Platelet Pool Research Articles

Microparticles from Aged Stored Platelets Impair Microvascular Pulmonary Endothelial Cell Barrier Function

BACKGROUNDTransfusion‐related acute lung injury (TRALI) has a mortality of 5–15% and an incidence ranging from 0.02–1%. TRALI causes chest infiltrates and hypoxia in part due to lung endothelial cell (EC) barrier failure and edema formation. Aged platelets which have a high risk of causing TRALI, accumulate microparticles (PMPs) throughout storage. PMPs have been shown to mediate pro‐inflammatory processes such as neutrophil priming.RESEARCH QUESTIONConsidering the role of ECs in TRALI, we set out to determine if aged platelet derived PMPs could mediate EC barrier disruption?HYPOTHESISSpecifically, we hypothesized that PMPs from aged but not fresh platelets will lead to EC monolayer disruption.METHODSC57BL/6 mouse platelets were stored for 1 (fresh) or 5 (aged) days duration prior to PMP isolation (centrifugation/filtration) and enumeration (flow cytometry). Human microvascular pulmonary ECs (passage 6–8) were cultured with PBS (sham) or 0.1 mg/ml LPS two hours prior to addition of PBS, 5×105 of fresh or aged PMPs, or PMP depleted supernatants from stored platelet pools. Transendothelial electrical resistance (TEER) readings were obtained every 30 minutes for 8 hours in triplicate. Alternatively aged 10 ml/kg PMPs (≈ 5×106) or saline (sham) was transfused into primed (2 mg/kg LPS intraperitoneal) BALB/c mice for assessment of lung tissue wet‐to‐dry (W/D) ratios 6 hours post transfusion.RESULTSPMPs were found to be increased more than 2 fold in aged as compared to fresh platelet storage pools. ECs exposed to LPS alone did not reveal decreases in TEER relative to PBS sham. When aged PMPs were added 2 hours after PBS there was a marked drop in TEER within 2 hours which became maximal (nearly 50% decrease relative to PBS alone) by 5 hours. Addition of LPS instead of PBS prior to aged PMPs led to similar decreases in TEER readings but with faster onset. Addition of platelet and PMP depleted supernatants (fresh or aged) or fresh PMPs with LPS did not show signs of decreased TEER readings relative to PBS sham. Beyond being more numerous, aged PMPs appear more injurious than fresh PMPs when delivered in our EC model at identical doses. Aged PMPs are able to reduce EC monolayer integrity within 2 hours and sustain this effect for up to 6 hours +/− LPS. LPS primed mice transfused aged PMPs had higher W/D ratios than LPS primed mice transfused saline (p<0.05).CONCLUSIONSPMPs may in part contribute to TRALI when aged platelets which are a rich source of PMPs are transfused. In our model aged PMPs were injurious, impairing EC barrier integrity in vitro and leading to increased W/D ratios in vivo. Targeting aged PMPs may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.Support or Funding InformationCanadian Institutes of Health Research (CIHR)Canadian Blood Services (CBS)

Prophylactic platelet transfusion plus supportive care versus supportive care alone in adults with dengue and thrombocytopenia: a multicentre, open-label, randomised, superiority trial

