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BACKGROUND Current limitations of platelet shelf life to 5 days have led to an increasingly greater demand for hemostatic agents with greater longevity. The objective of this study was to evaluate the function of a lyophilized platelet-derived hemostatic product (thrombosome [TS]) as a potential alternative to fresh platelets. METHODS Platelets were collected from whole blood from healthy donors. TSs were reconstituted with water and added to various configurations of reassembled whole blood (platelets, plasma, and RBCs); measures included rotational thromboelastometry (ROTEM), optical aggregometry, mitochondrial function, calibrated automated thrombogram, collagen adhesion under flow (shear flow assay), and flow cytometry. RESULTS In ROTEM, no differences were observed between maximum clot formation values for contact pathway activation thromboelastometry tests with TSs or platelet samples. Significantly decreased aggregation was observed in the TSs versus platelets (p

BACKGROUND: The impact of donor biology on blood component storability is increasingly appreciated as a determinant of the storage lesion and post-transfusion performances. Platelet metabolism is affected by age and it is critical to platelet responses to activating stimuli in an age-dependent manner. Sex has been previously highlighted as a contributing factor to the platelet proteomics lesion. However, little is known about the impact of donor sex and age on stored platelet metabolism and post-transfusion capacity to circulate. MATERIALS AND METHODS: Apheresis platelets were donated via apheresis by 21 healthy volunteers (12 males and 9 females; ages 20 to 59). Metabolomics analyses were performed at day 0 and after 5 days of storage at 22+2 °C, along with autologous post-transfusion recovery and survival studies with (51)Cr and (111)In. RESULTS: Sex and age significantly impacted platelet metabolism at baseline and upon storage. Platelets from older, male donors were characterised by higher levels of Krebs cycle metabolites, pentose phosphate pathway intermediates and byproducts, deaminated purines and long chain fatty acids. These metabolites ranked amongst the top significant correlates to post-transfusion recoveries. Glutathione homeostasis and sphingosine 1-phosphate were the top positive correlates to long term survival, which was lower in platelets from older, male donors – without reaching statistical significance. DISCUSSION: In this study we report that donor sex and age have a significant impact on platelet metabolism. Novel metabolic correlates to platelet post-transfusion performances (24 h recovery and long-term survival) were identified through high-resolution, stable isotope-labeled internal standard-assisted metabolomics approach.

Background The short dating period of room temperature-stored platelets (PLTs; 5-7 days) limits their availability at far-forward combat facilities and at remote civilian sites in the United States. PLT cryopreservation in 6% DMSO and storage for up to 2 years may improve timely availability for bleeding patients. Study design and methods A dose escalation trial of DMSO-cryopreserved PLTs (CPPs) compared to standard liquid-stored PLTs (LSPs) was performed in bleeding patients with thrombocytopenia. Within each of four cohorts, six patients received escalating doses of CPP (0.5 unit, 1 unit, and sequential transfusions of 2 and 3 units) and one received a LSP transfusion. Patients were monitored for adverse events (AEs), coagulation markers, PLT responses, and hemostatic efficacy. Results Patients with a World Health Organization bleeding score of 2 or more received from 0.5 to 3 units of CPP (n = 24) or 1 unit of LSP (n = 4). There were no related thrombotic or other serious AEs experienced. Mild transfusion-related AEs of chills and fever (n = 1), transient increased respiratory rate (n = 1), DMSO-related skin odor (n = 2), and headache (n = 1) were observed after CPP transfusion. Among CPP recipients 14 of 24 (58%) had improved bleeding scores, including three of seven (43%) patients who had intracerebral bleeding. CPP posttransfusion PLT increments were significantly less than those of LSPs; however, days to next transfusion were the same. After transfusion, the CPP recipients had improvements in some variables of thrombin generation tests and thromboelastography. Conclusion Cryopreserved PLT transfusions appear to be safe and effective when given to bleeding patients with thrombocytopenia.

BACKGROUND Pathogen reduction (PR) of whole blood (WB) may increase blood safety when applied before component separation. This study evaluates the in vivo performance of red blood cells (RBCs) derived from WB treated with the riboflavin and ultraviolet (UV) light PR (Mirasol) system. STUDY DESIGN AND METHODS This was a prospective, two-center, single-blind, randomized, two-period, crossover clinical trial designed to evaluate autologous 51Cr/99mTc-radiolabeled recovery and survival of RBCs derived from Mirasol-treated WB compared to untreated WB. RBCs were stored in AS-3 for 21 days at 1 to 6°C. In vitro RBC variables were characterized. Frequency and severity of treatment-emergent adverse event (TEAE) and neoantigenicity were determined. RESULTS Twenty-four healthy adult volunteers (n = 12 per site) were evaluated. The Mirasol 24-hr RBC recoveries were 82.5 ± 3.9% with one-sided 95% lower confidence limit of 80.9%, meeting US Food and Drug Administration acceptance criteria, albeit at lower level than controls (91.7 ± 6.8%, p

BACKGROUND In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of 530 subjects became clinically refractory (CR) to platelets (PLTs) without lymphocytotoxicity assay (LCA)-detectable anti-HLA antibodies. The LCA only detects complement-binding antibodies and is less sensitive than newer assays. Utilizing a more sensitive bead-based assay that does not distinguish between complement-binding versus non–complement-binding antibodies, we have previously shown that while many LCA-negative (LCA–) patients do have anti-HLA antibodies, these low- to moderate-level antibodies do not predict refractoriness. As complement can contribute to PLT rejection, we assessed if previously undetected complement-binding antibodies account for refractoriness among LCA– patients. STUDY DESIGN AND METHODS Samples from 169 LCA– (69 CR, 100 non-CR) and 20 LCA-positive (LCA+; 10 CR, 10 non-CR) subjects were selected from the TRAP study serum repository. Anti-Class I HLA immunoglobulin (Ig)G and C1q-binding antibodies were measured in serum or plasma with bead-based detection assays. Levels of C1q-binding antibodies were compared between CR and non-CR subjects and correlated with corrected count increments (CCIs). RESULTS While some of the LCA– subjects had detectable C1q-binding anti-Class I HLA antibodies, and some LCA+ subjects did not, levels were significantly higher among LCA+ subjects. C1q-binding anti-Class I HLA antibody levels did not differ significantly between CR and non-CR among either the LCA– or the LCA+ subjects. Furthermore, there was no significant correlation observed between CCIs and either C1q-binding or any anti-HLA IgG antibodies. CONCLUSIONS This work confirms that low- to moderate-level anti-Class I antibodies do not drive PLT rejection, suggesting a role for antibody-independent mechanisms.

Background There continues to be uncertainty about the optimal approach to documenting bleeding data in platelet transfusion trials, with a desire to apply a common assessment tool across all trials. With this in mind, a consensus bleeding assessment tool (BAT) has been developed by the Biomedical Excellence for Safer Transfusion (BEST) collaborative, based on review of data collection forms used in published randomized trials and following content validation with a range of healthcare professionals at seven haematology centres through BEST members. This study aimed to evaluate reliability and reproducibility of the consensus BAT. Methods Replicated clinical assessments of bleeding were undertaken by participants with haematological malignancies recruited at four haematology centres in an international, multicentred, observational study. Concordance of repeat assessments was calculated for agreement in site and grade of bleeding observed. Results Forty patients consented to participate, and 13 trained bleeding assessors collected these data. Bleeding assessments were carried out on 113 separate days. Of all 225 bleeding assessments, 204 were compared for grade concordance, and 160 were compared for site concordance. There was very good grade concordance (83%, 95% confidence interval 74–93%) and good bleeding site concordance (69%, 95% confidence interval 57–79%) in observations of bleeding. Discordance was primarily in relation to assessing skin bleeding. Conclusions Alongside a structured training programme, levels of concordance for a consensus BAT were high. Researchers using assessment tools for bleeding need to balance comprehensive data collection against potential loss of accuracy for some types of bleeding, such as skin findings.

Background The current 5-day storage time of room temperature (22°C)-stored platelets (RSPs) severely limits platelet (PLT) availability. Extended cold (4°C)-stored PLTs (CSPs) are currently being investigated for actively bleeding patients. However, we currently do not know how to best store PLTs in the cold for extended periods of time. In this study, we investigate how storage in plasma and PLT additive solutions (PASs) affects PLT viability in vivo. Study design and methods Twenty normal subjects had a 2-unit hyperconcentrated apheresis PLT collection. One unit was stored at 4°C in plasma for 3 days ("control unit"), and the CSP "test" unit was stored for 10 or 15 days in plasma or 10 days in 35% plasma with either 65% Intersol or Isoplate. After storage, all units were radiolabeled and transfused into their donors. Results For 10-day storage, both the plasma and the Intersol units had significantly better PLT recoveries than the Isoplate units (24% ± 8% vs. 11% ± 3% [55% ± 11% vs. 21% ± 8% as percentage of control data], p = 0.002; and 18% ± 4% vs. 11% ± 3% [43% ± 6% vs. 21% ± 8% as percentage of control data], p = 0.004, respectively). There was a trend for lower PLT recoveries with Intersol compared to plasma (p = 0.056). PLT survivals and most in vitro measurements did not differ significantly among the units. Conclusions While the in vitro variables suggest largely comparable results between plasma and PASs, in vivo recoveries were higher with plasma compared with both Intersol and Isoplate (p = 0.057 and p = 0.002, respectively). Whether this difference leads to clinically relevant differences in hemostatic efficacy remains to be determined.

