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1.  Increased Fluidity of Human Platelet Membranes during Complement-Mediated Immune Platelet Injury 
Journal of Clinical Investigation  1978;61(3):582-589.
Complement appears to be involved in the destruction of platelets in certain clinical disorders, such as quinidine purpura and post-transfusion purpura. In both disorders, the classical complement sequence is activated by antigen-antibody complexes. It has been suggested that the terminal components of the complement sequence insert into the hydrophobic core of cell surface membranes and that this process leads to cell lysis. Fluidity is a fundamental property of lipids within the membrane's hydrophobic core. To examine the interaction of complement with membranes, we investigated the effect of complement activation on the fluidity of human platelet membranes. Complement was fixed to platelets using a post-transfusion purpura antibody, and membrane lipid fluidity was assessed in terms of fluorescence anisotropy using two fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene and 9-(12-anthroyl) stearic acid. Microviscosity, expressed in poise, was derived from the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.
Post-transfusion purpura antibody plus complement made platelet membranes more fluid as evidenced by a 21% decrease in anisotropy and a 35% decrease in microviscosity of platelets at 37°C, and this was associated with platelet lysis (51Cr release). Complement damage to platelets was accompanied by a 10-15% increase in ΔE, the fusion activation energy for microviscosity, indicating that complement not only decreased membrane microviscosity but also made membrane lipids less ordered. These changes were consistent and rapid, with platelet lysis and the reduction in microviscosity being half-maximal by 6 min. They were prevented by inactivation of complement with heat or with EDTA, and they were not observed when C5-deficient plasma was used as the complement source. Qualitatively similar changes in platelet membrane fluidity were observed when complement was fixed to platelets by a quinidine-dependent anti-platelet antibody rather than by post-transfusion purpura antibody. Post-transfusion purpura antibody plus complement also decreased the microviscosity of isolated platelet membranes. Moreover, the lipids extracted from platelets lysed by complement had a 22% decrease in microviscosity (P < 0.01), with no associated changes in the amount of cholesterol relative to phospholipid or in the amounts of the various phospholipids.
These studies demonstrate that lipids within the hydrophobic core of platelet membranes damaged by complement become more fluid, and this is associated with platelet lysis. These findings are consistent with the concept that the insertion of the terminal complement components into the platelet membrane bilayer perturbs lipid-lipid interactions within the membrane's hydrophobic core.
PMCID: PMC372570  PMID: 641139
2.  CTLA4-Ig Prevents Alloantibody Production and BMT Rejection in Response to Platelet Transfusions in Mice 
Transfusion  2012;52(10):2209-2219.
Background
Platelet transfusions can induce humoral and cellular alloimmunity. Anti-HLA antibodies can render patients refractory to subsequent transfusion, and both alloantibodies and cellular alloimmunity can contribute to subsequent bone marrow transplant rejection. Currently, there are no approved therapeutic interventions to prevent alloimmunization to platelet transfusions other than leukoreduction. Targeted blockade of T cell costimulation has shown great promise in inhibiting alloimmunity in the setting of transplantation, but has not been explored in the context of platelet transfusion.
Study Design and Methods
We tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig would prevent alloreactivity against major and minor alloantigens on transfused platelets. BALB/c (H-2d) mice and C57BL/6 (H-2b) mice were used as platelet donors and transfusion recipients, respectively. Alloantibodies were measured by indirect immunofluorescence using BALB/c platelets and splenocytes as targets. Bone marrow transplants were carried out under reduced intensity conditioning using BALB/b (H-2b) donors and C57BL/6 (H-2b) recipients to model HLA identical transplants. Experimental groups were given CTLA4-Ig (before or after platelet transfusion) with control groups receiving isotype matched antibody.
Results
CTLA4-Ig abrogated both humoral alloimmunization (anti-H-2d antibodies) and transfusion induced bone marrow transplant rejection. Whereas a single dose of CTLA4-Ig at time of transfusion prevented alloimmunization to subsequent platelet transfusions, administration of CTLA4-Ig after initial platelet transfusion was ineffective. Delaying treatment until after platelet transfusion failed to prevent bone marrow transplant rejection.
Conclusions
These findings demonstrate a novel strategy using an FDA approved drug that has the potential to prevent the clinical sequela of alloimmunization to platelet transfusions.
doi:10.1111/j.1537-2995.2011.03550.x
PMCID: PMC4284104  PMID: 22321003
3.  Cross-match-compatible platelets improve corrected count increments in patients who are refractory to randomly selected platelets 
Blood Transfusion  2014;12(2):180-186.
Background
Cross-match-compatible platelets are used for the management of thrombocytopenic patients who are refractory to transfusions of randomly selected platelets. Data supporting the effectiveness of platelets that are compatible according to cross-matching with a modified antigen capture enzyme-linked immunosorbent assay (MAC-ELISA or MACE) are limited. This study aimed to determine the effectiveness of cross-match-compatible platelets in an unselected group of refractory patients.
Materials and methods
One hundred ABO compatible single donor platelet transfusions given to 31 refractory patients were studied. Patients were defined to be refractory if their 24-hour corrected count increment (CCI) was <5×109/L following two consecutive platelet transfusions. Platelets were cross-matched by MACE and the CCI was determined to monitor the effectiveness of platelet transfusions.