Dengue is the commonest vector-borne infection worldwide. It is often associated with thrombocytopenia, and prophylactic platelet transfusion is widely used despite the dearth of robust evidence. We aimed to assess the efficacy and safety of prophylactic platelet transfusion in the prevention of bleeding in adults with dengue and thrombocytopenia. We did an open-label, randomised, superiority trial in five hospitals in Singapore and Malaysia. We recruited patients aged at least 21 years who had laboratory-confirmed dengue (confirmed or probable) and thrombocytopenia (≤20 000 platelets per μL), without persistent mild bleeding or any severe bleeding. Patients were assigned (1:1), with randomly permuted block sizes of four or six and stratified by centre, to receive prophylactic platelet transfusion in addition to supportive care (transfusion group) or supportive care alone (control group). In the transfusion group, 4 units of pooled platelets were given each day when platelet count was 20 000 per μL or lower; supportive care consisted of bed rest, fluid therapy, and fever and pain medications. The primary endpoint was clinical bleeding (excluding petechiae) by study day 7 or hospital discharge (whichever was earlier), analysed by intention to treat. Safety outcomes were analysed according to the actual treatment received. This study was registered with ClinicalTrials.gov, number NCT01030211, and is completed. Between April 29, 2010, and Dec 9, 2014, we randomly assigned 372 patients to the transfusion group (n=188) or the control group (n=184). The intention-to-treat analysis included 187 patients in the transfusion group (one patient was withdrawn immediately) and 182 in the control group (one was withdrawn immediately and one did not have confirmed or probable dengue). Clinical bleeding by day 7 or hospital discharge occurred in 40 (21%) patients in the transfusion group and 48 (26%) patients in the control group (risk difference -4·98% [95% CI -15·08 to 5·34]; relative risk 0·81 [95% CI 0·56 to 1·17]; p=0·16). 13 adverse events occurred in the transfusion group and two occurred in the control group (5·81% [-4·42 to 16·01]; 6·26 [1·43 to 27·34]; p=0·0064). Adverse events that were possibly, probably, or definitely related to transfusion included three cases of urticaria, one maculopapular rash, one pruritus, and one chest pain, as well as one case each of anaphylaxis, transfusion-related acute lung injury, and fluid overload that resulted in serious adverse events. No death was reported. In adult patients with dengue and thrombocytopenia, prophylactic platelet transfusion was not superior to supportive care in preventing bleeding, and might be associated with adverse events. National Medical Research Council, Singapore.

Dose- and time-related platelet response with apheresis platelet concentrates and pooled platelets

This study was carried out to compare the post-transfusion platelet increment between the apheresis platelet concentrate (n=74) and pooled platelets (n=54). Pre- and post-transfusion platelet count of the recipient were carried out by automated hematology analyzer. In apheresis platelet concentrate group, the mean 24 hours post-transfusion platelet increment was 47 x 109/L which was statistically significant (p<0.001). On the other hand, in pooled platelets group, the mean 24 hours post–transfusions platelet count increment was 11.0 x 109/L which was also statistically significant (p<0.001). This study concluded that the transfusion of apheresis platelet concentrate was more useful than the transfusion of pooled platelets in terms of platelet count increment and requirement of donor.

Invited commentary

The multilevel disease seen in patients with critical limb ischemia (CLI) is difficult to address using endovascular therapy or open bypass because of anatomic constraints, lack of appropriate conduit, and significant comorbidities. Therapeutic arteriogenesis using cellular therapy has been the focus of research because single growth factor therapy has failed to improve outcomes in randomized controlled trials. Mesenchymal stromal cells (MSCs) are of particular interest because of their ability to secrete multiple angiogenic and arteriogenic factors and to stimulate both angiogenesis and arteriogenesis in vitro and in vivo in small animal models of hindlimb ischemia. However, there are two major limitations to using MSCs, harvest and generation of sufficient numbers of MSCs in a clinically relevant time frame and functional capacity of autogenous MSCs in unfavorable conditions common to patients with CLI, hypertension, hyperlipidemia, ischemia, and diabetes. The current article addresses these concerns by showing that use of pooled human platelet lysate as a human serum supplement rather than the more traditional fetal bovine serum significantly improved proliferation of MSCs harvested from the tibia of patients with CLI with or without concomitant diabetes undergoing amputation, although it was still worse than that seen in MSCs from healthy donors. The authors further demonstrate that the isolated CLI MSCs secrete similar levels of angiogenic and arteriogenic growth factors and stimulate angiogenesis in an in vitro coculture sprouting assay to MSCs from healthy donors. What remains lacking is the translational component back into humans, demonstrating that these isolated MSCs expanded using pooled human platelet lysate can stimulate therapeutic arteriogenesis in the unfavorable milieu of a CLI patient as opposed to a healthy animal subjected to experimental arterial ligation. However, this work is an important first step toward that translation and a tantalizing window into a true cell-based therapy for limb salvage. Expansion and angiogenic potential of mesenchymal stem cells from patients with critical limb ischemiaJournal of Vascular SurgeryVol. 65Issue 3PreviewCritical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1% to 10% of the population with peripheral arterial disease. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However, no randomized, controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. Full-Text PDF Open Archive

A Good Manufacturing Practice–grade standard protocol for exclusively human mesenchymal stromal cell–derived extracellular vesicles

Background aimsExtracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs. MethodsDepletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit. ResultsBone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV. ConclusionsThe 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice–grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.