Bleeding remains a significant problem for many thrombocytopenic hematology/oncology patients in spite of platelet transfusions. Factors that might contribute to bleeding were analyzed for 16 320 patient-days on or after their first platelet transfusion in 1077 adult patients enrolled in the Platelet Dose (PLADO) trial. All patients had a greatly increased risk of bleeding at platelet counts of ≤5 × 109/L (odds ratio [OR], 3.1; 95% confidence interval [CI], 2.0-4.8) compared with platelet counts ≥81 × 109/L. Platelet counts between 6 × 109/L and 80 × 109/L were also associated with a somewhat elevated bleeding risk in patients receiving allogeneic stem cell transplants (SCTs) or chemotherapy but not in those undergoing autologous SCTs. Other significant laboratory predictors of bleeding were hematocrit ≤25% (OR, 1.29; 95% CI, 1.11-1.49), activated partial thromboplastin time (aPTT) 30 to ≤50 seconds (OR, 1.40; 95% CI, 1.08-1.81; P = .01), aPTT >50 seconds (OR, 2.34; 95% CI, 1.54-3.56), international normalized ratio (INR) 1.2 to 1.5 (OR, 1.46; 95% CI, 1.17-1.83), and INR >1.5 (OR, 2.05; 95% CI, 1.43-2.95). Transfusion of either platelets or red blood cells (RBCs) on days with bleeding was often not sufficient to change bleeding outcomes on the following day. Because bleeding occurred over a wide range of platelet counts among patients undergoing allogeneic SCT or chemotherapy and because platelet transfusions may not prevent bleeding, other risk factors may be involved. These may include low hematocrit and coagulation abnormalities. This trial was registered at www.clinicaltrials.gov as #NCT00128713.

BACKGROUND How platelet (PLT) product characteristics such as dose, source (whole blood-derived (WBD) vs. apheresis), storage duration, and ABO matching status affect the risks of transfusion-related adverse events (TRAEs) is unclear. Similarly, more information is needed to define how recipient characteristics affect the frequency of TRAEs following PLT transfusion.

Background Platelet (PLT) concentrates (PCs) prepared from whole blood in the United States are made using the PLT-rich plasma method. The PCs must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, PCs are made using the buffy coat (BC) method from whole blood held overnight at 22°C and storage times may be up to 7 days. Our studies were designed to determine how long BC PLTs can be stored in plasma or Plasmalyte while meeting the FDA's poststorage viability criteria. Study Design and Methods Normal subjects donated whole blood that was stored at 22°C for 22 ± 2 hours before preparation of BC PLTs. PLTs were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65 or 80%. Radiolabeled autologous stored versus fresh PLT recoveries and survivals were assessed as well as poststorage in vitro assays. Results BC PLTs stored in either plasma or 65% Plasmalyte met FDA poststorage PLT recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored PLT recoveries and survivals correlated with the same donor's fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted poststorage PLT recoveries, while annexin V binding best predicted PLT survivals. Conclusion BC PLTs stored in either plasma or 65% Plasmalyte meet FDA's poststorage viability criteria for 6 days.

To evaluate the poststorage viability of apheresis platelets stored for up to 18 days in 80% platelet additive solution (PAS)/20% plasma, 117 healthy subjects donated platelets using the Haemonetics MCS+, COBE Spectra (Spectra), or Trima Accel (Trima) systems. Control platelets from the same subjects were compared with their stored test PAS platelets by radiolabeling their stored and control platelets with either (51)chromium or (111)indium. Trima platelets met Food and Drug Administration poststorage platelet viability criteria for only 7 days vs almost 13 days for Haemonetics platelets; ie, platelet recoveries after these storage times averaged 44 ± 3% vs 49 ± 3% and survivals were 5.4 ± 0.3 vs 4.6 ± 0.3 days, respectively. The differences in storage duration are likely related to both the collection system and the storage bag. The Spectra and Trima platelets were hyperconcentrated during collection, and PAS was added, whereas the Haemonetics platelets were elutriated with PAS, which may have resulted in less collection injury. When Spectra and Trima platelets were stored in Haemonetics' bags, poststorage viability was significantly improved. Platelet viability is better maintained in vitro than in vivo, allowing substantial increases in platelet storage times. However, implementation will require resolution of potential bacterial overgrowth during storage.

Background Both leukoreduction and ultraviolet (UV) light treatment of blood products have been shown to reduce the incidence of HLA antibody development in recipients, but the impact of these treatments on the magnitude and persistence of the antibody response is less clear. Study Design and Methods Longitudinal samples from 319 subjects taken from four different study cohorts were evaluated for HLA antibodies to determine the effects of leukoreduction and UV treatment on HLA antibody generation and persistence. Results Subjects receiving leukoreduced or UV-treated blood products were less likely to generate Class I HLA antibodies, and those receiving leukoreduced blood were also less likely to generate Class II HLA antibodies. Among those receiving nonleukoreduced blood, 55% developed Class I HLA antibodies and 51% developed Class II HLA antibodies compared with 28% (Class I) and 15% (Class II) for those receiving leukoreduced blood and 36% (Class I) and 54% (Class II) for those receiving UV-treated blood. Among alloimmunized subjects, leukoreduction resulted in a significant twofold reduction in the magnitude of Class I HLA antibodies, and UV treatment resulted in a significant threefold reduction in the magnitude of Class II HLA antibodies. Both treatments resulted in shorter persistence of Class I HLA antibodies. Conclusions These data demonstrate that leukoreduction and UV treatment of blood products results not only in a reduction in the incidence of HLA antibody production, but also in lower and more transient HLA antibody levels among sensitized transfusion recipients.

Background Many platelet (PLT) transfusion trials now use bleeding as a primary outcome; however, previous studies have shown a wide variation in the amount (5%-70%) and type of bleeding documented. Differences in the way bleeding has been identified, recorded, and graded may account for some of this variability. This study's aim was to compare trials' method to document and grade bleeding. Study Design and Methods Data were collected via three methods: a review of study publications, study case report forms, and a questionnaire sent to the authors. Authors of randomized controlled trials of PLT transfusion that used bleeding as an outcome measure were identified from the searches reported by two recent systematic reviews. Twenty-four authors were contacted, and 13 agreed to participate. Data submitted were reviewed and summarized. Results More recent studies with trained bleeding assessors, detailed documentation, and expanded grading systems have reported higher overall levels of bleeding. The World Health Organization grading system was widely used to grade bleeding, but there was no consistency in the bleeding grade definitions. For example, bleeding classified as Grade 2 in some studies (spreading petechiae) was classified as Grade 1 in other studies. Conclusions This study has highlighted differences in the method of recording and grading bleeding, which may account for some of the variation in reported bleeding rates. To ensure that differences between studies can be attributed to trial interventions or types of participant included, this study group is developing consensus bleeding definitions, a standardized approach to record and grade bleeding, and guidance notes to educate and train bleeding assessors.

Human lymphocyte antigen alloimmunization to filter leukoreduced (F-LR) platelets occurs in about 18% of immunosuppressed thrombocytopenic hematology/oncology patients and represents a significant challenge for effective chemotherapy. In a dog platelet transfusion model, we have evaluated other methods of preventing alloimmune platelet refractoriness and demonstrated that successful methods in our dog model are transferable to man. In the present study, donor/recipient pairs were dog lymphocyte antigen DR-B incompatible (88% of the pairs), and recipient dogs received up to 8 weekly treated transfusions from a single donor (a highly immunogenic stimulus), or until platelet refractoriness. Continued acceptance of F-LR platelets occurred in 6 of 13 recipients (46%), but neither γ-irradiation (γ-I; 0 of 5) nor Mirasol pathogen reduction (MPR; 1 of 7) treatment of donor platelets prevented alloimmune platelet refractoriness. Combining γ-I with F-LR was associated with only 2 of 10 (20%) recipients accepting the transfused platelets. Surprisingly, F-LR platelets that then underwent MPR were accepted by 21 of 22 (95%) recipients (P < .001 vs F-LR + γ-I recipients). Furthermore, 7 of 21 (33%) of these accepting recipients demonstrated specific tolerance to 8 more weekly donor transfusions that had not been treated. In addition, platelet concentrates prepared from F-LR + MPR whole blood were also nonimmunogenic; that is, 10 of 10 (100%) recipients accepted donor platelets. Overall, 31 of 32 (97%) recipients accepted F-LR + MPR platelets; none developed antibodies to donor lymphocytes. These data are the highest rate of acceptance for platelet transfusions reported in either animals or man. This approach to platelet transfusion may be particularly important when supporting patients with intact immune systems, such as in myelodysplastic syndromes.

Age-group analyses were conducted of patients in the prophylactic platelet dose trial (PLADO), which evaluated the relation between platelet dose per transfusion and bleeding. Hospitalized patients with treatment-induced hypoproliferative thrombocytopenia were randomly assigned to 1 of 3 platelet doses: 1.1 × 1011, 2.2 × 1011, or 4.4 × 1011 platelets/m2 per transfusion, given for morning counts of ≤ 10 000 platelets/μL. Daily hemostatic assessments were performed. The primary end point (percentage of patients who developed grade 2 or higher World Health Organization bleeding) was evaluated in 198 children (0-18 years) and 1044 adults. Although platelet dose did not predict bleeding for any age group, children overall had a significantly higher risk of grade 2 or higher bleeding than adults (86%, 88%, 77% vs 67% of patients aged 0-5 years, 6-12 years, 13-18 years, vs adults, respectively) and more days with grade 2 or higher bleeding (median, 3 days in each pediatric group vs 1 day in adults; P < .001). The effect of age on bleeding differed by disease treatment category and was most pronounced among autologous transplant recipients. Pediatric subjects were at higher risk of bleeding over a wide range of platelet counts, indicating that their excess bleeding risk may be because of factors other than platelet counts. This trial was registered at www.clinicaltrials.gov as #NCT00128713.