Results
The clinical sensitivity, specificity, positive predictive value and negative predictive value of the MACE-cross-matched platelets for post-transfusion CCI were 88%, 54.6%, 39.3% and 93.2%, respectively. The difference between adequate and inadequate post-transfusion 24-hour CCI for MACE cross-matched-compatible vs incompatible single donor platelet transfusions was statistically significant (p=0.000). The 24-hour CCI (mean±SD) was significantly higher for cross-match-compatible platelets (9,250±026.6) than for incompatible ones (6,757.94±2,656.5) (p<0.0001). Most of the incompatible cross-matches (73.2%) were due to anti-HLA antibodies, alone (55.3% of cases) or together with anti-platelet glycoprotein antibodies (17.9%).
Discussion
The clinical sensitivity and negative predictive value of platelet cross-matching by MACE were high in this study and such tests may, therefore, be used to select compatible platelets for refractory patients. A high negative predictive value demonstrates the greater chance of an adequate response with cross-matched-compatible platelets.
doi:10.2450/2013.0064-13
PMCID: PMC4039699  PMID: 24333069
crossmatching; corrected count increment; human platelet antigens; platelet refractoriness; predictive value of test
4.  Dose of Prophylactic Platelet Transfusions and Prevention of Hemorrhage 
The New England journal of medicine  2010;362(7):600-613.
BACKGROUND
We conducted a trial of prophylactic platelet transfusions to evaluate the effect of platelet dose on bleeding in patients with hypoproliferative thrombocytopenia.
METHODS
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 (1.1×1011, 2.2×1011, or 4.4×1011 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).
RESULTS
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 (9.25×1011) than in the medium-dose group (11.25×1011) or the high-dose group (19.63×1011) (P = 0.002 for low vs. medium, P<0.001 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; P<0.001 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 (P<0.001).
CONCLUSIONS
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 1.1×1011 and 4.4×1011 platelets per square meter, the number of platelets in the prophylactic transfusion had no effect on the incidence of bleeding. (ClinicalTrials.gov number, NCT00128713.)
doi:10.1056/NEJMoa0904084
PMCID: PMC2951321  PMID: 20164484
5.  Alterations of platelet function and clot formation kinetics following in vitro exposure to anti-A and -B antibodies 
Transfusion  2012;53(2):382-393.
Background
ABO mismatched platelets are commonly transfused despite reported complications. We hypothesized that because platelets possess A and B antigens on their surface, ABO mismatched transfused or recipient platelets could become activated and/or dysfunctional after exposure to anti-A or -B antibodies in the transfused or recipient plasma. We present here in vitro modeling data on the functional effects of exposure of platelets to ABO antibodies.
Methods
Platelet functions of normal platelets of all ABO types were assessed before and after incubation with normal saline, ABO identical plasmas, or O plasmas with varying titers of anti-A and anti-B (anti-A/B) antibodies. Assays used for this assessment include: platelet aggregation, clot kinetics, thrombin generation, platelet cytoskeletal function, and mediator release.
Results
Exposure of antigen bearing platelets to O plasma with moderate to high titers of anti-A/B antibodies significantly inhibits aggregation, prolongs PFA-100 epinephrine closure time, disrupts clot formation kinetics, accelerates thrombin generation, reduces total thrombin production, alters platelet cytoskeletal function, and influences pro-inflammatory and pro-thrombotic mediator release.
Conclusions
Our findings demonstrate a wide range of effects that anti-A/B antibodies have on platelet function, clot formation, thrombin generation, platelet cytoskeletal function, and mediator release. These data provide potential explanations for clinical observations of increased red cell utilization in trauma and surgical patients receiving ABO non-identical blood products. Impaired hemostasis caused by anti-A/B antibodies interacting with A and B antigens on platelets, soluble proteins, and perhaps even endothelial cells is a potential contributing factor to hemorrhage in patients receiving larger volumes of ABO non-identical transfusions.
doi:10.1111/j.1537-2995.2012.03718.x
PMCID: PMC3566315  PMID: 22624532
Platelet transfusion; Transfusion complications; Platelet aggregation; Thromboelastography; Thrombin generation
6.  Alloimmunization to transfused platelets requires priming of CD4+ T cells in the splenic microenvironment in a murine model 
Transfusion  2011;52(4):849-859.
Background
Alloantibodies are a clinically significant sequelae of platelet transfusion, potentially rendering patients refractory to ongoing platelet transfusion support. These antibodies are often IgG class switched, suggesting the involvement of CD4+ T cell help; however, platelet specific CD4+ T cells have not been visualized in vivo and specifics of their stimulation are not completely understood.
Study Design and Methods
A murine model of alloimmunization to transfused platelets was developed to allow in vivo assessment and characterization of CD4+ T cells specific for platelet MHC alloantigen. Platelets were harvested from BALB/c mice, filter leukoreduced, and transfused into C57BL/6 recipients. Platelet specific CD4+ T cell responses were visualized by using a TCR transgenic mouse that detects peptide from donor MHC I presented on recipient MHC II. Antibody responses were determined by indirect immunofluorescence using BALB/c donor targets.
Results
C57BL/6 recipients of BALB/c leukoreduced platelet transfusions produced anti-BALB/c antibodies, with proliferation of antigen specific CD4+ T cells seen in the spleen but not lymph nodes or liver. Depletion of recipient CD4+ cells or splenectomy independently abrogated the alloantibody response.