Polymorphism of collection, transfusion and effectiveness of donor platelets concentrates

There were compared the results of 520 platelets transfusions in 8 centers. In the surveyed organizations part of prophylactic platelet transfusions ranged from 10% to 91%. On average platelet transfusions for the prevention of bleeding in 3,6 times more often than to stop the bleeding. 67,5% ofrecipients ofplatelets have oncology diseases. Target platelet concentrations for preventive transfusions seems high compared to modern evidence-based value - 10 @. 10⁰/l. 25% ofprophylactic transfusions included in the study were performed in patients with platelet count of 30 @. 10⁰l or more. In 76,7% of transfusions used platelet concentrates prepared by apheresis. Single whole blood- derived-units are used at 3,7 times more often than pooled platelets.

A Look at Platelet Count in Chronic Hepatitis C Infection

A complete blood count performed in chronic hepatitis C virus (HCV) infected patients can identify thrombocytopenia or an increased number of platelets, the cause of which must be established. Most of these patients are predisposed to develop thrombocytopenia as the disease progresses due to a lower thrombopoietin production, increased platelet pooling in the spleen, viral bone marrow suppression, or interferon-based therapy. However, a severe thrombocytopenia can have an autoimmune aetiology, which is very probable at values <15×103/mm3. Thrombopoietin analogues are useful both in patients with primary immune thrombocytopenia and in those who will begin the treatment with pegylated interferon and ribavirin before surgery. The common causes of an increased number of platelets in chronic HCV infected patients are splenectomy, ribavirin treatment, liver transplantation, and hepatocellular carcinoma. However, thrombocytosis can also be hereditary, reactive, or malignant, especially in essential thrombocythaemia or other myeloproliferative diseases that can be associated. A hepatic blood flow obstruction present in chronic HCV infected patients must draw attention to a possible associated myeloproliferative disorder (which frequently manifests in thrombocytosis) that represents its aetiology in two-thirds of cases and which can evolve with a constant or an intermittent increase in platelet count. The role of the JAK-STAT signalling mechanism is presented in both chronic hepatitis C patients and in those with essential thrombocythaemia. It was suggested that STAT3 could have a role in HCV RNA replication. In addition, the HCV core protein is involved in the modulation of fibrogenetic gene expression in hepatic stellate cells through a JAK2-STAT3 dependent pathway. Ruxolitinib (a JAK1/JAK2 inhibitor) can have beneficial effects in essential thrombocythaemia and is a subject of research in chronic hepatitis C. The discovery of the aetiology of thrombocytopenia or an increased number of platelets can contribute to a more complete diagnosis and appropriate treatment. The identification of associated disorders in chronic HCV infected patients is of vital importance for them.

Flow cytometry analysis of platelet populations: usefulness for monitoringthe storage lesion in pooled buffy-coat platelet concentrates.

Early detection of the platelet storage lesion is still a challenge in transfusion practice. Using flow cytometry, we evaluated the appearance of the storage lesion, based on the expression of platelet activation markers, in total platelets and platelet populations. Buffy-coat-derived platelet concentrates were stored under standard conditions for 5 days. The expression of activation antigens CD42b, CD36, CD62p and phosphatidylserine on total platelets and populations of small, medium-sized and large platelets was analysed by flow cytometry on storage days 1, 3 and 5. The activation/lesion on total platelets and each platelet population was detected on storage day 3, by the increased expression of CD36. On the same day, increased expression of CD42b and CD62p was detected, but only on large platelets. Small and medium-sized platelets had increased CD62p expression only on day 5. Externalisation of phosphatidylserine was not detected. Evaluation of the level of expression of various activation markers on different platelet populations could be an additional valid analysis in cell quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation.