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.

BACKGROUND: The Food and Drug Administration (FDA) requires that red blood cells must be refrigerated within 8 hours of whole blood collection. Longer storage of whole blood at 22°C before component preparation would have many advantages. STUDY DESIGN AND METHODS: Two methods of holding whole blood for 20 to 24 hours at room temperature were evaluated, refrigerated plates or a 23°C incubator. After extended whole blood storage, platelet (PLT) concentrates were prepared from PLT-rich plasma on Day 1 postdonation, and the PLTs were stored for 6 more days. On Day 7 of PLT storage, blood was drawn from each subject to prepare fresh PLTs. The stored and fresh PLTs were radiolabeled and transfused into their donor. RESULTS: Eleven subjects' whole blood was stored using refrigerated butanediol plates (Compocool, Fresenius), and 10 using an incubator. Poststorage PLT recoveries averaged 47 ± 13% versus 53 ± 11% and survivals averaged 4.6 ± 1.7 days versus 4.7 ± 0.9 days for Compocool versus incubator storage, respectively (p = NS). With all results, poststorage PLT recoveries averaged 75 ± 10% of fresh and survivals 57 ± 13% of fresh; PLT recoveries met FDA guidelines for poststorage PLT viability but not survivals. CONCLUSION: Seven-day poststorage PLT viability is comparable when whole blood is stored for 22 ± 2 hours at 22°C using either refrigerated plates or an incubator to maintain temperature before preparing PLT concentrates.

In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of 530 subjects became clinically refractory (CR) to platelets (PLTs) without lymphocytotoxicity assay (LCA)-detectable anti-HLA antibodies. The LCA only detects complement-binding antibodies and is less sensitive than newer assays. Utilizing a more sensitive bead-based assay that does not distinguish between complement-binding versus non-complement-binding antibodies, we have previously shown that while many LCA-negative (LCA-) patients do have anti-HLA antibodies, these low- to moderate-level antibodies do not predict refractoriness. As complement can contribute to PLT rejection, we assessed if previously undetected complement-binding antibodies account for refractoriness among LCA- patients.Samples from 169 LCA- (69 CR, 100 non-CR) and 20 LCA-positive (LCA+; 10 CR, 10 non-CR) subjects were selected from the TRAP study serum repository. Anti-Class I HLA immunoglobulin (Ig)G and C1q-binding antibodies were measured in serum or plasma with bead-based detection assays. Levels of C1q-binding antibodies were compared between CR and non-CR subjects and correlated with corrected count increments (CCIs).While some of the LCA- subjects had detectable C1q-binding anti-Class I HLA antibodies, and some LCA+ subjects did not, levels were significantly higher among LCA+ subjects. C1q-binding anti-Class I HLA antibody levels did not differ significantly between CR and non-CR among either the LCA- or the LCA+ subjects. Furthermore, there was no significant correlation observed between CCIs and either C1q-binding or any anti-HLA IgG antibodies.This work confirms that low- to moderate-level anti-Class I antibodies do not drive PLT rejection, suggesting a role for antibody-independent mechanisms.

In April 2010, a working group sponsored by the National Heart, Lung, and Blood Institute was assembled to identify research strategies to improve our understanding of alloimmunization caused by the transfusion of allogeneic blood components and to evaluate potential approaches to both reduce its occurrence and manage its effects. Significant sequelae of alloimmunization were discussed and identified, including difficulties in maintaining chronic transfusion of red blood cells and platelets, hemolytic disease of the newborn, neonatal alloimmune thrombocytopenia, and rejection of transplanted cells and tissues. The discussions resulted in a consensus that identified key areas of future research and developmental areas, including genetic and epigenetic recipient factors that regulate alloimmunization, biochemical specifics of transfused products that affect alloimmunization, and novel technologies for high-throughput genotyping to facilitate extensive and efficient antigen matching between donor and recipient. Additional areas of importance included analysis of unappreciated medical sequelae of alloimmunization, such as cellular immunity and its effect upon transplant and autoimmunity. In addition, support for research infrastructure was discussed, with an emphasis on encouraging collaboration and synergy of animal models biology and human clinical research. Finally, training future investigators was identified as an area of importance. In aggregate, this communication provides a synopsis of the opinions of the working group on the above issues and presents both a list of suggested priorities and the rationale for the topics of focus. The areas of research identified in this report represent potential fertile ground for the medical advancement of preventing and managing alloimmunization in its different forms and mitigating the clinical problems it presents to multiple patient populations.

Background Using bacterial detection or pathogen reduction, extended platelet storage may be licensed if platelet viability is maintained. FDA's post-storage platelet acceptance guidelines are that autologous stored platelet recoveries and survivals should be ≥66% and ≥58%, respectively, of each donor's fresh platelet data.

A variety of patientand product-related factors influenced the outcome of 6379 transfusions given to 533 patients in the Trial to Reduce Alloimmunization to Platelets (TRAP). Responses measured were platelet increments, interval between platelet transfusions, and platelet refractoriness. Patient factors that improved platelet responses were splenectomy and increasing patient age. In contrast, at least 2 prior pregnancies, male gender, splenomegaly, bleeding, fever, infection, disseminated intravascular coagulation, increasing height and weight, lymphocytotoxic antibody positivity, an increasing number of platelet transfusions, or receiving heparin or amphotericin were associated with decreased posttransfusion platelet responses. Platelet factors that were associated with improved platelet responses were giving ABO-compatible platelets, platelets stored for 48 hours or less, and giving large doses of platelets while ultraviolet B (UV-B) or gamma irradiation decreased platelet responses. However, in alloimmunized lymphocytoxic antibody-positive patients, the immediate increment to UV-B-irradiated platelets was well maintained, whereas all other products showed substantial reductions. Refractoriness to platelet transfusions developed in 27% of the patients. Platelet refractoriness was associated with lymphocytotoxic antibody positivity, heparin administration, fever, bleeding, increasing number of platelet transfusions, increasing weight, at least 2 pregnancies, and male gender. The only factors that reduced platelet refractoriness rates were increasing the dose of platelets transfused or transfusing filtered apheresis platelets.

The effectiveness of different methods of leukoreduction in preventing alloimmune platelet refractoriness was evaluated in a canine model. Platelets from a random donor dog were administered for up to 8 weeks or until platelet refractoriness. Standard (STD; unmodified) platelets were accepted by 14% of recipients (n = 7) compared with 14% for centrifuge leukoreduced (C-LR) platelets (n = 21) and 31% for filter leukoreduced (F-LR) platelets (n = 13; no significant differences). Surprisingly, using both F-LR and C-LR platelets was highly effective (87% acceptance, n = 15). Transfusing F-LR/C-LR red blood cells (n = 4) or F-LR/C-LR plasma (n = 4), along with F-LR/C-LR platelets, did not affect platelet acceptance (100% acceptance). Overall acceptance of F-LR/C-LR platelets was 91% (n = 23; P ≤ .05 versus STD, C-LR, or F-LR platelets). F-LR/C-LR transfusions also induced tolerance to subsequent STD platelet transfusions from the same donor (82% acceptance, n = 19) as well as to donor skin grafts without recipient immunosuppression (57% acceptance, n = 7). To evaluate mechanisms of tolerance induction, F-LR/C-LR platelets were γ-irradiated. Although the γ-irradiated F-LR/C-LR platelets were uniformly accepted (n = 6), tolerance to STD platelets was lost. These data suggest that some allostimulatory white cells are filter adherent, whereas others escape filtration but can be removed by centrifugation and tolerance requires a residual functioning white cell.

To characterize recombinant human macrophage-colony stimulating factor (rhM-CSF)-associated thrombocytopenia (TCP), in vivo studies were performed in dogs, including the biodistributions and recoveries of radiolabelled autologous and allogeneic platelets. rhM-CSF induced a reversible, dose-dependent decrease in platelet counts. The number of megakaryocytes in spleen and marrow of rhM-CSF-treated dogs was increased two to threefold. Recoveries of allogeneic platelets transfused from rhM-CSF-treated donors into tolerized recipients (n = 3) were not significantly different from allogeneic baseline studies (93 +/- 10% of baseline values at 24 h and 90 +/- 1% at 40 h), whereas autologous platelets infused back into rhM-CSF-treated donors had decreased recoveries (45 +/- 2% of baseline values at 24 h, P = 0.03 and 20 +/- 4% at 40 h, P = 0.001). Platelet biodistribution studies showed increased accumulation of radiolabelled platelets over the spleens and livers of rhM-CSF-treated dogs. Histochemistry showed increased levels of platelet-specific antigen (CD41; glycoprotein IIb) associated with Kupffer cells. The sensitivity of platelets from rhM-CSF-treated dogs to activation from thrombin, as measured by expression of P-selectin (CD62P), was not significantly different when compared with baseline studies (P = 0.18; n = 4). These results support the concept that rhM-CSF induces an activation of the monocyte-macrophage system (MMS), which causes a reversible TCP in a dog model.

