alloimmunization to HLA/HPA. 2.8. Statistical analysis The proportional differences of anti-platelet antibody formations and clinical PTRs between the BSLR and the ULR group were evaluated by the chi-square test. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for the ULR group relative to the BSLR group. A multivariate logistic regression model was used to estimate the risk factors associated with anti-platelet antibody production. To predict antibody production by this model, we selected 8 variables; age, sex, history of pregnancy (yes/ no), number of platelet transfusion donors, number of RBC transfusion donors, periods of transfusion, history of allogeneic stem cell transplantation (yes/no), and method of leukocyte reduction (BSLR vs. ULR). All 955 cases, including 73 antibody developing ones, were applied to the model calculation. Stepwise selection was not applied, because of their different proportion in the group with antiplatelet antibody formation (Tables 2 and 3A), as well as their well-known impacts on HLA alloimunization and PTRs. Model fit was evaluated using –2 log likelihood and Hosmer– Lemeshow statistics. A two-sided P value less than 0.05 was considered significant. A multivariate logistic regression analysis was performed with SPSS statistics ver. 19 (IBM, Armonk, NY, USA). To evaluate the difference of platelet transfusion effectiveness and CCI values between HLAand/or HPA-matched platelets and non-matched random donor derived platelets, the two-sample Mann–Whitney U test was used. 3. Results 3.1. Patients’ clinico-pathological features A total of 995 hematological patients (Table 1A) were divided into those receiving platelet transfusions in the period prior to (1999–2003) and after (2006–2010) the implementation of ULR. The former group was nominated as BSLR group (n = 409), since they had received platelet products leukoreduced at the bedside, and the latter, as ULR

Table 1 Background of the patients. A

Age, year Gender

Male Female Transfusion donor PLT RBC Transfusion units PLT RBC Transfusion period, days

BSLR (n = 409)

ULR (n = 586)

51 (15–84) 262 (64) 147 (36) 14 (1–298) 11 (0–210) 150 (10–3505) 18 (0–341) 271 (1–5523)

59 (16–89) 350 (60) 236 (40) 14 (1–285) 10 (0–172) 140 (10–3140) 18 (0–327) 173 (1–5523)

B

Disease (%) AML ALL CML CLL Lymphoma MM MDS AA ATLL MPD SCT (%) Autologous, no. Allogeneic, no.

BSLR (n = 409)

ULR (n = 586)

95 (23) 55 (13) 48 (12) 6 (1) 103 (25) 12 (3) 61 (15) 22 (5) 5 (1) 2 (0.5)

171 (29) 59 (10) 13 (2) 3 (0.5) 209 (36) 34 (6) 56 (10) 31 (5) 6 (1) 4 (0.7)

8 (2) 160 (39)

35 (6) 160 (27)

Each value is shown as median (minimum-maximum) except for gender (No. (%)). The number of patients with each disease is shown. The number of patients who underwent autologous or allogeneic stem cell transplantation is also shown. Abbreviations: AML; acute myeloid leukemia, ALL; acute lymphoid leukemia, CML; chronic myeloid leukemia, CLL; chronic lymphoid leukemia, MM; multuple myeloma, MDS; myelodysplastic syndrome, AA; aplastic anemia, ATLL; adult T-cell leukemia/lymphoma, MPD; myeloproliferative disease, SCT; stem cell transplantation.

group (n = 586). Cases transfused in the period 2004– 2005 were excluded from this study, which was transitional period from BSLR to ULR. A great diversity of hematological disease was observed in both groups (Table 1B). They included acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoid leukemia (CLL), lymphoma, multiple myeloma, myelodysplastic syndromes (MDS), aplastic anemia, adult T-cell leukemia/lymphoma (ATLL) and myeloproliferative disease (MPD). 3.2. Higher risk of alloimmunization after implementation of universal leukoreduction The antibody detection rate in the BSLR group was 5.4% (22/409), compared with the 8.7% (51/586) in the ULR (Table 2), and the difference was found statistically significant (P = 0.048, odds ratio (OR) 1.68, 95% CI = 1.003–2.80) Also, the immunological PTR rate was higher in the ULR group compared to the BSLR one (7.3% vs. 3.2%), and the difference was statistically significant (P = 0.0051, OR 2.41, 95% CI = 1.28–4.54). To elucidate this apparently paradoxical result, we analyzed the other clinical parameters that might have influenced alloimmunization after transfusion (Table 3A).

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Table 2 Antibody detection rate and PTR rate.