Platelet-Rich Plasma Powder: A New Preparation Method for the Standardization of Growth Factor Concentrations

Background: Platelet-rich plasma (PRP) is widely used in sports medicine. Available PRP preparations differ in white blood cell, platelet, and growth factor concentrations, making standardized research and clinical application challenging. Purpose: To characterize a newly standardized procedure for pooled PRP that provides defined growth factor concentrations. Study Design: Controlled laboratory study. Methods: A standardized growth factor preparation (lyophilized PRP powder) was prepared using 12 pooled platelet concentrates (PCs) derived from different donors via apheresis. Blood samples and commercially available PRP (SmartPrep-2) served as controls (n = 5). Baseline blood counts were analyzed. Additionally, single PCs (n = 5) were produced by standard platelet apheresis. The concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor AB (PDGF-AB), transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), interleukin (IL)–1α, IL-1β, and IL-1 receptor agonist (IL-1RA) were analyzed by enzyme-linked immunosorbent assay, and statistical analyses were performed using descriptive statistics, mean differences, 95% CIs, and P values (analysis of variance). Results: All growth factor preparation methods showed elevated concentrations of the growth factors VEGF, bFGF, PDGF-AB, and TGF-β1 compared with those of whole blood. Large interindividual differences were found in VEGF and bFGF concentrations. Respective values (mean ± SD in pg/mL) for whole blood, SmartPrep-2, PC, and PRP powder were as follows: VEGF (574 ± 147, 528 ± 233, 1087 ± 535, and 1722), bFGF (198 ± 164, 410 ± 259, 151 ± 99, and 542), PDGF-AB (2394 ± 451, 17,846 ± 3087, 18,461 ± 4455, and 23,023), and TGF-β1 (14,356 ± 4527, 77,533 ± 13,918, 68,582 ± 7388, and 87,495). IGF-1 was found in SmartPrep-2 (1539 ± 348 pg/mL). For PC (2266 ± 485 pg/mL), IGF-1 was measured at the same levels of whole blood (2317 ± 711 pg/mL) but was not detectable in PRP powder. IL-1α was detectable in whole blood (111 ± 35 pg/mL) and SmartPrep-2 (119 ± 44 pg/mL). Conclusion: Problems with PRP such as absent standardization, lack of consistency among studies, and black box dosage could be solved by using characterized PRP powder made by pooling and lyophilizing multiple PCs. The new PRP powder opens up new possibilities for PRP research as well as for the treatment of patients. Clinical Relevance: The preparation of pooled PRP by means of lyophilization may allow physicians to apply a defined amount of growth factors by using a defined amount of PRP powder. Moreover, PRP powder as a dry substance with no need for centrifugation could become ubiquitously available, thus saving time and staff resources in clinical practice. However, before transferring the results of this basic science study to clinical application, regulatory issues have to be cleared.

Improved Survival of Older Patients with Mantle Cell Lymphoma (MCL) with Front-Line Cytarabine-Based Immunochemotherapy

IntroductionWhile the role of cytarabine-based immunochemotherapy (ICT) and autologous stem cell transplantation (ASCT) in untreated younger patients with MCL is well established, the utility of 'more intense' approaches in older patients is uncertain. Despite a median age at presentation of 60 years or more there are few therapeutic trials focussing on older patients. With the development of targeted agents, the need to define the optimal ICT strategy for comparison is pressing.MethodsThe primary aim of this study was to compare the safety and efficacy of first line ICT based therapy in patients with MCL aged over 60 years. Treatment and outcome data were collected for patients treated between 2000-2015 across a combined health-care network servicing the south-eastern, south-western and central corridor of the Australian state of Victoria (catchment area >2 million patients encompassing four major academic tertiary referral centers). Eligible patients had MCL diagnosed according to WHO 2008 criteria, confirmed by either fluorescence in situ hybridization for t(11;14) translocation or immunohistochemistry for cyclin D1 expression. Only cases with adequate information, including baseline characteristics, treatment regimens and outcome, were included. Overall survival (OS) and progression free survival (PFS) were modelled using Cox regression. For ICT comparison, patients undergoing ASCT were censored at the time of transplant. Differences in hospitalization and transfusion were analyzed using the Mann-Whitney U test.Results52 patients met inclusion criteria with a median age of 69 (range 60-91; M/F >3:1), of which 23 patients were >70 years. Most patients had advanced disease on staging, >1 extra-nodal site and an intermediate-high MIPI score. All received rituximab with initial therapy. Treatment regimens included: R-CHOP-like (31), alternating R-CHOP/R-DHAC (10), R-HyperCVAD/R-MA (7) and other (4). Eleven patients underwent ASCT. The median follow up for surviving patients was 40 months.Type of chemotherapy and up-front ASCT were the only variables influencing PFS and OS. Cytarabine-based ICT was associated with an improved median PFS (not reached vs 35 months, HR 0.27 [0.08-0.93], p=0.018) and OS (not reached vs 54 months, HR 0.28 [0.08-0.94], p=0.039) compared to R-CHOP-like regimens. Patients undergoing ASCT were younger (mostly < 70 years) and demonstrated improved median OS (not reached vs 46 months, HR 0.124 [0.017-0.921], p=0.041). No statistically significant difference in efficacy between R-HyperCVAD/R-MA and R-CHOP/R-DHAC could be determined due to low numbers. Both group of patients receiving cytarabine demonstrated durable response over time. However, those receiving R-HyperCVAD/R-MA experienced greater toxicity with increased hospitalisation: median 54 [31-72] v 20 [6-77] days, p=0.023 and transfusion requirements: median 13 [7-24] v 2 [0-5] red cell units, p=0.001; median 6 [2-15] v. 0 [0-1] platelet pools, p=0.001.ConclusionIn older patients with previously untreated MCL, cytarabine-based ICT resulted in a significant improvement in OS, with a 72% reduction in the risk of death relative to CHOP-like therapy. Cytarabine-based ICT is highly efficacious and well tolerated and should serve as a benchmark with which to compare targeted therapies. ASCT should also be considered in selected older patients. [Display omitted] [Display omitted] DisclosuresQuach:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Opat:Roche: Consultancy, Honoraria, Other: Provision of subsidised drugs, Research Funding.