Conclusion
We report a novel model to study antigen-specific CD4+ T cells during alloimmunization to platelet transfusion. The presented data support a critical role for CD4+ T cell help in the humoral response to platelet transfusion and establish the spleen as a required microenvironment for effective CD4+ T cell priming against donor platelet derived MHC I.
doi:10.1111/j.1537-2995.2011.03346.x
PMCID: PMC3257367  PMID: 21981241
7.  Platelet antibody detection by flow cytometry: an effective method to evaluate and give transfusional support in platelet refractoriness 
Background
Immune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80-90% of cases) and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness.
Objective
The aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness.
Methods
A group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity.
Results
Seventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years) were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% and a negative predictive value of 85.71%. The accuracy of the method was 80.26%.
Conclusion
This study shows that the flow cytometry platelet immunofluorescence test has a high correlation with the anti-human leukocyte antigen antibodies. Despite a few limitations, the method seems to be efficient, fast and feasible as the initial screening for platelet antibody detection and a useful tool to crossmatch platelets for the transfusional support of patients with immune platelet refractoriness.
doi:10.5581/1516-8484.20130062
PMCID: PMC3789429  PMID: 24106442
Blood platelets; Antigens, human leukocyte; Flow cytometry; Histocompatibility; Antigens, human platelet
8.  Measurement of the absolute immature platelet number reflects marrow production and is not impacted by platelet transfusion 
Transfusion  2012;53(6):1201-1204.
BACKGROUND
The ability to distinguish increased platelet destruction from platelet hypo-production is important in the care of patients with bone marrow failure syndromes and patients receiving high dose chemotherapy. The measurement of immature circulating platelets based on RNA content using an automated counter is now feasible. This study evaluated the impact of recent platelet transfusion on measurement of immature platelet parameters.
STUDY DESIGN AND METHODS
The immature platelet fraction (IPF) and absolute immature platelet number (AIPN) were measured using the Sysmex XE-5000 analyzer prior to and following platelet transfusion in 9 transfusion-dependent patients with marrow failure secondary to aplastic anemia, myelodysplasia or transplantation conditioning. IPF and AIPN were also measured serially over 5 days of storage in 3 plateletpheresis components collected from normal donors.
RESULTS
Platelet transfusion did not significantly change the mean AIPN in transfused patients. In contrast, IPF decreased significantly from 6.6 ±4.6% at day -1 to 2.3 ±1.4% at day 0 before returning to 4.3 ±2.3% at day +1. In the platelet component, AIPN and IPF% increased significantly over 5 days of storage, most likely due to an artifact of the staining and detection process for stored platelets, no longer detected in vivo once the platelets were transfused.
CONCLUSION
Platelet transfusion decreases the IPF due to the resultant increase in circulating platelet count. However, platelet transfusion does not change the circulating absolute immature platelet number (AIPN), validating this assay as a reflection of ongoing platelet production by the bone marrow in various clinical settings, regardless of proximity to platelet transfusion.
doi:10.1111/j.1537-2995.2012.03918.x
PMCID: PMC3543490  PMID: 23043309
9.  Platelets protect from septic shock by inhibiting macrophage-dependent inflammation via the cyclooxygenase 1 signaling pathway 
Nature communications  2013;4:2657.
While it has been long known that patients with sepsis often have thrombocytopenia and that septic patients with severe thrombocytopenia have a poor prognosis and higher mortality, the role of platelets in the pathogenesis of sepsis is poorly understood. Here we report a protective role of platelets in septic shock. We show that experimental thrombocytopenia by intraperitoneal injection of an anti-glycoprotein Ibα monoclonal antibody increases mortality and aggravates organ failure whereas transfusion of platelets reduces mortality in Lipopolysaccharide-induced endotoxemia and a bacterial infusion mouse sepsis model. Plasma concentrations of proinflammatory cytokines TNF-α and IL-6 are elevated by thrombocytopenia and decreased by platelet transfusion in septic mice. Furthermore, we identify that platelets protect from septic shock by inhibiting macrophage-dependent inflammation via the COX1/PGE2/EP4 dependent pathway. Thus, these findings demonstrate a previously unappreciated role for platelets in septic shock and suggest that platelet transfusion may be effective in treating severe septic patients.
doi:10.1038/ncomms3657
PMCID: PMC4217311  PMID: 24150174
10.  Identification of platelet refractoriness in oncohematologic patients 
Clinics  2011;66(1):35-40.
OBJECTIVES:
To identify the occurrence and the causes of platelet refractoriness in oncohematologic patients.
INTRODUCTION:
Platelet refractoriness (unsatisfactory post-transfusion platelet increment) is a severe problem that impairs the treatment of oncohematologic patients and is not routinely investigated in most Brazilian services.
METHODS:
Forty-four episodes of platelet concentrate transfusion were evaluated in 16 patients according to the following parameters: corrected count increment, clinical conditions and detection of anti-platelet antibodies by the platelet immunofluorescence test (PIFT) and panel reactive antibodies against human leukocyte antigen class I (PRA-HLA).
RESULTS:
Of the 16 patients evaluated (median age: 53 years), nine (56%) were women, seven of them with a history of pregnancy. An unsatisfactory increment was observed in 43% of the transfusion events, being more frequent in transfusions of random platelet concentrates (54%). Platelet refractoriness was confirmed in three patients (19%), who presented immunologic and non-immunologic causes. Alloantibodies were identified in eight patients (50%) by the PIFT and in three (19%) by the PRA-HLA. Among alloimmunized patients, nine (64%) had a history of transfusion, and three as a result of pregnancy (43%). Of the former, two were refractory (29%). No significant differences were observed, probably as a result of the small sample size.