Many patients receiving dose-intensive chemotherapy acquire thrombocytopenia and need platelet transfusions. A study was conducted to determine whether platelets harvested from healthy donors treated with thrombopoietin could provide larger increases in platelet counts and thereby delay time to next platelet transfusion compared to routinely available platelets given to thrombocytopenic patients. Community platelet donors received either 1 or 3 microg/kg pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo and then donated platelets 10 to 15 days later. One hundred sixty-six of these platelet concentrates were then transfused to 120 patients with platelets counts 25 x 10(9)/L or lower. Pretransfusion platelet counts (11 x 10(9)/L) were similar for recipients of placebo-derived and PEG-rHuMGDF-derived platelets. Early after transfusion, the median platelet count increment was higher in patients receiving PEG-rHuMGDF-derived platelets: 19 (range, -12-66) x 10(9)/L, 41 (range, 5-133) x 10(9)/L, and 82 (range, -4-188) x 10(9)/L for placebo-, 1-microg/kg-, and 3-micro/kg-derived platelets, respectively. This difference was maintained 18 to 24 hours after transfusion. Transfusion-free intervals were 1.72, 2.64, and 3.80 days for the recipients of the placebo-, 1-microg/kg-, and 3-micro/kg-derived platelets, respectively. The rate of transfusion-related adverse events was not different in recipients of placebo-derived and PEG-rHuMGDF-derived platelets. Therefore, when transfused into patients with thrombocytopenia, platelets collected from healthy donors undergoing thrombopoietin therapy were safe and resulted in significantly greater platelet count increments and longer transfusion-free intervals than platelets obtained from donors treated with placebo.

Availability of platelets (plts) is severely limited by shelf life in some military as well as civilian settings. Additionally, some bleeding, thrombocytopenic patients do not have a therapeutic response to a standard plt transfusion. Methods for cryopreservation of apheresis plts for up to two years in 6% DMSO at Eligibility criteria were hospitalized thrombocytopenic hematology/oncology patients with active World Health Organization (WHO) Grade 2 or greater bleeding that was, in most patients, uncontrolled by standard plts, no history of unprovoked thrombotic events, and no indication of active DIC. Fifty-nine patients consented and 28 were transfused with ½ CPP unit (n=5), one CPP unit (n=7), two CPP units (n=6), and three CPP units (n=6). One thawed single apheresis CPP unit contained 2.5 x 1011 ± 4.2 x 1010 plts in 50 ± 4 ml. In addition, one standard apheresis plt unit was randomly given to patients enrolled in each cohort (n=4). The study conformed to the Declaration of Helsinki and was listed on clinical trials.gov (NCT02078284). Patients were monitored for six days post-transfusion for adverse events (AEs) including clinical assessments for signs or symptoms of thrombosis and specific laboratory assays: prothrombin time (PT), partial thromboplastin time (aPTT), D-dimer, fibrinogen, prothrombin fragments 1 + 2 (F1+2), antithrombin, thrombin antithrombin (TAT), thrombin generation (TGT), and thromboelastography (TEG). All safety data were reviewed by an independent data safety monitoring committee prior to escalation to the next higher dose cohort. No thrombotic events occurred after transfusion of CPP units. Five serious AEs were reported, and none were associated with the CPP transfusion but, rather, were related to worsening of the patients' underlying medical conditions. Of 38 AEs, 5 were, at least, possibly related to a CPP transfusion and included DMSO skin odor following a ½ CPP unit and three CPP units (n=2), mild fever and chills in the same patient after one CPP unit (n=2), and moderate headache the next day following transfusion of three CPP units (n=1). As expected in this clinically-ill patient population, D-dimer, fibrinogen, F1+2, aPTT, and TAT averaged higher than the upper limit of normal prior to transfusion and remained similar following transfusion. TGT and TEG were suppressed pre-transfusion and were improved towards normal levels following transfusion of either CPP or standard plts. There was no induction of a post-transfusion hypercoagulable state in any patient based on laboratory results. Modest increases in corrected plts count increments (x 103/mm3) were observed following CPP transfusion (one CPP unit gave CCI's of 2.3 ± 3.5; two CPP units 4.2 ± 2.8; and three CPP units 5.6 ± 2.3) compared with 21.1 ± 3.6 after one unit of standard apheresis plts. Notably, all patients had stabilization or improvement of their bleeding following a CPP transfusion including one patient with Grade 4 CNS bleeding who had resolution of neurologic symptoms with no further plt transfusions, and four patients (17%) had WHO bleeding downgraded. In conclusion, the infusion of up to three sequential units of CPP in patients with severe thrombocytopenia and active bleeding was safe without any evidence of thrombotic complications despite CPP having a procoagulant phenotype resulting from the cryopreservation process. CPP may be efficacious to stop bleeding in thrombocytopenic patients as suggested by stabilization or downgrading of WHO bleeding grades. Phase 2/3 efficacy clinical trials are now indicated. Disclosures Slichter: NHLBI / NIH: Research Funding; Genentech: Research Funding; Cerus Corporation: Research Funding; Terumo BCT: Research Funding; Cellphire: Research Funding; Department of Defense / US Army Medical Research and Material Command: Research Funding; Megakaryon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Stock options. Dumont:US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding. Cancelas:New Health Sciences, Inc.: Membership on an entity's Board of Directors or advisory committees; US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding; National Institutes of Health: Research Funding; Terumo BCT: Research Funding; Cerus Corporation: Research Funding; Haemonetics, Inc.: Research Funding; Citra Labs, Inc.: Research Funding; Cellphire, Inc.: Membership on an entity's Board of Directors or advisory committees; William & Lawrence Hughes Foundation: Research Funding; Leukemia & Lymphoma Society of North America: Research Funding. Gernsheimer:Department of Defense: Research Funding; NHLBI / NIH: Research Funding. Szczepiorkowski:Fresenius Kabi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Grifols: Consultancy, Research Funding; Terumo BCT: Consultancy; Cerus Corporation: Research Funding; Erydel: Research Funding; Citra Labs: Research Funding; US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding; American Association of Blood Banks: Other: President-Elect. Dunbar:Verax Biomedical: Membership on an entity's Board of Directors or advisory committees; US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding. Jones:US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding. Rugg:US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding. Prakash:US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding. Hmel:US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding. Ransom:US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding.

The MpL ligand (ML) is a potent stimulus for thrombocytopoiesis. To create an in vivo model of ML deficiency, we injected dogs with a recombinant human ML (rhML) to determine whether cross-reacting antibodies would develop and cause thrombocytopenia. RhML was administered subcutaneously for 8 weeks to three normal dogs (mean platelets, 197 ± 5.5 × 103/μL). Within 5 days their platelet counts were twice baseline and greater than 4 times baseline by day 21. Then, uniformly, chronic thrombocytopenia developed. At 1 week after terminating rhML, mean platelets were 0.5 times baseline and at 2 months 0.25 times baseline. Early in treatment, marrow biopsies showed increased megakaryocyte number and ploidy, which decreased as platelets declined. Paralleling these changes, high titer anti-rhML antibodies developed. Autologous 51Cr-labeled platelet recovery and survival measurements indicated that the thrombocytopenia was principally due to decreased production. Infusion of plasma from the thrombocytopenic dogs into two normal dogs and one dog previously made thrombocytopenic with rhML caused platelet counts to fall gradually. These studies show that dogs with anti-rhML antibodies develop thrombocytopenia, presumably because the cross-reacting antibodies neutralize endogenous canine ML. The results strongly suggest that ML plays an essential role in maintaining normal platelet levels.

Platelet (PLT) concentrates (PCs) prepared from whole blood in the United States are made using the PLT-rich plasma method. The PCs must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, PCs are made using the buffy coat (BC) method from whole blood held overnight at 22 °C and storage times may be up to 7 days. Our studies were designed to determine how long BC PLTs can be stored in plasma or Plasmalyte while meeting the FDA's poststorage viability criteria.Normal subjects donated whole blood that was stored at 22 °C for 22 ± 2 hours before preparation of BC PLTs. PLTs were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65 or 80%. Radiolabeled autologous stored versus fresh PLT recoveries and survivals were assessed as well as poststorage in vitro assays.BC PLTs stored in either plasma or 65% Plasmalyte met FDA poststorage PLT recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored PLT recoveries and survivals correlated with the same donor's fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted poststorage PLT recoveries, while annexin V binding best predicted PLT survivals.BC PLTs stored in either plasma or 65% Plasmalyte meet FDA's poststorage viability criteria for 6 days.

How platelet (PLT) product characteristics such as dose, source (whole blood derived [WBD] vs. apheresis), storage duration, and ABO matching status affect the risks of transfusion-related adverse events (TRAEs) is unclear. Similarly, more information is needed to define how recipient characteristics affect the frequency of TRAEs after PLT transfusion.In the multicenter Platelet Dose ("PLADO") study, pediatric and adult hematology-oncology patients with hypoproliferative thrombocytopenia were randomized to receive low-dose (LD), medium-dose (MD), or high-dose (HD) PLT prophylaxis for a pretransfusion PLT count of not more than 10 × 10(9) /L. All PLT units (apheresis or WBD) were leukoreduced. Post hoc analyses of PLADO data were performed using multipredictor models.A total of 5034 PLT transfusions to 1102 patients were analyzed. A TRAE occurred with 501 PLT transfusions (10.0%). The most common TRAEs were fever (6.6% of transfusions), allergic or hypersensitivity reactions (1.9%), and sinus tachycardia (1.8%). Patients assigned HD PLTs were more likely than LD or MD patients to experience any TRAE (odds ratio for HD vs. MD, 1.50; 95% confidence interval, 1.10-2.05; three-group comparison p = 0.02). PLT source and ABO matching status were not significantly related to overall TRAE risk. Compared to a patient's first PLT transfusion, subsequent PLT transfusions were less likely to have a TRAE reported, primarily due to a lower risk of allergic or hypersensitivity reactions.The most important PLT unit characteristic associated with TRAEs was PLT dose per transfusion. HD PLTs may increase the risk of TRAEs, and LD PLTs may reduce the risk.