Incidence and Outcomes of Catheter Related Thrombosis in Acute Leukemia: Single Centre Experience

▪IntroductionCatheter related thrombosis (CRT) is a common complication of central venous catheters (CVCs) in patients with hematological and solid organ cancers. However, little is known about the incidence of CRT in acute leukemia patients and the optimal approach to anticoagulant therapy especially in the presence of concurrent and often prolonged thrombocytopenia. Some of these patients might receive sub-therapeutic anticoagulation with a potentially higher risk of recurrent CRT, device failure and thromboembolic events.MethodWe conducted a retrospective chart review of adult patients with newly diagnosed acute leukemia at Vancouver General Hospital (VGH), Canada from January 2014 to December 2014. The study aimed to determine the incidence of CRT and review the current practice of CRT management. The outcomes were symptomatic thrombotic and bleeding events, catheter complications (e.g. infection), and blood product use. Only objectively confirmed CRT cases were included. The follow up period was from CRT diagnosis to last known date of follow-up.ResultsA total of 151 patients were identified, of whom 116 received CVC insertion. Eleven patients (78.5%) received more than one CVC (median 2, range 1-4). Fourteen patients (12%) were diagnosed with symptomatic CRT (table). All CRT events occurred within 30 days (range 4-30 days) after CVC insertion and 85.7% within the first 6 months (range 0.26-6 months) after their diagnosis of acute leukemia. Median follow up of these 14 patients was 447 patient-days (range 13-759). Two patients were lost to follow-up at 13 and 23 days due to transfer of care to other hospitals. Three out of 14 patients died during follow up (at 72, 86 and 372 days, respectively) due to leukemia.Eleven patients (78.5%) were treated with therapeutic anticoagulation using low molecular weight heparin (LMWH). Three untreated patients had CRT confined to basilic vein. Nine out of 11 patients required LMWH dose reduction due to thrombocytopenia according to our local protocol (1). Full dose weight-based LMWH was administered if platelet count could be maintained above 50 × 109/L (with or without platelet transfusion in acute phase, i.e. within 1 month of CRT diagnosis). For platelet counts between 20 and 50 × 109/L, half-dose LMWH was administered with close monitoring. LMWH was withheld if platelet count was lower than 20 x 109/L in subacute phase, i.e. after 1 month. No patient had recurrent CRT or pulmonary embolism during anticoagulation and follow up.Bleeding events occurred in 3 (27%) patients during anticoagulation. There was one major bleeding (spontaneous retroperitoneal hematoma), one clinically relevant non-major bleeding (adenovirus induced cystitis and hematuria requiring hospital admission) and one minor bleeding (bruise) event. Catheter-related infection was documented in 4 (36%) patients. No deaths occurred due to CRT or its treatment.Patients were treated for a minimum of 3 months or longer if the implicated catheter was still in situ. Median treatment time was 3 months (range 2 weeks-7 months). Three patients stopped anticoagulation prior to 3 months (palliation and lost follow-up). None of the CVC was removed as a result of CRT, but 4 were removed for infection, 3 for the completion of chemotherapy, 2 non tunnelled catheters were replaced with long term CVCs, 1 was accidentally dislodged and 1 was removed for logistic reason. The median CVC dwell time was 30 days (range 4-173).Among the 11 treated patients, 8 patients received platelet transfusions during treatment with LMWH. Eighty seven doses of pooled platelets (or its equivalent) and 64 packed red cells were transfused. Sixteen doses (18%) of platelet transfusions were given to keep platelet count above 20-50 for anticoagulation during the first month of CRT; the remainder were given for clinical indications. Only 4 units (6%) of packed red cells were transfused for major bleeding in one patient while 60 units were used for routine support when hemoglobin was <80 g/L.ConclusionTwelve percent of newly diagnosed acute leukemia patients developed CRT. Anticoagulation with LMWH using a dose reduction approach for thrombocytopenia appeared safe and effective with no recurrent VTE events and few bleeding events.1) Lee AY, Peterson EA. Treatment of cancer-associated thrombosis. Blood. 2013 Oct 3; 122:2310-7 [Display omitted] DisclosuresLee:LEO: Consultancy, Honoraria; Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Implications of a switch to a 100% apheresis platelet supply for patients and for blood donors: a risk benefit analysis.