CONCLUSION:
The high rate of unsatisfactory platelet increment, refractoriness and alloimmunization observed support the need to set up protocols for the investigation of this complication in all chronically transfused patients, a fundamental requirement for the guarantee of adequate management.
doi:10.1590/S1807-59322011000100007
PMCID: PMC3044569  PMID: 21437433
Transfusion; CCI; Alloimmunization; PIFT; HLA
11.  Platelet Transfusion – The New Immunology of an Old Therapy 
Platelet transfusion has been a vital therapeutic approach in patients with hematologic malignancies for close to half a century. Randomized trials show that prophylactic platelet transfusions mitigate bleeding in patients with acute myeloid leukemia. However, even with prophylactic transfusions, as many as 75% of patients, experience hemorrhage. While platelet transfusion efficacy is modest, questions and concerns have arisen about the risks of platelet transfusion therapy. The acknowledged serious risks of platelet transfusion include viral transmission, bacterial sepsis, and acute lung injury. Less serious adverse effects include allergic and non-hemolytic febrile reactions. Rare hemolytic reactions have occurred due to a common policy of transfusing without regard to ABO type. In the last decade or so, new concerns have arisen; platelet-derived lipids are implicated in transfusion-related acute lung injury after transfusion. With the recognition that platelets are immune cells came the discoveries that supernatant IL-6, IL-27 sCD40L, and OX40L are closely linked to febrile reactions and sCD40L with acute lung injury. Platelet transfusions are pro-inflammatory, and may be pro-thrombotic. Anti-A and anti-B can bind to incompatible recipient or donor platelets and soluble antigens, impair hemostasis and thus increase bleeding. Finally, stored platelet supernatants contain biological mediators such as VEGF and TGF-β1 that may compromise the host versus tumor response. This is particularly of concern in patients receiving many platelet transfusions, as for acute leukemia. New evidence suggests that removing stored supernatant will improve clinical outcomes. This new view of platelets as pro-inflammatory and immunomodulatory agents suggests that innovative approaches to improving platelet storage and pre-transfusion manipulations to reduce toxicity could substantially improve the efficacy and safety of this long-employed therapy.
doi:10.3389/fimmu.2015.00028
PMCID: PMC4313719
platelets; transfusion; immune response; transfusion reaction; storage; thrombosis; bleeding
12.  Clinical use of recombinant human activated factor VII (rFVIIa) in the prevention and treatment of bleeding episodes in patients with Glanzmann’s thrombasthenia 
Glanzmann’s thrombasthenia (GT) is a congenital qualitative platelet disorders due to the deficiency or defect of platelet membrane GPIIb/IIIa (integrin αIIbβ3). The standard treatment for bleeding is platelet transfusion but repeated transfusion may result in the development of anti-platelet antibodies (to HLA and/or GPIIbIIIa) rendering future platelet transfusion ineffective. Alternative effective agent(s) are needed. There are increasing reports documenting efficacy of high dose rFVIIa in GT patients with adverse events uncommon. The efficacy is supported by evidence that high concentration FVIIa binds to activated platelet surface and improves thrombin generation to enhance deposition (adhesion) and aggregation of platelets lacking GPIIb/IIIa. While there are increasing clinical experiences, evidence-based clinical data are not available. There is a need for more clinical studies, particularly clinical trials, to further assess the efficacy, safety (particularly thrombotic events) and optimal regimen of rFVIIa in GT patients, either singly or in combination with other hemostatic agents such as platelet transfusion. In the absence of this data, for treatment of severe bleeding in GT patients with platelet antibodies and platelet refractoriness, rFVIIa at dose 90 μg/kg every 2 h for 3 or more doses could be considered. This more “optimal regimen” derived from a recent International Survey needs confirmation with larger studies. What the optimal regimen for surgical coverage is remains unresolved.
PMCID: PMC2291310  PMID: 18078017
Glanzmann’s thrombasthenia; recombinant human activated factor VII (rFVIIa); bleeding; surgery; platelet transfusion; GPIIb/IIIa
13.  Platelets from WAS patients show an increased susceptibility to ex vivo phagocytosis 
Platelets  2012;24(4):288-296.
The thrombocytopenia of Wiskott–Aldrich syndrome (WAS) is thought to be due to both reduced platelet production and accelerated platelet consumption. We have previously demonstrated that platelets from WASP-deficient mice are consumed more rapidly in vivo than are WT platelets, and that opsonization accelerates their uptake by bone marrow- derived macrophages more than it does that of WT platelets. Here we asked whether platelets from WAS patients show similar features. We show that ex vivo phagocytosis by activated THP-1 cells of DIO-labeled platelets from a series of WAS or XLT patients is increased in comparison to that of normal control platelets. Using a numerical analysis method, we distinguish this effect from a concurrent effect on the amount of detectable fluorescent signal transferred to the macrophage per phagocytosed platelet. We show that the latter quantity is reduced by platelet WASP deficiency, as might be expected if the fluorescence transferred from these smaller platelets is more rapidly quenched. We are unable to detect a differential effect of opsonization with anti-CD61 antibody on the uptake of WASP(−) vs. WT platelets. However, the high probability of phagocytosis per adsorbed WASP(−) platelet could limit the sensitivity of the assay in this case. We also see no effect of sera from WAS patients on the uptake of normal control platelets, suggesting that in vivo opsonization is not the cause of increased uptake of WASP(−) platelets. Finally, we show little, if any, increase in the reticulated platelet fraction in WAS patients, suggesting that impaired production of reticulated platelets contributes to the thrombocytopenia. Our findings suggest that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. They also demonstrate the feasibility of routinely performing functional assays of phagocytosis of small numbers of platelets obtained at remote locations, a method which should be applicable to the study of other types of thrombocytopenia such as ITP.