Both leukoreduction and ultraviolet (UV) light treatment of blood products have been shown to reduce the incidence of HLA antibody development in recipients, but the impact of these treatments on the magnitude and persistence of the antibody response is less clear.Longitudinal samples from 319 subjects taken from four different study cohorts were evaluated for HLA antibodies to determine the effects of leukoreduction and UV treatment on HLA antibody generation and persistence.Subjects receiving leukoreduced or UV-treated blood products were less likely to generate Class I HLA antibodies, and those receiving leukoreduced blood were also less likely to generate Class II HLA antibodies. Among those receiving nonleukoreduced blood, 55% developed Class I HLA antibodies and 51% developed Class II HLA antibodies compared with 28% (Class I) and 15% (Class II) for those receiving leukoreduced blood and 36% (Class I) and 54% (Class II) for those receiving UV-treated blood. Among alloimmunized subjects, leukoreduction resulted in a significant twofold reduction in the magnitude of Class I HLA antibodies, and UV treatment resulted in a significant threefold reduction in the magnitude of Class II HLA antibodies. Both treatments resulted in shorter persistence of Class I HLA antibodies.These data demonstrate that leukoreduction and UV treatment of blood products results not only in a reduction in the incidence of HLA antibody production, but also in lower and more transient HLA antibody levels among sensitized transfusion recipients.

In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of 530 participants became refractory to platelet transfusions without evidence of HLA or human platelet antigen (HPA) antibodies. We used a more sensitive bead-based assay to detect and quantify HLA antibodies and a qualitative solid-phase enzyme-linked immunosorbet assay for HPA to determine whether low-level antibodies could predict refractoriness in longitudinal panels from 170 lymphocytotoxicity assay (LCA)(-) and 20 LCA(+) TRAP participants. All TRAP recipients who previously tested LCA(+) were HLA antibody(+), using the bead-based system. Levels of HLA or HPA antibodies did not predict refractoriness among LCA(-) recipients, although higher levels of HLA antibodies were associated with refractoriness among LCA(+) recipients. These data demonstrate that weak to moderate HLA antibody levels detectable by modern binding assays are not associated with platelet refractoriness.

We performed a prospective, randomized trial in CMV seronegative marrow recipients to determine if filtered blood products were as effective as CMV-seronegative blood products for the prevention of transfusion- transmitted CMV infection after marrow transplant. Before transplant, 502 patients were randomized to receive either filtered or seronegative blood products. Patients were monitored for the development of CMV infection and tissue-documented CMV disease between days 21 and 100 after transplant. Infections occurring after day 21 from transplant were considered related to the transfusion of study blood products and, thus, were considered evaluable infections for the purpose of this trial. In the primary analysis of evaluable infections, there were no significant differences between the probability of CMV infection (1.3% v 2.4%, P = 1.00) or disease (0% v 2.4%, P = 1.00) between the seronegative and filtered arms, respectively, or probability of survival (P = .6). In a secondary analysis of all infections occurring from day 0 to 100 post-transplant, although the infection rates were similar, the probability of CMV disease in the filtered arm was greater (2.4% v 0% in the seronegative arm, P = .03). However, the disease rate was still within the prestudy clinically defined acceptable rate of < or = 5%. We conclude that filtration is an effective alternative to the use of seronegative blood products for prevention of transfusion- associated CMV infection in marrow transplant patients.

Autoimmune thrombocytopenic purpura (AITP) is generally a chronic disorder in affected adults. Twenty-five percent of these patients will become refractory to routine therapy (corticosteroids and splenectomy), as well as most other available agents. Intravenous pulse cyclophosphamide therapy was used to treat 20 patients with severe refractory AITP who had previously failed to achieve a sustained remission with a mean of 4.8 agents (range 2 to 8). Patients received 1 to 4 doses (mean 2.0) of 1.0 to 1.5 g/m2 intravenous cyclophosphamide per course. Of the 20 patients treated with pulse cyclophosphamide therapy, 13 patients (65%) achieved a complete response (CR), four (20%) a partial response (PR), and three patients (15%) failed to respond. Of the 13 complete responders, eight have remained in remission with stable platelet counts during followup intervals of 7 months to 7 years (median 2.5 years). Five patients developed recurrent AITP 4 months to 3 years following a CR. Of these, two patients responded to subsequent courses of pulse cyclophosphamide therapy with current remissions of 1 and 4 years. Of the four patients who obtained a PR, two remain in partial remission after 10 months and 4 years; one relapsed after 18 months and, after retreatment, is still in remission at 6 months. Of the patient characteristics examined, duration of disease was most strongly associated with response to pulse cyclophosphamide. Side-effects of treatment included neutropenia (three patients, one of whom developed staphylococcal sepsis), acute deep venous thrombosis (two patients), and psoas abscess (one patient). Intravenous pulse cyclophosphamide should be strongly considered in the treatment of patients with refractory AITP. There is a relatively low incidence of side-effects, and it can be administered easily on an out- patient basis.

Background: Patients treated with Ado-Trastuzumab-Emtansine (T-DM1) are at risk of thrombocytopenia with the reported rate of 13% in metastatic patients of >3 toxicity. A recent report supports that off-target uptake of T-DM1 into megakaryocyte precursors may result in thrombocytopenia. It is unclear if this is the only mechanism to explain the thrombocytopenia since many patients have lower grade effects on platelets and often report mild mucosal bleeding. In this study, we will test whether platelet half-life in circulation or platelet function are also affected by treatment with T-DM1. Trial design: We initiated a single institution Phase 1 study of T-DM1 in metastatic or unresectable breast cancer in any line of therapy. The general method is to collect autologous platelets, radiolabel the platelets ex vivo, and reinfuse them into patients to measure recovery (twice, at day 4 and again on day 7-9 days). The first 21-day cycle on protocol is to measure baseline platelet recovery (no T-DM1) followed by two cycles where platelet recovery is measured after T-DM1 infusion. Only, three radiolabeled platelet infusions occur on trial (2 with drug) after which disease response is assessed and patients can remain on drug at the discretion of the primary treating oncologist. In addition, patients will have platelet aggregation studies and bleeding times measured. Adult patients with Her2+ breast cancer are eligible for treatment provided they have adequate bone marrow (ANC > 1500 cells/mm3, Platelet >100,000/mm3, hemoglobin >9 g/dL), cardiac (Ejection fraction > 50%), kidney and liver function at baseline and have an ECOG performance score better than 2. Patients' prior treatment with chemotherapy or trastuzumab, if any, must have a >21 day washout period. Prior T-DM1 treatment is not permitted nor is a personal history of known platelet disorder (ITP, von Willebrand's disease). Patients with brain metastases are permitted to participate provided the disease is treated and is stable for >30 days. Statistical methods: The primary goal is to measure platelet T1/2 following T-DM1 exposure and to compare this to baseline recovery. The proposed sample size of 20 participants is selected to insure that at least 15 patients will have complete data for all time points and study procedures. The first test performed will compare pre-therapy platelet lifespan to platelet lifespan on T-DM1 (cycles 1 and 2). Using platelet lifespan from normal controls to approximate pre-therapy values (mean 10.7 days, standard deviation 2.0), for 15 patients a two-sided paired t-test with alpha=0.05 would have 82% power to detect a 15% (1.605 days) decrease in platelet lifespan, assuming a correlation of 0.5 between paired values. The actual analysis will fit a linear mixed effects model, using a two-sided Wald test to compare pre-therapy to the two post-therapy values. Accrual: The study activated in January 2015. One patient has undergone all study procedures. 3 Additional patients have been screened and 2 of these patients chose not to participate citing that the study had too many procedures and one was discovered to have had prior T-DM1 treatment. The investigators are in the process of opening the study to Her2 positive non-breast cancer patients to accelerate accrual. Citation Format: Gadi VK, Butler BS, Corson J, Slichter SJ. Thrombokinetic studies of ado-trastuzumab emtansine (T-DM1). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT3-01-11.

Background and Objectives The purpose of our studies was to determine the effects of extended platelet storage on poststorage platelet viability. Materials and Methods Normal subjects were recruited to donate platelets using two different apheresis systems: either the COBE Spectra (n = 58) or the Haemonetics MCS+ (n = 84). Platelet recovery and survival data from the two systems were compared with each other and with in vitro measurements of the stored platelets. Results There were no significant differences in either platelet recoveries or survivals between the two machines between 1 and 8 days of storage. Combining the data from both machines, platelet recoveries decreased by 2·6% and survivals by 0·3 days/storage day. In vitro assays did not predict either platelet recoveries or survivals during storage for 5–8 days. After 9 days of storage, pHs were unacceptable (≤ 6·1), suggesting that 8 days will be the longest possible storage time. Conclusions These data suggest that, if stored platelet bacterial contamination issues are resolved, significant extension of platelet storage times is possible.