Anti-platelet antibody

+ −

Antibody detection rate (%) PTR + − PTR rate (%)

BSLR (n = 409)

ULR (n = 586)

22 387 5.4 13 396 3.2

51 535 8.7* 43 543 7.3**

* p = 0.048, odds ratio (OR) 1.68 95% CI (1.003–2.80). ** p = 0.0051, OR 2.41 95% CI (1.28–4.54). Each OR was calculated by comparing ULR cohort to BSLR cohort.

The median age of patients was lower in the BSLR group compared to the ULR one, especially in the group without anti-platelet antibody formation (51 vs. 60). On the contrary, among cases with anti-platelet antibodies, the female ratio and the history of pregnancy were higher in the ULR group than in the BSLR one (78.4 vs. 68.2 and 66.7 vs. 50, respectively). The median number of exposed donors for platelet and RBC and the periods of transfusion were almost similar (ULR vs. BSLR, 43 vs. 40.5, 28 vs. 22, 431 vs. 415, respectively). These three variables were 2–4 fold higher in the group with antibody formation compared to the group without antibody in both the ULR and the BSLR groups. The history of a prior allogeneic stem cell transplantation had positive impacts on antibody formation, especially in the ULR group (allo vs. auto vs. none, 14.4 vs. 5.7 vs. 6.6% in the ULR group; 6.3 vs. 0 vs. 5.0% in the BSLR group, respectively).

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3.3. Female gender and number of platelet donor exposure, but not the universal leukoreduction, were the important risk factors for alloimmunization Since these variables analyzed here are the presently known confounding factors that strongly influence antiplatelet antibody formation and the result of preliminary analysis by sub-classification with antibody formation shown in Table 3A seemed to support it, we next performed a multivariate analysis to reveal their real impact on alloantibody formation in our study population. The result of the multivariate logistic regression model using the above variables, including the method of leukoreduction (bedside vs. universal), without stepwise selection, is shown in Table 3B. Although the patients’ background related to diagnosis seemed to be somewhat different between the BSLR and the ULR groups (Table 1A), their contribution to the production of anti-platelet antibody was primarily excluded by a multivariate logistic regression model with stepwise selection (data not shown). The female gender and the number of exposed platelet donors were strongly associated with alloantibody formation (P = 0.0026 and 0.0063, OR (95% CI) = 3.94 (1.6–9.7) and 1.01 (1.003–1.02), respectively). On the contrary, the transfusion period and the ULR had no impact on alloimmunization (P = 0.232 and 0.313, respectively). While the history of pregnancy was also found to positively impact on alloantibody production in this model, it was not statistically significant (P = 0.085). In addition, the number of exposed RBC donors and prior allogeneic stem cell transplantation were found to be less important in terms of alloimmunization in this model (P = 0.107 and 0.108, respectively).

Table 3 Association between platelet alloimmunization, bedside vs. universal-leukoreduction and other clinical parameters. A Antibody (–)

Age, years Female Pregnancy history Transfusion donors Transfusion units Transfusion period, days SCT

PLT RBC PLT RBC Autologous Allogeneic

Antibody (+)

BSLR (n = 387)

ULR (n = 535)

BSLR (n = 22)

ULR (n = 51)

51 (26–84) 132 (34.1) 79 (20.4) 13 (1–298) 10 (0–210) 140 (10–3505) 18 (0–341) 253 (1–5523) 8 (2.0) 150 (38.8)

60 (16–89) 196 (36.6) 144 (26.9) 12 (1–285) 9 (0–172) 120 (10–3140) 16 (0–286) 165 (1–5523) 33 (6.2) 137 (25.6)

54 (28–71) 15 (68.2) 11 (50) 40.5 (5–136) 22 (1–103) 490 (50–1725) 38 (2–196) 415 (14–4110) 0 (0) 10 (45.5)

55 (18–84) 40 (78.4) 34 (66.7) 43 (2–185) 28 (1–167) 440 (20–2240) 54 (2–327) 431 (5–4110) 2 (3.9) 23 (45.1)

B

Age Female Pregnancy history PLT Transfusion donor RBC Transfusion donor Transfusion period Allogeneic SCT Universal leukocyte reduction

β

SD

X2 value

p value

OR (95% CI)

0.0055 1.375 0.746 0.012 0.011 0.00017 0.545 0.293

0.011 0.456 0.433 0.0043 0.0066 0.00015 0.339 0.291

0.252 9.10 2.965 7.472 2.599 1.430 2.579 1.017

0.616 0.0026 0.085 0.0063 0.107 0.232 0.108 0.313

1.006 (0.984–1.027) 3.956 (1.619–9.669) 2.109 (0.902–4.930) 1.012 (1.003–1.020) 1.011 (0.998–1.024) 1.0002 (0.9998–1.0005) 1.724 (0.887–3.353) 1.341 (0.758–2.369)

Each value is shown as median (minimum-maximum) except for female gender and pregnancy history and autologous SCT and allogeneic SCT (No. (%)). A multivariate logistic regression model evaluating risk of platelet alloimmunization.