A 100% apheresis platelet supply is considered to increase transfusion safety by lowering donor exposures for transfusion recipients. We performed a risk benefit analysis to contrast the reduction of donor exposures and the risk of contaminated blood products in the nation-wide inventory with the donor risks associated with a switch to a 100% apheresis platelet supply in Germany. Donor exposures and the number of contaminated blood products resulting from HIV-like, HBV-like, HCV-like pathogens and two theoretical agents with infection rates of 10 and 1000 in 100000, respectively, were calculated for a 100% apheresis platelet supply in Germany based on the 2006-2012 hemovigilance reports. These numbers were compared with the current mixed platelet supply of pooled and apheresis platelets. Moreover, additional donation time and apheresis donor complications resulting from a 100% apheresis platelet supply were estimated. Per million total blood products (red cells, platelets, fresh frozen plasma), a 100% apheresis platelet supply would reduce donor exposures by 87100 and the number of contaminated blood products ranging from 0·8 to 871·1. On the other hand, this requires additional 29 478 apheresis donations, 3·4years additional donor time, and would be associated with 58 additional donor complications, respectively. A 100% apheresis platelet supply would reduce donor exposures and the number of contaminated blood products in the inventory, but would increase apheresis complications in donors. Potential risks for patients must be carefully weighed against the risks for donors, dependent on the specific pathogen scenario.

Removal process of prion and parvovirus from human platelet lysates used as clinical-grade supplement for ex vivo cell expansion

Background aimsPooled human platelet lysate (HPL) is becoming the new gold standard as supplement for ex vivo cell culture for clinical protocols. However, the risk of pathogen contamination of HPL increases with the platelet pool size. We hypothesized that hollow fiber anion exchange membrane chromatography using QyuSpeed D (QSD) could remove resistant and untested bloodborne pathogens, such as parvoviruses and prions, from HPL-supplemented growth media without substantially affecting their capacity to support ex vivo cell expansion. MethodsFrozen or thawed platelet concentrates were serum-converted and centrifuged for obtaining HPL that was added to various growth media (ca. 100 mL), filtered through a 0.6-mL QSD membrane and characterized for proteins, growth factors and chemical composition. Capacity to expand Chinese hamster ovary, periodontal ligament, gingival fibroblast cells and Wharton's jelly mesenchymal stromal cells was studied. Removal of porcine parvovirus (PPV) and of the 263K prion strain of hamster-adapted scrapie was studied by spiking experiments following international guidelines. ResultsQSD had minimal impact on HPL-supplemented medium composition in proteins, growth factors and chemical content, nor capacity to expand and differentiate cells. In addition, QSD could remove ≥5.58 log10 [TCID50/mL] and ≥3.72 log10 of PPV and the 263K prion, respectively. ConclusionsQSD hollow fiber chromatography can be used to improve the virus and prion safety of HPL-supplemented media to safely expand cells for clinical protocols. These data bring new perspectives for increasingly safer use of pooled HPL in cell therapy and regenerative medicine applications.