Background: Trauma surgeons have suggested that the optimal transfusion support for actively bleeding patients may be a 1:1:1 ratio of red cells, plasma and platelets (plts). Therefore, the only reason to give component therapy - rather than whole blood (WB) - for these patients may be because of the poor post-transfusion survivals of plts stored at 4°C. However, for trauma/surgical patients, even short plt survivals may be sufficient to allow the surgeon enough time to repair the injury. We report the "first ever" data on the post-storage viability of plts stored at 4°C beyond 3 days. In the studies to be reported here, plts were stored within WB for up to 22 days at 4°C. Study Design/Methods: Normal subjects donated a unit of WB that was stored at 4°C either for 12 days with mixing only at the end of storage or for 10, 15, or 22 days with end-over-end rotation of the WB throughout storage. After storage, a plt concentrate was prepared from the WB using standard centrifugation procedures. At the end of WB storage, the donor donated a 40 ml blood sample from which fresh plts were prepared. The autologous stored and fresh plts were radiolabeled with 111In or 51Cr, respectively, before simultaneous transfusion into their donor. Serial post-transfusion blood samples were collected to determine stored versus fresh plt recoveries and survivals. Our pre-defined acceptance criteria were: 1) stored plt recoveries should average ≥50% of the same donor's fresh plt recoveries; and 2) plt survivals should average ≥1 day. Results/Findings: When the WB was mixed only at the end of storage, plt yields in the WB were just 48% of baseline values with average plt counts of 4.6 ± 2.0 x 1010. This amount of plt loss was considered unacceptable (Table). In contrast, with continuous end-over-end rotation of the WB during storage, plt yields after 15 days were 76% of baseline values and plt counts averaged 7.6 ± 1.4 x 1010 (well within FDA guidelines that require 5.5 x 1010 plts/concentrate prepared from WB). Radiolabeled autologous plt recoveries averaged 27 ± 11% (49 ± 16% of fresh) and survivals averaged 1.2 ± 0.4 days (16 ± 1% of fresh); i.e., our pre-defined plt acceptance criteria were met. Storage for 22 days gave unacceptable results. Conclusion: Plt concentrates stored at 4°C for up to 3 days have been FDA approved for use in thrombocytopenic patients since the 1970's. These plts had autologous plt recoveries of 40 ± 5% and survivals of 1.0 ± 0.1 days (S.E.) (Br J Haematol 1976;34:403-419). The FDA has recently approved the use of 3-day 4°C stored apheresis plts for actively bleeding patients. Our data may suggest that plts stored within WB at 4°C for up to 15 days may provide similar post-storage plt viability as plt concentrates or apheresis plts stored for ≤3 days. Red cells and plasma derived from CP2D WB stored for 21 days at 4°C are already licensed. Therefore, for actively bleeding patients who need all three components (red cells, plasma, and plts), WB stored for up to 15 days may provide effective quantities of all the needed components. Table 1. AUTOLOGOUS YIELDS, RECOVERIES, AND SURVIVALS OF PLATELETS STORED IN WHOLE BLOOD AT 4°C Storage Time (Days) N Rotation WB PLT COUNTS x 1010 Post-Storage Yield(% of Baseline) PLT RECOVERIES (%) PLT SURVIVALS (Days) Baseline Post-Storage Fresh Stored % of Fresh Fresh Stored % of Fresh 12 7 Mixed at end of storage* 9.4 ± 2.4 4.6 ± 2.0 48 ± 12 50 ± 15 22 ± 8 48 ± 29 8.3 ± 1.6 1.8 ± 0.6 23 ± 10 10 10 Continuously during storage** 9.5 ± 2.1 7.2 ± 1.6 77 ± 10 50 ± 10 26 ± 7 51 ± 7 8.1 ± 1.1 1.3 ± 0.3 16 ± 4 15 10 Continuously during storage** 10.0 ± 1.0 7.6 ± 1.4 76 ± 10 55 ± 11 27 ± 11 49 ± 16 8.0 ± 0.1 1.2 ± 0.4 16 ± 1 22 3 Continuously during storage** 12.9 ± 1.9 9.2 ± 0.7 73 ± 15 56 ± 12 14 ± 4 25 ± 5 7.7 ± 0.6 0.8 ± 0.3 10 ± 3 Data reported as average ±1 S.D. *WB units were hand mixed only at the end of storage before plt counting. **WB units were continuously rotated end-over-end during storage. Disclosures Slichter: Cellphire, Inc.: Research Funding; Cerus Corporation: Research Funding; Terumo BCT: Research Funding; Department of Defense: Research Funding; Genentech: Research Funding.

Immunomodulation following transfusion of cellular blood products presents as a generalized immunosuppression and/or production of alloantibody. The immunological effects of transfusion in immunocompetent subjects were evaluated in patients undergoing cardiac surgery with cardio-pulmonary by-pass. Subjects were consented for this IRB-approved study and were randomized to receive unmodified (STD) red blood cells (rbc), leukoreduced (LR) rbc or LR rbc that were g-irradiated (g-I). All platelet transfusions were collected LR and they were also g-I for the third arm. Patients were transfused only when clinically indicated. Subjects receiving no transfusions during the study served as a fourth control arm. Peripheral blood samples collected prior to surgery were controls for samples collected after surgery (day 10 +/- 4 and day 30 +/-5). HLA antibody production was evaluated in 245 subjects using single-antigen multiarray assays for antibody to both HLA class I and class II, a summary of results in shown in Table 1. For subjects with HLA antibody in their pre-surgery sample, these assays allowed evaluation of change in antibody specificity or levels. For these pre-sensitized subjects, there was no significant difference in HLA antibody production between the transfused study arms: even those not transfused showed an increase, most likely due to activation of memory responses. For subjects without HLA antibody in their pre-surgery sample, recipients of LR products showed little increase supporting prior studies showing the efficacy of LR in reducing antibody. However the protection afforded by LR was abrogated by g-I. This was an unexpected finding. We evaluated cellular and cytokine mediators of immunosuppression in the first 139 consecutive subjects of the 245 subjects enrolled in this study. This part of the study was powered for hypothesis generation, not statistical significance. Mononuclear cells (MNC) were isolated from each sample, frozen, and all samples from a subject assayed together. We assayed these cells for production of TGFB1 using ELISPOT. We used flow cytometry phenotyping for markers of CD4+25+ T regulatory cells (CTLA4, FoxP3) and for intracellular cytokines for CD4+ and CD4- NKT cells (IFNG, IL-4, IL-10). As shown in Table 2, recipients of standard rbc showed an increase (following transfusion and surgery) in TGFB1 producing mononuclear cells with a lower increase in recipients of LR rbc and those who were not transfused, but further reduced in the group receiving g-I LR products. Recipients of standard rbc or of LR rbc showed an increase of T lymphocytes expressing FoxP3 or CTLA4, and of CD4+ NKT cells producing IL-4. In contrast, subjects whose LR products were g-I showed a markedly lower increase in all three measures. These data identify an increase in mediators of immunosuppression in recipients of standard rbc and LR rbc that was reduced in recipients of g-I and LR rbc and platelets. Surgery and bypass alone increased the numbers of TGFB1+ MNC and IL4+ NKT cells suggesting a role for the innate immune system in immunosuppression following transfusion. This study is the first to exam the immunomodulatory effects of transfusion by combining measurement of immunosuppression in ex vivo surrogate assays and measurement of HLA antibody production inclusive of pre-sensitized subjects. Our observations of differences in responses to blood products prepared with g-I plus LR compared to LR alone were unexpected and require confirmation. It suggests that the reduction in immunosuppressive cells and cytokines in the recipients of g-I and LR products contributed to the increased allosensitization we observed in that arm compared to recipients of LR products. It may be possible to manipulate immunomodulation after transfusion to reach a desired goal for specific patient populations. Disclosures Slichter: Department of Defense: Research Funding; Terumo BCT: Research Funding; Cerus Corporation: Research Funding; Cellphire, Inc.: Research Funding; Genentech: Research Funding.

Patients with acute myelogenous leukemia undergoing induction chemotherapy have significant decreases in alloimmune platelet refractoriness if they receive filter-leukoreduced or UV-B–irradiated vs standard platelet transfusions (3%-5% vs 13%, respectively; P ≤ .03) with no differences among the treated platelet arms (Trial to Reduce Alloimmunization to Platelets). Therefore, measuring antibody persistence might identify the best platelets for transfusion. Lymphocytotoxic (LCT) antibody duration was evaluated for association with patient age, sex, prior transfusion and pregnancy history, study-assigned platelet transfusions, and percentage LCT panel reactive antibodies. During the Trial to Reduce Alloimmunization to Platelets, 145 patients became antibody positive; and 81 (56%) of them subsequently became antibody negative. Using Kaplan-Meier estimates, projected antibody loss was 73% at 1 year. Major factors associated with antibody persistence were prior pregnancy and percentage panel reactive antibody positivity, whereas neither the assigned type of platelets transfused during the 8 weeks of the trial nor prior transfusion history was predictive. After 5 to 8 weeks, the number and type of blood products transfused had no effect on either antibody development or loss. A majority of patients with acute myelogenous leukemia who develop LCT antibodies during induction chemotherapy will lose their antibodies within 4 months regardless of the type or number of blood products they receive.

We conducted a trial of prophylactic platelet transfusions to evaluate the effect of platelet dose on bleeding in patients with hypoproliferative thrombocytopenia.We randomly assigned hospitalized patients undergoing hematopoietic stem-cell transplantation or chemotherapy for hematologic cancers or solid tumors to receive prophylactic platelet transfusions at a low dose, a medium dose, or a high dose (1.1x10(11), 2.2x10(11), or 4.4x10(11) platelets per square meter of body-surface area, respectively), when morning platelet counts were 10,000 per cubic millimeter or lower. Clinical signs of bleeding were assessed daily. The primary end point was bleeding of grade 2 or higher (as defined on the basis of World Health Organization criteria).In the 1272 patients who received at least one platelet transfusion, the primary end point was observed in 71%, 69%, and 70% of the patients in the low-dose group, the medium-dose group, and the high-dose group, respectively (differences were not significant). The incidences of higher grades of bleeding, and other adverse events, were similar among the three groups. The median number of platelets transfused was significantly lower in the low-dose group (9.25x10(11)) than in the medium-dose group (11.25x10(11)) or the high-dose group (19.63x10(11)) (P=0.002 for low vs. medium, P0.001 for high vs. low and high vs. medium), but the median number of platelet transfusions given was significantly higher in the low-dose group (five, vs. three in the medium-dose and three in the high-dose group; P0.001 for low vs. medium and low vs. high). Bleeding occurred on 25% of the study days on which morning platelet counts were 5000 per cubic millimeter or lower, as compared with 17% of study days on which platelet counts were 6000 to 80,000 per cubic millimeter (P0.001).Low doses of platelets administered as a prophylactic transfusion led to a decreased number of platelets transfused per patient but an increased number of transfusions given. At doses between 1.1x10(11) and 4.4x10(11) platelets per square meter, the number of platelets in the prophylactic transfusion had no effect on the incidence of bleeding. (ClinicalTrials.gov number, NCT00128713.)