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Table 4 Specificity of the anti-platelet antibodies.

BSLR (n = 409)

ULR (n = 586)

Antibody(-) HLA HPA HLA + HPA Total Antibody(-) HLA HPA HLA + HPA Total

Female

%

Male

%

132 15 0 0 147 196 37 1 2 236

89.8 10.2 0 0

255 7 0 0 262 339 9 2 0 350

97.3 2.7 0 0

83.1 15.7 0.4 0.8

96.9 2.6 0.6 0

3.4. Specificity of the anti-platelet antibodies According to the specificity of anti-platelet antibodies, only anti-HLA antibodies were detected in the BSLR group (15 in female, 7 in male), whereas in the ULR group, both anti-HLA and anti-HPA antibodies were found. In the latter, among females, 37 had only anti-HLA, 1 had only antiHPA, and 2 had anti-HLA and HPA antibodies. On the other hand, among males, 9 had only anti-HLA, and 2 had only anti-HPA antibody (Table 4). 3.5. Effectiveness of HLA/HPA-matched platelet transfusion In general, HLA/HPA-matched platelets are indicated for the prevention of PTR due to anti-platelet antibodies. For cases with anti-platelet antibodies, single donor HLA-A, B-matched platelets or HPA-matched platelets, and ABOcompatible, whenever possible, are provided from the JRCBC. Whereas in the ULR group 84.3% (43/51) of the cases had received HLA/HPA-matched platelets, in the BSLR group, only 59.1% (13/22) had received compatible ones (data not shown). The HLA/HPA-matched platelets were effective in the majority of the cases. 3.6. Transfusion effectiveness and platelet increment count Table 5 shows the rate of platelet transfusion effectiveness, according to the type of the detected anti-platelet antibodies, i.e., anti-HLA antibody only, anti-HPA antibody only, or both. Matched platelet transfusions were significantly more effective than random platelet transfusions in all three groups (55.6% vs. 42.4% in cases with anti-HLA

Table 5 Effectiveness of platelet transfusion in patients with anti-platelet antibody. Platelet transfusion effectiveness

Anti-HLA antibody Anti-HLA antibody and Anti-HPA antibody Anti-HPA antibody

Matched-platelets

Random-platelets

179 / 322 (55.6%) 29 / 30 (96.7%)

184 / 434 (42.4%)* 13 / 40 (32.5%) *

2 / 2 (100%)

2 / 13 (15.4%) *

* p < 0.01. The rates of platelet transfusion effectiveness were compared between matched and random platelet transfusions in patients with anti-HLA antibody, anti-HPA antibody or both. Each platelet transfusion was evaluated as effective when CCI 16–24 hours after transfusion was over 4.5 × 109/L.