Platelet-derived factor V promotes angiogenesis in a mouse hind limb ischemia model

Coagulation factor V (FV) is distributed in plasma and platelet pools, which are distinguished by physical and functional differences. FV has been extensively studied for its roles in coagulation. The roles of FV in other physiologic pathways remain understudied. Hind limb ischemia was produced in transgenic mice by femoral artery ligation, with different levels of FV gene expression restricted to the plasma or platelets. Hind limb blood flow perfusion in mice with higher platelet FV was significantly increased. The expression of major angiogenesis-related factors was correlated with the level of FV during ischemia. Furthermore, a platelet depletion and transfusion procedure showed that the transfusion of platelets with higher levels of FV into transgenic mice with undetectable platelet FV significantly rescued the ischemia-mediated impairments in blood flow perfusion. Immunohistochemistry analysis also indicated markedly increased capillary formation in the ischemic muscle of mice with higher platelet FV. Moreover, thrombin activity was significantly higher in the mice with higher platelet FV. Platelets expressing higher levels of FV stimulated increased endothelial cell migration. Hind limb blood flow perfusion was significantly blocked by thrombin inhibitor. These findings suggest that platelet-derived FV contributes to the control of angiogenesis and is likely associated with thrombin generation.

Abstract 536: Platelet-derived Factor V Promotes Angiogenesis in a Mouse Hindlimb Ischemia Model

Objective: Coagulation factor V (fV) is distributed in plasma and platelet pools distinguished by physical and functional differences. fV has been extensively studied for its roles in coagulation. The roles of fV in other physiological pathways remain understudied. In the current study, we report that platelet fV is critical for the regulation of angiogenesis in a mouse model of hind-limb ischemia. Methods and Results: Hindlimb ischemia was produced in mice by femoral artery ligation in transgenic mice, with different level of fV gene expression restricted to either the plasma or platelets. The hindlimb blood flow perfusion in mice with higher platelet fV was significantly greater. Furthermore, using a platelet depletion/transfusion procedure, transfusion of platelets with higher level of fV into transgenic mice with undetectable platelet fV significantly rescued the ischemia-mediated impairments in blood flow perfusion. Immunohistochemical analysis also indicated markedly increased capillary formation in ischemic muscle of mice with higher platelet fV. Moreover, thrombin activity was significantly higher in the mice with higher platelet fV. Platelet with higher level of fV demonstrated significantly higher pro-migratory activity in an endothelial cells migration assay. Conclusion: These findings suggest that platelet-derived fV contributes to the control of angiogenesis, in addition to its role in hemostasis. The stimulation is likely associated with thrombin generation due to platelet-derived fV.

The pathophysiology of thrombocytopenia in chronic liver disease.

Thrombocytopenia is the most common hematological abnormality encountered in patients with chronic liver disease (CLD). In addition to being an indicator of advanced disease and poor prognosis, it frequently prevents crucial interventions. Historically, thrombocytopenia has been attributed to hypersplenism, which is the increased pooling of platelets in a spleen enlarged by congestive splenomegaly secondary to portal hypertension. Over the past decade, however, there have been significant advances in the understanding of thrombopoiesis, which, in turn, has led to an improved understanding of thrombocytopenia in cirrhosis. Multiple factors contribute to the development of thrombocytopenia and these can broadly be divided into those that cause decreased production, splenic sequestration, and increased destruction. Depressed thrombopoietin levels in CLD, together with direct bone marrow suppression, result in a reduced rate of platelet production. Thrombopoietin regulates both platelet production and maturation and is impaired in CLD. Bone marrow suppression can be caused by viruses, alcohol, iron overload, and medications. Splenic sequestration results from hypersplenism. The increased rate of platelet destruction in cirrhosis also occurs through a number of pathways: increased shear stress, increased fibrinolysis, bacterial translocation, and infection result in an increased rate of platelet aggregation, while autoimmune disease and raised titers of antiplatelet immunoglobulin result in the immunologic destruction of platelets. An in-depth understanding of the complex pathophysiology of the thrombocytopenia of CLD is crucial when considering treatment strategies. This review outlines the recent advances in our understanding of thrombocytopenia in cirrhosis and CLD.

A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization

Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4–1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. Statement of SignificanceInnovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel’s desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs’ angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.