Canine granulocyte-macrophage colony-stimulating factor (caGM-CSF) was cloned and expressed to allow further investigation of GM-CSF in a large animal model. The cDNA is 850 base pairs (bp) long and encodes a peptide of 144 amino acids. The nucleotide and amino acid sequence homology between caGM-CSF and human GM-CSF (hGM-CSF) is 80% and 70%, respectively. A mammalian expression vector pCMV/CAGM was constructed and used to transfect COS cells for expression of caGM-CSF. Supernatant from transfected COS cells enriched with caGM-CSF was shown to have significant stimulating activity in granulocyte-macrophage colony forming unit (CFU-GM) assays of canine marrow. caGM-CSF, expressed from bacteria, was used to treat seven dogs at varying doses twice daily subcutaneously (sc) for 14 to 16 days. Circulating blood neutrophils and monocytes increased significantly. The increase in circulating eosinophils was variable. Thrombocytopenia developed during administration of caGM-CSF but corrected rapidly after cessation of treatment. Evaluation of survival times of 51Cr-labeled autologous platelets suggested increased consumption as the primary reason for thrombocytopenia. A species-specific GM-CSF will be a useful tool for hematologic or immunologic studies in dogs.

In a group of stable, nonthrombocytopenic leukemia patients awaiting bone marrow transplantation, results of paired allogeneic radiolabeled platelet kinetic measurements were correlated with the results of several different platelet and lymphocytotoxic antibody tests to determine which parameters could be used to identify patients who were alloimmunized to platelets. Seven patients with acute leukemia who had been transfused during induction therapy were used as the test group, and, as a control group, five untransfused patients with chronic myelogenous leukemia were also studied. Concurrent fibrinogen survival measurements were performed in all patients to assess whether hemostatic factor consumption (ie, disseminated intravascular coagulation) was present. Allogeneic platelet survival measurements were reduced from normal in all 12 study patients. In 8 of 12 patients, fibrinogen and platelet survival measurements were comparably reduced, suggesting disease-related platelet consumption. In four heavily transfused patients with acute leukemia, allogeneic platelet survivals were markedly reduced to less than or equal to 2.1 days, compared with the 3.5- to 7.4-day platelet survival measurements found in the other eight patients. The disproportionately short platelet survivals compared with fibrinogen survival measurements in these four patients, combined with documented positive antibody tests to their donors' platelets in the three patients with evaluable tests, suggested that these patients had become alloimmunized to platelets because of their prior transfusions. There was substantial concordance between the two radiolabeled allogeneic donor platelet survival measurements performed in each of these patients, suggesting that host rather than donor factors have a major influence on transfusion outcome (r = .93, P less than .001). The platelet cross-match tests, using the radiolabeled protein Staph A assay combined with the IgG enzyme-linked immunosorbent assay test, had the best correlation with the posttransfusion recovery and survival of the donors' platelets.

Patients with severe thrombocytopenia are presumed to be at increased risk for bleeding, and consequently it has been standard practice for the past four decades to give allogeneic platelet transfusions to severely thrombocytopenic patients as supportive care. Platelet transfusions may be given either prophylactically to reduce the risk of bleeding, in the absence of clinical hemorrhage (prophylactic transfusions), or to control active bleeding when present (therapeutic transfusions). While no one would argue with the need for platelet transfusions in the face of severe bleeding, important questions remain about what constitutes clinically significant bleeding and whether a strategy of prophylactic platelet transfusions is effective in reducing the risk of bleeding in clinically stable patients. It is now uncommon for patients undergoing intensive chemotherapy or bone marrow transplantation to die of hemorrhage, but it is open to debate as to what degree platelet transfusions have been responsible for this change in outcome, given the many other advances in other aspects of supportive care. If a prophylactic strategy is followed, the optimal transfusion trigger or quantity of platelets to be transfused prophylactically per transfusion episode needs to be addressed in adequately powered clinical trials, but these remain highly controversial issues. This is because, until recently, there have been few high-quality, prospective, randomized clinical trial (RCT) data for evaluating the relative effects of different platelet transfusion regimens or platelet doses on clinical outcomes. Moreover, most of these RCTs have not used bleeding as the primary outcome measure. Two such studies on platelet dose have now been undertaken, the PLADO (Prophylactic PLAtelet DOse) and the SToP (Strategies for the Transfusion of Platelets) trials. Data from these RCTs are not contained in this overview, as these data have not yet been completely analyzed or submitted for peer review publication. In addition to the above, several recent observational studies have raised the possibility that there is not a clear association between the occurrence of a major clinical bleeding episode and the platelet count in thrombocytopenic patients. Such findings have led to the questioning of the efficacy of prophylactic platelet transfusions in all clinically stable patients, and whether a policy of therapeutic transfusions used only when patients have clinical bleeding might be as effective and safe for selected patients. At least two RCTs evaluating the relative value of prophylactic versus therapeutic platelet transfusions have been initiated in thrombocytopenic patients with hematological malignancies. One such study, known as the TOPPS (Trial of Prophylactic Platelets Study) study, is currently underway in the U.K.

Cold storage of platelets for transfusion is desirable to extend platelet storage times and to prevent bacterial growth. However, the rapid clearance of cold-stored platelets prevents their use. A novel method for preventing the rapid clearance of cold-stored platelets has previously been developed in a murine model. Cold storage induces the clustering and recognition of exposed β-N-acetylglucosamine (βGlcNAc) on platelet surfaces. Glycosylation of βGlcNAc residues with uridine 5′-diphosphogalactose (UDP-galactose) results in the normal survival of short-term (2 h) 0°C-stored murine platelets. Based on this finding, we developed a similar glycosylation process by adding UDP-galactose to human apheresis platelets. A phase 1 clinical trial was conducted transfusing radiolabeled autologous apheresis platelets stored for 48 hours at 4°C with or without pretreatment with UDP-galactose. In contrast to the murine study, galactosylation of human platelets did not prevent the accelerated platelet clearance routinely observed after 4°C storage. We next developed a murine model of platelet storage for 48 hours at 4°C and showed that UDP-galactose treatment of murine platelets also did not prevent their rapid clearance, in agreement with the human platelet study. We conclude that different mechanisms of clearance may exist for short- and long-term cold-stored platelets.

Transfused platelets (plts) are either pooled random-donor platelet (plt) concentrates or single-donor apheresis plts. When stored for 5 days, all of these products are equally efficacious.A 10,000/μL prophylactic plt transfusion trigger has been documented to be both hemostatically efficacious and cost effective in reducing plt transfusion requirements. The optimal plt dose/transfusion is being evaluated in an ongoing clinical trial. Therapeutic plt transfusions to control or prevent bleeding with trauma or surgical procedures require higher transfusion triggers of 100,000/μL for neurosurgical procedures and between 50,000/μL and 100,000/μL for other invasive procedures or trauma.Leukoreduction has been documented to reduce plt alloimmunization rates, cytomegalovirus (CMV) transmission by transfusion, and febrile transfusion reactions. Whether it reduces immunomodulatory effects of transfusion (i.e., decreases infection rates and cancer recurrence) is still controversial, as is universal leukoreduction.Poor responses to plt transfusions are often multifactorial. For alloimmune plt refractoriness, HLA matching, cross-matching, and identification of the specificity of the patient’s antibodies with avoidance of mismatched donor antigens are all equally effective in identifying compatible plts for transfusion. Other causes of poor plt responses are splenomegaly, ABO mismatching, females with 2 or more pregnancies and males, use of heparin or amphotericin, bleeding, fever, graft-vs-host disease (GVHD), and vaso-occlusive disease (VOD).

BACKGROUND: Recently, bleeding has been used in platelet (PLT) trials rather than surrogate outcomes. The purpose of this study was to provide a descriptive summary of data from PLT studies conducted in four countries and exploratory analyses to determine the relationship between bleeding and PLT count. STUDY DESIGN AND METHODS: A descriptive analysis was performed on original data from the Italian trigger study, the US TRAP study, the Canadian febrile reaction study, and a clinical chart review from one hospital site in the United Kingdom. The relationship between bleeding and PLT count was explored with the Italian data. RESULTS: A total of 897 patients with acute leukemia received 10,506 PLT transfusions. Grade 3 or Grade 4 WHO bleeding frequency was 28.1 percent (252/897) but varied by country: Italy, 10.8 percent (27/250); United States, 36.4 percent (217/598); Canada, 18.9 percent (7/37); and the United Kingdom, 8.3 percent (1/12). Grade 1 or Grade 2 bleeding was reported only in the Italian study (46.4%[116/250] and 11.6%[29/250], respectively). The relative rates of WHO Grade 2, 3, or 4 bleeding for PLT counts in the ranges of 0 to 4, 5 to 9, 10 to 14, and 15 to 19 (x10(9)/L) were 8.8, 1.9, 1.8, and 1.2, respectively, compared to those counts within the range of 20 to 29 (x10(9)/L). CONCLUSION: The study provides descriptive data on PLT use and frequency of bleeding. When PLT counts were 0x10(9) to 4x10(9) per L there was an eightfold increase in bleeding and a twofold risk increase when counts were 5x10(9) to 14x10(9) per L compared to the 20x10(9) to 29x10(9) per L reference range. The increased rate of bleeding at low counts occurred despite PLT therapy.