antibody only, 96.7% vs. 32.5% in those with anti-HLA plus anti-HPA antibody, 100% vs. 15.4% in those with anti-HPA antibody group). The distribution of CCI is shown in Fig. 1. Among the patients receiving antigen-matched platelets, the CCI was the highest in those with anti-HPA antibody only (25737.6 ± 3784.9), followed by those with anti-HLA plus anti-HPA antibody (15912.0 ± 5529.2), and those with antiHLA antibody only (8782.7 ± 9897.4). The effectiveness of the antigen-matched transfusion seemed to be higher in patients with anti-HPA only or anti-HPA plus anti-HLA, compared to those with anti-HLA antibody only. In some cases, HLA-matched platelet transfusions failed to achieve effective CCI (i.e. > 4.5). Considering the stringent HLA matching strategy of the JRCBC in terms of donor selection, causes other than the sub-optimal HLA matching, such as the undiagnosed clinical background of the patients, leading to non-immunological PTR, may be involved. 4. Discussion The vulnerability to platelet refractoriness is often the case with patients with hematological malignancies, because of the repeated platelet transfusions to prevent or treat the severe and persistent thrombocytopenia resultant from the chemotherapeutic treatments, which lead to bone marrow aplasia and damage to the endothelial regions. ULR has been previously proven to effectively reduce the frequency and severity of non-hemolytic febrile transfusion reactions [26–29], the risk of cytomegaloviral transmission [30] and the risk of HLA-alloimmunization and platelet refractoriness (PTR) [18,31]. But in fact, PTR cannot be adequately prevented or corrected in a relatively small but not negligible proportion of recipients. The ULR of blood products in Japan was implemented gradually, starting in November 2004 with platelet concentrates (PC) and, in 2007, it was achieved for all blood products. Prior to the start of the ULR of platelet concentrates, i.e., before November 2004, in our institution, leukocyte reduction filters were routinely used at the bedside for the prevention of PTR, especially for those multitransfused onco-hematological patients. As for platelets, ULR of RBC products was started in January 2007, thus in the period 1999–2006, bedside leukoreduced RBC transfusions were given to all onco-hematological patients. Therefore, all patients in the first cohort (1999 to Dec 2003) received bedside leukoreduced RBC, whereas in the second cohort, a part of the cases (Jan to Dec 2006) received bedside leukoreduced RBC, and the other part (Jan 2007 to 2010) received ULR RBC. Due to this historical background of the use of bedside filtration, we were not able to analyze the effectiveness of the ULR in preventing alloimmunization. Therefore, we designed a retrospective observational assessment of alloimmunization, comparing those patients who received bedside leukoreduction (BSLR group) with those transfused in the period after the full implementation of universal leukoreduction (ULR group). Many reports have demonstrated the effectiveness of ULR in preventing alloimmunization to platelets, as well as PTR. Seftel et al. demonstrated that ULR of PC effectively reduced alloimmunization, platelet refractoriness, and the number of patients requiring matched platelets in patients with

Please cite this article in press as: Yuko Mishima, Nelson H. Tsuno, Mika Matsuhashi, Tetsuichi Yoshizato, Tomohiko Sato, Toshiyuki Ikeda, Naoko WatanabeOkochi, Yutaka Nagura, Shinji Sone, Mineo Kurokawa, Hitoshi Okazaki, Effects of universal vs bedside leukoreductions on the alloimmunization to platelets and the platelet transfusion refractoriness, Transfusion and Apheresis Science (2014), doi: 10.1016/j.transci.2014.11.001

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7

Fig. 1. Platelet count increment according to the antibody specificity and the type of platelets transfused. The CCIs were calculated, and the mean values are shown according to the specificity of the antibodies, and the type of platelet transfusion (random or matched) (n = 322 for matched platelet transfusions to patients with anti-HLA antibody, n = 434 for random platelet transfusions to those with anti-HLA antibody, n = 30 for matched platelet transfusions to those with anti-HLA antibody and anti-HPA antibody, n = 40 for random platelet transfusions to those with anti-HLA antibody and anti-HPA antibody, n = 2 for matched platelet transfusions to those with anti-HPA antibody, n = 13 for random platelet transfusions to those with anti-HPA antibody). Data are mean ± SD. *p < 0.01.

hematological malignancy with similar clinico-pathological background to ours [21]. The effectively leukoreduced RBCs and apheresis-platelets in their study contained less than 5 × 106 white blood cells per bag, a number quite higher than the Japanese standard which is less than 1 × 106 cells per bag. Compared to their data, however, the alloimmunization rate (7% vs. 8.7%), the PTR rate (4% vs. 7.3%) and the rate of patients requiring matched platelets (5% vs. 7.3%) were almost comparable in the ULR group, whereas in the nonULR (or the BSLR) group, the alloimmunization rate (19% vs. 5.4%), the PTR rate (14% vs. 3.2%), and the rate of patients requiring matched platelets (14% vs. 3.2%) were reduced. Thus, in terms of ULR, the effectiveness in our series is comparable to those previous reported, but in terms of non-leukoreduced transfusion, the direct comparison could not be performed, because our control cases had received bedside leukoreduced platelets. Intriguingly, however, when we compared the antiplatelet antibody formation rate and the PTR rate between BSLR and ULR groups of our series, both rates were higher in the latter (5.4% vs. 8.7%, and 3.2% vs. 7.3%, respectively; Table 2), meaning that ULR had no additional benefit, at least in terms of alloimmunization, compared to BSLR. Although post-storage leukoreduction by bedside filtration has been widely applied for the prevention of PTR in the 1980– 1990s period, the evidence of its usefulness in various hemato-oncological disorders is limited, depending on the implementation of ULR in Western countries in the early 2000s. In a prospective randomized controlled trial,