doi:10.3109/09537104.2012.693991
PMCID: PMC3529772  PMID: 22812495
Wiskott–Aldrich syndrome; phagocytosis; numerical analysis; thrombocytopenia
14.  PERSISTENCE OF LYMPHOCYTOTOXIC ANTIBODIES IN PATIENTS IN THE TRIAL TO REDUCE ALLOIMMUNIZATION TO PLATELETS: IMPLICATIONS FOR USING MODIFIED BLOOD PRODUCTS 
Transfusion medicine reviews  2011;25(2):102-110.
AML patients undergoing induction chemotherapy have significant decreases in alloimmune platelet refractoriness if they receive filter-leukoreduced or UV-B irradiated versus standard platelet transfusions (3% to 5% versus 13%, respectively, p≤0.03) with no differences among the treated platelet arms (TRAP Trial). 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 percent LCT panel reactive antibodies (PRA). During the TRAP trial, 145 patients became antibody-positive, and 81 (56%) of them subsequently became antibody-negative. Using Kaplan-Meier estimates, projected antibody loss was 73% at one year. Major factors associated with antibody persistence were prior pregnancy and percent PRA positivity, while neither the assigned type of platelets transfused during the 8 weeks of the trial nor prior transfusion history were 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 AML patients 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.
doi:10.1016/j.tmrv.2010.11.002
PMCID: PMC3073712  PMID: 21345638
Platelet Transfusions; UV-B Irradiation; Leukoreduction; Platelet Refractoriness; Platelet Alloimmunization; Lymphocytotoxic Antibodies
15.  Evaluation of platelet cross-matching in the management of patients refractory to platelet transfusions 
Blood Transfusion  2014;12(2):187-194.
Background
Cross-match-compatible platelets are used to support thrombocytopenic patients who are refractory to randomly selected platelets. However, few studies have addressed the efficacy of using this strategy for patients requiring intensive platelet transfusion therapy. The aim of this study was to determine the effectiveness of cross-match-compatible platelets in an unselected group of patients refractory to platelets from random donors.
Materials and methods
A total of 406 cross-match-compatible platelet components were administered to 40 evaluable patients who were refractory to random-donor platelets. A solid-phase red cell adherence method was used for platelet cross-matching. The corrected count increment was used to monitor the effectiveness of each platelet transfusion. Multivariate analysis was performed to detect whether any variables could predict the response to transfusion.
Results
Statistically significant improvements were found in the mean corrected count increment when comparing cross-match-compatible platelets with randomly selected and incompatible platelets (p<0.001 for each). Compatible platelet transfusions were associated with a good response in 72.9% of cases while incompatible platelets were associated with a poor response in 66.7% of transfusion events (p<0.001). In the presence of clinical factors or alloimmunisation, compatible platelets were associated with good responses in 67.9% and 28.0% respectively vs 100% and 93.3% in their absence (p=0.009, p<0.001). Multivariate analysis revealed that cross-matching and alloimmunisation were the strongest predictors of transfusion response at 1 hour, while ABO compatibility, type of units received, followed by alloimmunisation then clinical factors were predictors at 24 hours.
Discussion
Platelet cross-matching using the solid-phase red cell adherence technique is an effective and rapid first-line approach for the management of patients refractory to platelet transfusions.
doi:10.2450/2014.0120-13
PMCID: PMC4039700  PMID: 24931840
platelets; cross-match; refractoriness; solid-phase red cell adherence method; corrected count increment
16.  Refractoriness to platelet transfusion after single-donor consecutive platelet transfusions and its relationship to platelet antibodies. 
In thirty patients with acute leukemia and severe aplastic anemia receiving random single donor platelet transfusions, the development of refractoriness by consecutive platelet transfusions with cytapheresis and its relationship to the appearance of anti-platelet antibodies were investigated. The median number of platelet transfusions inducing refractoriness was 13 times, and 20% of the patients remained unrefractory despite of the repeated multiple platelet transfusions up to 20 to 25 times. The results of anti-platelet antibody tasts by the enzyme-linked immunosorbent assay(ELISA) and immunofluorescent techniques(IFT) showed no statistically significant relationship with the refractoriness (p greater than 0.1). Although there was significant correlation between the results of ELISA and IFT, both tests were insufficient to find out refractoriness even with the use of pooled platelets from multiple donors as target cells. This study shows that 13 single donor platelet transfusions result in refractoriness, that both ELISA and IFT are insufficient to detect refractoriness despite of their significant correlation, and that other methods than these are needed in order to detect alloimmunization.
PMCID: PMC3053673  PMID: 3267362
17.  Post-transfusion purpura in an African-American man due to human platelet antigen-5b alloantibody: a case report 
Introduction
Post-transfusion purpura is a rare immunohematological disorder characterized by severe thrombocytopenia following transfusion of blood components and induced by an alloantibody against a donor platelet antigen. It occurs primarily in women sensitized by pregnancy and is most commonly caused by anti-human platelet antigen-1a antibodies. Here, we describe what we believe to be the first documented case of an African-American man who developed post-transfusion purpura due to an anti-human platelet antigen-5b alloantibody after receiving multiple blood products.