The Relationship Between Preoperative Hemoglobin Concentration, Use of Hospital Resources, and Outcomes in Cardiac Surgery.

Preoperative anemia is an established risk factor associated with adverse perioperative outcomes after cardiac surgery. However, limited information exists regarding the relationship between preoperative hemoglobin concentration and outcomes. The aim of this study was to investigate how outcomes are affected by preoperative hemoglobin concentration in a cohort of patients undergoing cardiac surgery. A retrospective, observational cohort study. A single-center tertiary referral hospital. The study comprised 1,972 adult patients undergoing elective and nonelective cardiac surgery. The independent relationship of preoperative hemoglobin concentration was explored on blood transfusion rates, return to the operating room for bleeding and/or cardiac tamponade, postoperative intensive care unit (ICU) and in-hospital length of stay, and mortality. The overall prevalence of anemia was 32% (629/1,972 patients). For every 1-unit increase in hemoglobin (g/dL), blood transfusion requirements were reduced by 11%, 8%, and 3% for red blood cell units, platelet pools, and fresh frozen plasma units, respectively (adjusted incident rate ratio 0.89 [95% CI 0.87-0.91], 0.92 [0.88-0.97], and 0.97 [0.96-0.99]). For each 1-unit increase in hemoglobin (g/dL), the probability (over time) of discharge from the ICU and hospital increased (adjusted hazard ratio estimates 1.04 [1.00-1.08] and 1.12 [1.12-1.16], respectively). A lower preoperative hemoglobin concentration resulted in increased use of hospital resources after cardiac surgery. Each g/dL unit fall in preoperative hemoglobin concentration resulted in increased blood transfusion requirements and increased postoperative ICU and hospital length of stay.

Mini-Bentall Procedure: The “French Cuff” Technique

This article describes how I perform a minimally invasive aortic root replacement, also known as the Mini-Bentall procedure. In particular, it highlights the details on howto perform the "French Cuff" technique to ensure anabsolute proximal annular hemostasis. Six consecutive patients underwent the Mini-Bentall procedure using the"French Cuff" technique. Five patients did not haveany transfusion and one patient had two pooled platelets. Five patients were extubated on table in the operating room and one patient was extubated 12 hours after the procedure. All patients were discharged home within 7 days after the procedure without any surgical complications.

Effectiveness of Pooled Platelet Transfusion in Concordant and Discordant Groups among Dengue Patients.

Dengue affects more than 50 million people per year and is one of the most common causes of severe thrombocytopaenia. Thrombocytopaenia is a common complication of dengue and other viral fevers apart from malaria, typhoid, leptospirosis, leukaemia and megaloblastic anaemia. A platelet count of <20,000/μl is characteristically seen in dengue haemorrhagic fever and dengue fever. It results from immune complex mediated platelet destruction or bone marrow suppression. Severe thrombocytopaenia <10,000/μl is one of the indications for prophylactic platelet transfusion therapy to prevent haemorrhage. To evaluate the effectiveness of transfusion of ABO compatible and ABO incompatible pooled platelet units in severe thrombocytopaenia cases. In this study ABO compatible and incompatible pooled platelet units were transfused to serologically confirmed dengue cases having thrombocytopaenia with or without bleeding manifestations. Each of the adult patients received 4-6 units of pooled platelet concentrates prepared from random donor whole blood suspended in plasma for severe thrombocytopaenia. Pre and post transfusion platelet counts were compared. Children aged less than 12 years, pregnant women and patients with splenomegaly those on ayurvedic and homeopathic therapy, recipients of packed red cells on the same day of platelet transfusion and recipients of multiple platelet transfusions within 24 hours were excluded from the study. The median post transfusion platelet increments (PPI) and corrected count increments (CCI) at 4hour post transfusion were 25,000/μL (5,000-80,000/μL) and 18,000/μL (range 8,000/μL- 47,500/μL) respectively among the responders. Median PPI and CCI at 24 hours were 45,000/μL and 28,863/μL among the responders. The median CCI at 4 hour post transfusion among the non-responders was 850/μL and at 24hours was 1,425/μL. At 24 hours responders showed significantly higher PPI as compared to non responders. The average platelets transfused were 4units in case of responders and 8 units in case of non-responders. ABO identical and compatible pooled platelet transfusions were more successful in increasing the post transfusion platelet counts as compared to ABO incompatible pooled platelets.