Introduction: In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of the 530 subjects became clinically refractory (CR) to platelet transfusions in the absence of detectable antibodies against HLA as measured by the lymphocytotoxicity assay (LCA). Using more sensitive bead-based detection methods we have previously demonstrated that while many of these LCA- patients do have anti-HLA antibodies, that these low to moderate level antibodies do not predict refractoriness. In addition to being less sensitive then bead based methods, the LCA screen only detects complement-binding antibodies. As these antibodies could be important for platelet rejection, we assessed if previously undetected complement-binding antibodies could account for some of the refractoriness seen in LCA- patients. Methods: 169 LCA- (69 CR, 100 non-CR) and 20 LCA+ (10 CR, 10 non-CR) subjects were selected from the TRAP study. Anti-class I HLA IgG and C1q binding antibodies were measured in serum or plasma using two different multi-analyte, semi-quantitative, bead-based fluorescent antibody detection assays: the LabScreen mixed Luminex assay, and the LabScreen single antigen class I assay with or without added EDTA (One Lambda). Groups were compared using an unpaired t-test, a=0.05, and correlation between variables was also assessed. Results: New measurements of anti-class I HLA IgG antibodies reliably reproduced earlier data with a strong correlation between the old and new measurements (R2=0.9736, p Conclusions: While complement-binding anti-class I HLA antibody levels were higher in the LCA+ subjects, higher levels of these antibodies were not seen in CR LCA+ patients as compared with non-CR LCA+ patients. Complement-binding anti-class I HLA antibodies do not account for refractoriness seen among the LCA- TRAP subjects. This work confirms that low to mid level anti-class I antibodies do not drive refractoriness to platelets, and suggests that antibody-independent mechanisms cause refractoriness in patients lacking higher levels of anti-HLA antibodies. Disclosures No relevant conflicts of interest to declare.

We report a transfusion trial of platelets photochemically treated for pathogen inactivation using the synthetic psoralen amotosalen HCl. Patients with thrombocytopenia were randomly assigned to receive either photochemically treated (PCT) or conventional (control) platelets for up to 28 days. The primary end point was the proportion of patients with World Health Organization (WHO) grade 2 bleeding during the period of platelet support. A total of 645 patients (318 PCT and 327 control) were evaluated. The primary end point, the incidence of grade 2 bleeding (58.5% PCT versus 57.5% control), and the secondary end point, the incidence of grade 3 or 4 bleeding (4.1% PCT versus 6.1% control), were equivalent between the 2 groups (P =.001 by noninferiority). The mean 1-hour posttransfusion platelet corrected count increment (CCI) (11.1 x 10(3) PCT versus 16.0 x 10(3) control), average number of days to next platelet transfusion (1.9 PCT versus 2.4 control), and number of platelet transfusions (8.4 PCT versus 6.2 control) were different (P

Abstract 271 Background: The largest transfusion (tx) trial to evaluate methods of preventing platelet (plt) alloimmunization (TRAP Trial; NEJM 1997;337:1861) demonstrated residual alloimmunization rates of 17% to 21% in AML patients (pts) undergoing induction chemotherapy despite receiving either filter-leukoreduced (F-LR) or UV-B irradiated (UV-BI) blood products, respectively. Our pre-clinical dog plt tx studies, the basis for testing UV-BI in the TRAP Trial, demonstrated this model was able to predict pt results; i.e., prevention of alloimmunization was 45% in the dog but 79% in pts. The greater effectiveness in pts was probably because they had chemotherapy-induced immunosuppression compared to the immunocompetent dogs. Our current dog plt tx studies have focused on evaluating F-LR to remove antigen-presenting WBCs (APCs) or pathogen-reduction (PRT) (Mirasol treatment) to inactivate APCs. Methods: For pts, plts are obtained using either apheresis procedures or as plt concentrates prepared from whole blood (WB). To re-duplicate these types of plts in our dog model, we prepared plt-rich-plasma (PRP) from WB which would be equivalent to non-leukoreduced apheresis plts. The PRP was then either unmodified, F-LR, PRT, or the treatments were combined. Because the success rates were very poor with the single treatments of PRP (see table), the WB studies evaluated only combined F-LR and PRT treatments. In clinical practice, the treated WB would then be used to prepare a plt concentrate. The WB studies assessed either PRT of the WB followed by F-LR of PRP made from the WB or, conversely, F-LR of the WB using a plt-sparing filter (Terumo Immuflex WB-SP) followed by PRT of the WB and then preparation of PRP. After completion of all treatments, PRP from each study was centrifuged to prepare a plt concentrate, the plts were radiolabeled with 51Cr, injected into a recipient, and samples were drawn from the recipient to determine recovery and survival of the donor's (dnr's) plts. Dnr and recipient pairs were selected to be DLA-DRB incompatible and crossmatch-negative. Eight weekly dnr plt txs were given to the same recipient or until the recipient became refractory to the dnr's plts defined as ≤5% of the dnr's plts still circulating in the recipient at 24-hours post-tx following 2 sequential txs. Results: The table shows the percent of recipients who accepted 8 weeks of dnr plts and the total number of dnr plts and WBC injected. Using either filter, there was equal reduction in WBCs to 105/tx. Acceptance of unmodified dnr plts was 1/7 recipients (14%), PRT 1/8 recipients (13%), PL1-B filter 1/5 recipients (20%), and PLS-5A filter 4/6 recipients (66%). None of these differences were statistically significant. In contrast, combining F-LR of the PRP followed by PRT of the PRP was effective in 21/22 recipients (95%), regardless of the filter used. WB studies showed dnr plts were accepted by 2/5 recipients (40%) when WB was first treated with PRT followed by F-LR of the PRP made from the WB. Conversely, if the WB was first F-LR followed by PRT of the WB, 5/6 (83%) accepted dnr plts; more of these studies are in progress. Data are given as average ±1 S.D. Conclusions: F-LR of PRP or WB followed by PRT of the same PRP or WB is highly-effective in preventing alloimmune plt refractoriness in our dog plt tx model. These data suggest that most of the APCs must be removed by filtration before PRT can eliminate the activity of any residual APCs. Based on the high rate of success of this combined approach in our immunocompetent dog model, similar results should be achieved in pts even those who are not immunocompetent as were the AML pts receiving chemotherapy in the TRAP Trial. Disclosures: Slichter: Terumo BCT: Research Funding.

Abstract 3343 Background: The TRAP Trial (NEJM 1997;337:1861) evaluated filtration leukocyte-reduction (F-LR) and UV-B irradiation (UV-BI) to prevent plt alloimmunization in AML patients (pts) receiving chemotherapy. Inclusion of UV-BI was based on a 45% rate of preventing plt alloimmunization in our dog plt tx model. UV-BI was 79% successful in the TRAP Trial. The lower success rate in the dog was probably because we use normal immunocompetent recipient dogs versus chemotherapy-induced immunosuppressed pts. The residual rate of alloimmunization in the TRAP Trial using either F-LR or UV-BI was still 17% to 21%, suggesting that better prevention methods are needed. Methods: In our dog plt tx model, we evaluated two leukoreduction filters to remove antigen presenting WBCs (APCs) and γ-irradiation (γ-I) or UV-I combined with riboflavin (Mirasol pathogen reduction technology) to inactivate APCs. Crossmatch-negative, DLA-DRB incompatible donor/recipient pairs were used. Txs from the same donor were given weekly for up to 8 weeks or until the donor's 51Cr-labeled plts were rejected based on two sequential txs with donor plt recoveries ≤5% at 20 hours post-tx. Results: Residual WBC counts for standard, γ-l, or Mirasol-treated plts averaged 2,860/μl ± 1,800/μl, and, for F-LR plts using the PL1-B or the PLS-5A filter, they were 80/μl ± 10/μl and 110/μl ± 270/μl, respectively. Residual WBCs were significantly reduced after F-LR compared to non-leukoreduced plts (p Conclusions: Except for the results with the PLS-5A filter, a single modification of the donor dog's plts is only minimally successful. Although residual WBC counts were similar for both filters, the better results achieved with the PLS-5A filter compared to the PL1-B filter (66% versus 20% success rate, respectively) suggest that the types of WBCs removed may be as important as simply a quantitative reduction in WBCs. Unfortunately, adding γ-l to F-LR did not improve the results using either filter. In contrast, Mirasol treatment markedly improved the results with both filters, suggesting that the effects of F-LR and Mirasol treatment are synergistic with F-LR removing APCs, while Mirasol treatment inactivates residual APCs. The effect is particularly striking for the PL1-B filter that is only minimally effective when used alone. The fact that a combined F-LR Mirasol treatment approach is so successful in our immunocompetent dog model using both good and poor performing filters may suggest two things: 1) the combined approach will be equally successful in both immunosuppressed as well as non-immunosuppressed pts; and 2) using any available leukoreduction filter combined with Mirasol treatment will give results similar to the two filters already tested. Based on our prior experience with UV-BI, data from our dog model may be directly transferable to man. Disclosures: Slichter: CaridianBCT Biotechnologies, LLC; Lakewood, CO: Research Funding; US Army Medical Research and Materiel Command/Dept of the Army – USAMRAA: Research Funding.