Case presentation
A 68-year-old African-American man initially admitted with atrial flutter was started on anticoagulation treatment, which was complicated by severe hematemesis. On days 4 and 5 of hospitalization, he received six units of packed red blood cells, and on days 4, 13 and 14 he received plasma. His platelet count began to drop on day 25 and on day 32 reached a nadir of 7 × 109/L. His platelet count increased after receiving intravenous immune globulin. An antibody with reactivity to human platelet antigen-5b was detected by a solid-phase enzyme-linked immunoassay. Our patient was homozygous for human platelet antigen-5a.
Conclusion
This case emphasizes the importance of including post-transfusion purpura in the differential diagnosis for both men and women with acute onset of thrombocytopenia following transfusion of blood products. The prompt recognition of this entity is crucial for initiation of the appropriate management.
doi:10.1186/1752-1947-6-420
PMCID: PMC3549824  PMID: 23234542
18.  Heterogeneity of antibody response to human platelet transfusion. 
Journal of Clinical Investigation  1976;58(2):432-438.
To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors.
PMCID: PMC333198  PMID: 956376
19.  Amphotericin B induced abnormalities in human platelets 
Clinical Molecular Pathology  1996;49(5):M301-M307.
Aims—To investigate in vitro the effect of amphotericin B on platelets in order to understand poor platelet recovery in patients receiving platelet transfusions and amphotericin B simultaneously.
Methods—Washed platelets were isolated from platelet concentrates and exposed to amphotericin B (4 μg/ml) for one hour. Platelet function was assessed by aggregation response to thrombin (0-0.6 U/ml), serotonin release, response to hypotonic stress, and mean platelet volume. The expression of surface membrane glycoprotein (GP) Ib-IX complex, GPIIb-IIIa complex and CD62P (P-selectin) was examined by flow cytometry using fluorescence labelled monoclonal antibodies. Heterotypic cell adhesion was measured in amphotericin B treated platelets coincubated with isolated, autologous polymorphonuclear leucocytes (PMN) by flow cytometric analysis.
Results—Amphotericin B induced platelet dysfunction. The rate of aggregation by thrombin, serotonin uptake and thrombin induced release of serotonin, and the response of platelets to hypotonic stress were inhibited. There was up to a two-fold increase in the mean platelet volume. The expression of platelet surface GPIb-IX and GPIIb-IIIa was not affected. P-selectin, normally expressed only on the surface of activated platelets, was also expressed on unactivated platelets. Amphotericin B increased platelet adherence to PMN and the number of platelets bound per PMN.
Conclusions—In vitro, amphotericin B induces P-selectin expression on the surface of unactivated platelets and increases platelet adhesion to PMN, which is exacerbated by storage. Platelet dysfunction resulting from exposure to amphotericin B may contribute to poor platelet recovery in vivo when amphotericin B is administered concomitantly with platelet transfusion.
PMCID: PMC408077  PMID: 16696093
amphotericin B; platelets; surface membrane glycoprotein; flow cytometry
20.  Red Blood Cell Transfusion and Mortality in Trauma Patients: Risk-Stratified Analysis of an Observational Study 
PLoS Medicine  2014;11(6):e1001664.
Using a large multicentre cohort, Pablo Perel and colleagues evaluate the association of red blood cell transfusion with mortality according to the predicted risk of death for trauma patients.
Please see later in the article for the Editors' Summary
Background
Haemorrhage is a common cause of death in trauma patients. Although transfusions are extensively used in the care of bleeding trauma patients, there is uncertainty about the balance of risks and benefits and how this balance depends on the baseline risk of death. Our objective was to evaluate the association of red blood cell (RBC) transfusion with mortality according to the predicted risk of death.
Methods and Findings
A secondary analysis of the CRASH-2 trial (which originally evaluated the effect of tranexamic acid on mortality in trauma patients) was conducted. The trial included 20,127 trauma patients with significant bleeding from 274 hospitals in 40 countries. We evaluated the association of RBC transfusion with mortality in four strata of predicted risk of death: <6%, 6%–20%, 21%–50%, and >50%. For this analysis the exposure considered was RBC transfusion, and the main outcome was death from all causes at 28 days. A total of 10,227 patients (50.8%) received at least one transfusion. We found strong evidence that the association of transfusion with all-cause mortality varied according to the predicted risk of death (p-value for interaction <0.0001). Transfusion was associated with an increase in all-cause mortality among patients with <6% and 6%–20% predicted risk of death (odds ratio [OR] 5.40, 95% CI 4.08–7.13, p<0.0001, and OR 2.31, 95% CI 1.96–2.73, p<0.0001, respectively), but with a decrease in all-cause mortality in patients with >50% predicted risk of death (OR 0.59, 95% CI 0.47–0.74, p<0.0001). Transfusion was associated with an increase in fatal and non-fatal vascular events (OR 2.58, 95% CI 2.05–3.24, p<0.0001). The risk associated with RBC transfusion was significantly increased for all the predicted risk of death categories, but the relative increase was higher for those with the lowest (<6%) predicted risk of death (p-value for interaction <0.0001). As this was an observational study, the results could have been affected by different types of confounding. In addition, we could not consider haemoglobin in our analysis. In sensitivity analyses, excluding patients who died early; conducting propensity score analysis adjusting by use of platelets, fresh frozen plasma, and cryoprecipitate; and adjusting for country produced results that were similar.