Williamson et al. failed to confirm the usefulness of bedside filtration in preventing HLA alloimmunization in transfused hemato-oncological patients (P = 0.07), except for acute myeloid leukemia (AML) patients, who had more prolonged exposure to blood products, or the more immunosuppressive regimes (P = 0.025) [17]. In a retrospective study from Japan, bedside filtration has been shown to significantly reduce HLA alloimmunization in transfused hemato-oncological patients with similar backgrounds to our cases [32]. Although we could not compare cases receiving leukoreduced transfusion with those receiving nonleukoreduced ones, the antibody detection rate was low in our cases, strongly suggestive of the effectiveness of leukoreduction, either universal or bedside, in preventing alloimmunization, and consequently PTR. Interestingly, it has been shown that the shorter the pre-filtration preservation time, the higher the CCI of bedside filtered platelet transfusions reported [33]. Although the accurate prefiltration preservation time of PC is not available, all bedside filtered platelets in our series were transfused within 72 hours after collection, which was the period of validity stipulated by the JRCBC for PC products at that time. The shorter period of validity of PC products in Japan, compared to other countries, may be one reason for the effectiveness of BSLR in preventing platelet alloimmunization observed in Japanese studies. To get a better understanding of this paradoxical result, the multivariate logistic regression model was applied, including the patients’ clinico-pathological variables and the

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leukoreduction method (bedside vs. universal) as parameters, and it was found that the reduction in the platelet alloimmunization rate was dependent on the differences of the clinico-pathological features between these two groups (Table 3A and 3B). The female gender and the number of platelet donor exposure, which were higher in the ULR group than in the BSLR one, were found to be the factors strongly associated with platelet alloimmunization. Thus, these factors were responsible for the higher incidence of platelet alloimunization in the ULR group. On the contrary, the method of leukoreduction and the transfusion period were found not associated with alloimmunization in the multivariate analysis. The history of pregnancy, the numbers of RBC donor exposure and the prior allogeneic stem cell transplantation showed marginal association with the platelet alloimmunization (P = 0.085, 0.107 and 0.108, respectively). Pregnancy is well known to be associated with alloimmunization, and anti-HLA antibodies are detected with higher incidence in women with pregnancy history. Accordingly, multiple pregnancies may be associated with higher rate of anti-HLA antibodies formation [12,34]. Antiplatelet antibody formation rate increases with increasing the number of pregnancies, and a previous report from Japan has shown 0.19% in their first pregnancy, 1.14% in the second pregnancy, 1.62% in the third pregnancy and 1.97% in the fourth or more [35]. Our data, however, suggested that the female gender might be an independent risk factor of platelet alloimmunization, stronger than the history of pregnancy, at least in our study population. The reason why history of pregnancy was not found significantly associated with alloimmunization is unclear, but there might be subsequent missed abortions and a potential dissociation between the patients’ understanding and the actual gravidity and parity, which might have impaired the accuracy of the data, and weakened the power of our study population for the statistical analysis. It is also possible that a gender-dependent alloimmunizationsusceptible HLA allele or haplotype may exist in the Japanese population. In the present study, we found a higher rate of HLA alloimmunization in female in the ULR group compared to the BSLR one (15.7 vs. 10.2%), although of the comparable male/female rate in both groups. Various autoimmune diseases have been reported to be more prevalent in female than male. Accumulated experimental and clinical evidences have shown the influence of sex hormones and sex chromosome-related genes on innate and pathologic immunological responses, and the mechanisms of their involvement in the onset of several autoimmune diseases have been confirmed [36–38]. Although a metaanalysis failed to prove the female-specific risk on RBC alloimmunization, independent from pregnancies [39], there may be some female-specific risk factors, related to sex hormones or sex chromosome genes, that may be associated with platelet alloimmunization. In addition to these evidences, the variability of HLA haplotypes is known to be more limited in Japanese compared to Caucasian population [40]. Thus, it is tempting to speculate that a specific alloimmunization-susceptible combination of HLA alleles or haplotypes may exist in Japan, leading to higher alloimmunization in female compared to male. Recent