Conclusions
The association of transfusion with all-cause mortality appears to vary according to the predicted risk of death. Transfusion may reduce mortality in patients at high risk of death but increase mortality in those at low risk. The effect of transfusion in low-risk patients should be further tested in a randomised trial.
Trial registration
www.ClinicalTrials.gov NCT01746953
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Trauma—a serious injury to the body caused by violence or an accident—is a major global health problem. Every year, injuries caused by traffic collisions, falls, blows, and other traumatic events kill more than 5 million people (9% of annual global deaths). Indeed, for people between the ages of 5 and 44 years, injuries are among the top three causes of death in many countries. Trauma sometimes kills people through physical damage to the brain and other internal organs, but hemorrhage (serious uncontrolled bleeding) is responsible for 30%–40% of trauma-related deaths. Consequently, early trauma care focuses on minimizing hemorrhage (for example, by using compression to stop bleeding) and on restoring blood circulation after blood loss (health-care professionals refer to this as resuscitation). Red blood cell (RBC) transfusion is often used for the management of patients with trauma who are bleeding; other resuscitation products include isotonic saline and solutions of human blood proteins.
Why Was This Study Done?
Although RBC transfusion can save the lives of patients with trauma who are bleeding, there is considerable uncertainty regarding the balance of risks and benefits associated with this procedure. RBC transfusion, which is an expensive intervention, is associated with several potential adverse effects, including allergic reactions and infections. Moreover, blood supplies are limited, and the risks from transfusion are high in low- and middle-income countries, where most trauma-related deaths occur. In this study, which is a secondary analysis of data from a trial (CRASH-2) that evaluated the effect of tranexamic acid (which stops excessive bleeding) in patients with trauma, the researchers test the hypothesis that RBC transfusion may have a beneficial effect among patients at high risk of death following trauma but a harmful effect among those at low risk of death.
What Did the Researchers Do and Find?
The CRASH-2 trail included 20,127 patients with trauma and major bleeding treated in 274 hospitals in 40 countries. In their risk-stratified analysis, the researchers investigated the effect of RBC transfusion on CRASH-2 participants with a predicted risk of death (estimated using a validated model that included clinical variables such as heart rate and blood pressure) on admission to hospital of less than 6%, 6%–20%, 21%–50%, or more than 50%. That is, the researchers compared death rates among patients in each stratum of predicted risk of death who received a RBC transfusion with death rates among patients who did not receive a transfusion. Half the patients received at least one transfusion. Transfusion was associated with an increase in all-cause mortality at 28 days after trauma among patients with a predicted risk of death of less than 6% or of 6%–20%, but with a decrease in all-cause mortality among patients with a predicted risk of death of more than 50%. In absolute figures, compared to no transfusion, RBC transfusion was associated with 5.1 more deaths per 100 patients in the patient group with the lowest predicted risk of death but with 11.9 fewer deaths per 100 patients in the group with the highest predicted risk of death.
What Do These Findings Mean?
These findings show that RBC transfusion is associated with an increase in all-cause deaths among patients with trauma and major bleeding with a low predicted risk of death, but with a reduction in all-cause deaths among patients with a high predicted risk of death. In other words, these findings suggest that the effect of RBC transfusion on all-cause mortality may vary according to whether a patient with trauma has a high or low predicted risk of death. However, because the participants in the CRASH-2 trial were not randomly assigned to receive a RBC transfusion, it is not possible to conclude that receiving a RBC transfusion actually increased the death rate among patients with a low predicted risk of death. It might be that the patients with this level of predicted risk of death who received a transfusion shared other unknown characteristics (confounders) that were actually responsible for their increased death rate. Thus, to provide better guidance for clinicians caring for patients with trauma and hemorrhage, the hypothesis that RBC transfusion could be harmful among patients with trauma with a low predicted risk of death should be prospectively evaluated in a randomised controlled trial.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001664.
This study is further discussed in a PLOS Medicine Perspective by Druin Burch
The World Health Organization provides information on injuries and on violence and injury prevention (in several languages)
The US Centers for Disease Control and Prevention has information on injury and violence prevention and control
The National Trauma Institute, a US-based non-profit organization, provides information about hemorrhage after trauma and personal stories about surviving trauma
The UK National Health Service Choices website provides information about blood transfusion, including a personal story about transfusion after a serious road accident
The US National Heart, Lung, and Blood Institute also provides detailed information about blood transfusions
MedlinePlus provides links to further resources on injuries, bleeding, and blood transfusion (in English and Spanish)
More information in available about CRASH-2 (in several languages)
doi:10.1371/journal.pmed.1001664
PMCID: PMC4060995  PMID: 24937305
21.  Prevalence of platelet reactive antibodies in patient's refractory to platelet transfusions 
Introduction & Aims:
Though platelet transfusions have greatly reduced the incidence of major haemorrhagic complications associated with the management of haematological and oncological disorders, refractoriness to infused platelets becomes a major clinical problem for many of these patients.
Materials and methods:
The present study was done to determine the percentage of platelet alloimmunisation due to platelet-reactive antibodies in 340 patients with hematologic or oncologic diseases who had received multiple transfusions (> 10) of blood and blood components and showed platelet refractoriness in 1-hour post transfusion sample.