technical advances in HLA antibody detection and HLA typing, which include the Luminex technology, may help elucidate these facts in the future. Anti-platelet antibodies can be distinguished into antiHLA class I antibodies and anti-HPA antibodies, the former being produced mainly by exposure to leukocytes, and the latter mainly to platelets (although some HPA antigens are also present on leukocytes; e.g., HPA-5). Thus, although the majority of PTR of immunological etiology is caused by antiHLA I antibodies, and anti-HPA antibodies comprise only a small proportion, the predominant antibody found in both groups is an important matter to be considered. Interestingly, in the BSLR group, only antibodies to HLA class I were detected, whereas in the ULR group, antibodies to both HLA class I and HPA were identified (Table 4). It is feasible that the leukoreduction performed immediately after the collection might have activated platelets, making them more immunogenic compared to those subjected to filtration after a period of storage. Additionally, in the ULR cohort, females showed higher incidence of anti-HLA antibodies than males, possibly due to the higher risk of alloimmunization to HLA antigens in females as discussed above. More detailed studies are warranted for the clarification of these points. In Japan, the HLA and HPA donor registry system of the JRCBC allows the use of matched platelets whenever required. The matched platelets were given to our PTR cases and both HLA- and HPA-matched platelets were more efficient than the random ones in terms of CCI. Interestingly, when we compared the effectiveness of HPA-matched platelets with the HLA-matched ones, the former were superior (Table 5 and Fig. 1). The finding seems to be reasonable, considering that platelets have a stronger HLA antigen expression than HPA antigen expression, and HPA fullmatching is usually more easily achieved, due to the lower polymorphism of HPA compared to HLA. In RBC transfusions, van de Watering et al. has shown that post-storage leukoreduction by filtration results in similar posttransfusion alloimmunization to pre-storage leukoreduction in cardiac surgery patients [41]. Likewise, our present data suggested that bedside leukoreduction would be as effective as universal leukoreduction, at least in terms of platelet alloimmunization and refractoriness. Although inflammatory cytokines or fragmented WBC containing soluble HLA molecules, not removed by poststorage BSLR, may have some influence on the recipient’s immunological responses and alloimmunization, their actual impacts in clinical situations remain to be confirmed. Platelets themselves can induce an immune response by the indirect antigen-presentation pathways or by expressing molecules, such as CD40L, which may significantly affect the recipient’s immunity [34,42]. In mice models, the recipient’s inflammatory state has been shown to affect RBC alloimmunization, even in case pre-storage leukoreduced RBC was transfused [43]. In clinical settings of platelet transfusion, most of the recipients may be immunomodulated or under an inflammatory state, caused by the disease itself or by the implemented treatment. In such a situation, the infused donor-derived inflammatory cytokines and/or the fragmented WBC containing soluble HLA would have only limited impacts on the whole immunological responses to the transfused alloantigens. Since ULR, in addition to

Please cite this article in press as: Yuko Mishima, Nelson H. Tsuno, Mika Matsuhashi, Tetsuichi Yoshizato, Tomohiko Sato, Toshiyuki Ikeda, Naoko WatanabeOkochi, Yutaka Nagura, Shinji Sone, Mineo Kurokawa, Hitoshi Okazaki, Effects of universal vs bedside leukoreductions on the alloimmunization to platelets and the platelet transfusion refractoriness, Transfusion and Apheresis Science (2014), doi: 10.1016/j.transci.2014.11.001

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preventing alloimmunization, has many other favorable effects, including prevention of FNHTR, prevention of leukocyte-mediated transmission of pathogens, such as CMV and Yersinia enterocolitica, and better blood flow property [44], it is an ideal alternative that we believe should be implemented whenever possible. However, the high cost of the infra-structure for ULR can be an obstacle for most developing countries. The bedside leukoreduction, as we have demonstrated here, seems to be comparably effective to ULR in terms of prevention of alloimmunization. Taken into account the previous data evaluating the effect of poststorage leukoreduction by filtration [17], although quality control of filters and administration discipline should be well-prepared [45], the bedside leukoreduction should be actively indicated as a realistic alternative, before the ULR is fully implemented.

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Please cite this article in press as: Yuko Mishima, Nelson H. Tsuno, Mika Matsuhashi, Tetsuichi Yoshizato, Tomohiko Sato, Toshiyuki Ikeda, Naoko WatanabeOkochi, Yutaka Nagura, Shinji Sone, Mineo Kurokawa, Hitoshi Okazaki, Effects of universal vs bedside leukoreductions on the alloimmunization to platelets and the platelet transfusion refractoriness, Transfusion and Apheresis Science (2014), doi: 10.1016/j.transci.2014.11.001