Results:
Platelet-reactive antibodies were detected in the sera of 127 out of 340 patients (37.35%) who received multiple transfusions (> 10) and showed platelet refractoriness.
Conclusion:
Platelet-reactive antibodies appear to be an important cause of platelet refractoriness in patients of acute leukaemia, aplastic anaemia, NHL, MDS and multiple myeloma receiving multiple platelet transfusions. Platelet refractoriness in patients of ITP and chronic leukaemia appears to be due to other causes and not due to platelet-reactive antibodies.
doi:10.4103/0973-6247.137453
PMCID: PMC4140056  PMID: 25161354
Platelet alloimmunization; platelet immunofluorescent test; refractoriness
22.  Role of thrombospondin in platelet aggregation. 
Journal of Clinical Investigation  1984;74(5):1764-1772.
Thrombospondin (TSP), the major alpha-granule protein of human platelets, binds to the activated platelet surface upon platelet stimulation. TSP has hemagglutinating (lectin-like) activity and forms a specific complex with fibrinogen. Based on these observations, it was postulated that the interaction of TSP and fibrinogen on the activated platelet surface may be an important step in the platelet aggregation process. To test this hypothesis, monospecific, affinity-purified anti-TSP Fab fragments were prepared and their effects on platelet aggregation and platelet fibrinogen binding were studied. Anti-TSP Fab caused significant interference with thrombin- and collagen-induced platelet aggregation, as monitored by both turbidometric aggregometry and particle counting measuring the disappearance of single platelets. Phase-contrast microscopy revealed that anti-TSP Fab caused a marked decrease in platelet macroaggregates and an increase in microaggregates and nonaggregated single platelets. Anti-TSP Fab did not affect the initial phase of ADP-induced platelet aggregation but caused rapid platelet disaggregation with the abolition of the secondary phase of aggregation. The effect of anti-TSP Fab was not mediated by a direct inhibition of platelet secretion. The effect of anti-TSP Fab on specific binding of labeled fibrinogen to thrombin-stimulated platelets was also studied. Anti-TSP Fab caused a marked decrease in the affinity of fibrinogen binding to the receptors on the activated platelet surface. Kinetic analyses revealed significant displacement of labeled fibrinogen by unlabeled fibrinogen in the presence of anti-TSP Fab, suggesting that TSP serves to stabilize fibrinogen binding to the activated platelet surface and reinforces the strength of interplatelet interactions. It is proposed that platelet aggregation is a dynamic, multistep process, governed initially by the platelet membrane glycoprotein IIb/IIIa-fibrinogen interaction, with the TSP-fibrinogen interaction playing an important role in determining the size and reversibility of platelet aggregates.
PMCID: PMC425356  PMID: 6501568
23.  P-Selectin Glycoprotein Ligand 1 (Psgl-1) Is Expressed on Platelets and Can Mediate Platelet–Endothelial Interactions in Vivo 
The Journal of Experimental Medicine  2000;191(8):1413-1422.
The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin–immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin–bound ligands were eluted with ethylenediaminetetraacetic acid. An ∼210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti–human PSGL-1 antibody and could be blotted with P-selectin–IgG. Under reducing conditions, both the predicted PSGL-1 ∼210-kD dimer and the ∼120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25–100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti–PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.
PMCID: PMC2193129  PMID: 10770806
P-selectin; endothelium; hemostasis; inflammation; adhesion
24.  Rapid Screening Method for Detection of Bacteria in Platelet Concentrates 
Journal of Clinical Microbiology  2004;42(5):1903-1908.
Public awareness has long focused on the risks of the transmission of viral agents through blood product transfusion. This risk, however, pales in comparison to the less publicized danger associated with the transfusion of blood products contaminated with bacteria, in particular, platelet concentrates. Up to 1,000 cases of clinical sepsis after the transfusion of platelet concentrates are reported annually in the United States. The condition is characterized by acute reaction symptoms and the rapid onset of septicemia and carries a 20 to 40% mortality rate. The urgent need for a method for the routine screening of platelet concentrates to improve patient safety has long been recognized. We describe the development of a rapid and highly sensitive method for screening for bacteria in platelet concentrates for transfusion. No culture period is required; and the entire procedure, from the time of sampling to the time that the final result is obtained, takes less than 90 min. The method involves three basic stages: the selective removal of platelets by filtration following activation with a monoclonal antibody, DNA-specific fluorescent labeling of bacteria, and concentration of the bacteria on a membrane surface for enumeration by solid-phase cytometry. The method offers a universal means of detection of live, nondividing, or dead gram-negative and gram-positive bacteria in complex cellular blood products. The sensitivity is higher than those of the culture-based methods available at present, with a detection limit of 10 to 102 CFU/ml, depending upon the bacterial strain.
doi:10.1128/JCM.42.5.1903-1908.2004
PMCID: PMC404662  PMID: 15131147
25.  Platelets and Infection – An Emerging Role of Platelets in Viral Infection 
Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis) are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia) and several platelet function disorders increase the risk of bleeding. Over the last years, more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients. Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favors platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen–antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies. All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count but also shape immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation, and platelet-mediated modulations of innate and adaptive immune responses.
doi:10.3389/fimmu.2014.00649
PMCID: PMC4270245  PMID: 25566260
platelets; viruses; thrombocytopenia; thrombosis; immune response

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