Allogeneic Platelet Research Articles

Clinical and Epidemiological Profile of Deep Second Degree Burn Patients Treated with Platelet Lysate

The wound healing process in burn victims is still a problem today despite the advances made in the management of these patients with the different resuscitation regimens, the advent of antibiotic therapy as well as the different local treatments used. The local application of a high concentration of growth factor through the use of halogenic platelet concentrates lyzed by freezing- thawing has been done in the past with the aim of accelerating the healing process of the various wounds. The aim of this work is to describe Clinical and epidemiological profile of deep second degree burn patients treated with platelet lysate. A longitudinal and prospective study was carried out in 30 adult patients with deep second-degree burns with boiling water with a burned body surface area ≤ 5%, treated locally with allogeneic platelet lysate in the Burn centre and Plastic Surgery Service of the General Hospital “Dr. Juan Bruno Zayas Alfonso” in Santiago de Cuba, during the period of February 2015 to February 2016.A total of 30 adult patients have been studied. The mean age was 33.93. Female sex predominated with a male/female ratio of 1/2. The most frequent location of burns was in the lower limbs accounting for 36.6% of patients, followed by the upper limbs and anterior trunk at 26.7% in both cases. For the study group, the epitelization time was less than 21 days and in the majority of cases (66.7%) this time was 7-13 days. Non-relevant side effects were observed in 80% of patients and included pruritus (50%), perilesional erythema (8.3%) and pain in 29.2% of patients. Apart from these side effects, no complications have been observed in our patients. The promising results reported in this study and in the literature warn that platelet lysate may become a potential therapeutic candidate for deep second- degree burns from boiling water.

Proteomics of human platelet lysates and insight from animal studies on platelet protein diffusion to hippocampus upon intranasal administration.

Human platelet lysates (HPLs) from allogeneic platelet concentrates (PCs) are biomaterials, which are rich in various trophic factors, increasingly used in regenerative medicine and biotherapy. Understanding how preparation methods influence the HPL protein profile, biological function, and clinical outcomes is crucial. Our study sheds light on the proteomes and functionality of different HPLs, with the aim of advancing their scientifically grounded clinical applications. To achieve this, PCs suspended in plasma underwent three distinct processing methods, resulting in seven HPL types. We used three characterization techniques: label-free proteomics and tandem mass tag (TMT)-based quantitative proteomics, both before and after the immunodepletion of abundant plasma proteins. Bioinformatic tools assessed the proteome, and western blotting validated our quantitative proteomics data. Subsequent pre-clinical studies with fluorescent labeling and label-free proteomics were used as a proof of concept for brain diffusion. Our findings revealed 1441 proteins detected using the label-free method, 952 proteins from the TMT experiment before and after depletion, and 1114 proteins from the subsequent TMT experiment on depleted HPLs. Most detected proteins were cytoplasmic, playing key roles in catalysis, hemostasis, and immune responses. Notably, the processing methodologies significantly influenced HPL compositions, their canonical pathways, and, consequently, their functionality. Each HPL exhibited specific abundant proteins, providing valuable insight for tailored clinical applications. Immunoblotting results for selected proteins corroborated our quantitative proteomics data. The diffusion and differential effects to the hippocampus of a neuroprotective HPL administered intranasally to mice were demonstrated. This proteomics study advances our understanding of HPLs, suggesting ways to standardize and customize their production for better clinical efficacy in regenerative medicine and biotherapy. Proteomic analyses also offered objective evidence that HPPL, upon intranasal delivery, not only effectively diffuses to the hippocampus but also alters protein expression in mice, bolstering its potential as a treatment for memory impairments.

Impact of Intraoperative Allogeneic Platelet Transfusion on Healthcare-Associated Infections in Cardiac Surgery: Insights From a Large Single-Center Cohort Study

ObjectivesDespite significant improvement in patient blood management-cardiac surgery remains a high hemorrhagic risk procedure. Platelet transfusion is commonly used to treat thrombocytopenia-induced perioperative bleeding. Allogeneic platelet transfusion may induce transfusion-related immunomodulation. However, its association with postoperative healthcare-associated infections is still a matter of debate. Our objective was to evaluate the impact of allogeneic platelet transfusion during cardiac surgery on postoperative HAI incidence. DesignRetrospective cohort study SettingTertiary referral academic center ParticipantsPatients undergoing cardiac surgery from 2012 to 2018. InterventionsNone Measurements and Main ResultsIntraoperative platelet transfusion was defined as exposure in a causal model. The primary outcome was the incidence of healthcare-associated infections-comprised of bloodstream infection-hospital-acquired pneumonia and surgical-site infection. Among 7662 included patients, 528 (6.8%) were exposed to intraoperative platelet transfusion and 329 (4.3%) developed 454 postoperative infections. Bloodstream infection affected 106 (1.4%) patients, hospital-acquired pneumonia 174 (2.3%) and surgical-site infection 148 (1.9%). Intraoperative platelet transfusion was associated with an increased risk of bloodstream infection after adjustment by multivariable logistic regression (OR 2.85; 95%CI (1.40-5.8); p=0.004; n=7662), propensity score matching (OR 3.95; 95%CI (1.57-12.0), p=0.007; n=766) and propensity score overlap weighting (OR 3.04; 95%CI (1.51-6.1), p=0.002; n=7762). Surgical-site infection and hospital-acquired pneumonia were not significantly increased by platelet transfusion. ConclusionsOur results suggest that intraoperative allogeneic platelet transfusion is a risk factor for bloodstream infection after cardiac surgery. These results support the development of patient blood management strategies aiming at minimizing perioperative platelet transfusion in cardiac surgery.

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

Polyinosinic: polycytidylic acid induced inflammation enhances while lipopolysaccharide diminishes alloimmunity to platelet transfusion in mice.

Alloimmune responses against platelet antigens, which dominantly target the major histocompatibility complex (MHC), can cause adverse reactions to subsequent platelet transfusions, platelet refractoriness, or rejection of future transplants. Platelet transfusion recipients include individuals experiencing severe bacterial or viral infections, and how their underlying health modulates platelet alloimmunity is not well understood. This study investigated the effect of underlying inflammation on platelet alloimmunization by modelling viral-like inflammation with polyinosinic-polycytidylic acid (poly(I:C)) or gram-negative bacterial infection with lipopolysaccharide (LPS), hypothesizing that underlying inflammation enhances alloimmunization. Mice were pretreated with poly(I:C), LPS, or nothing, then transfused with non-leukoreduced or leukoreduced platelets. Alloantibodies and allogeneic MHC-specific B cell (allo-B cell) responses were evaluated two weeks later. Rare populations of allo-B cells were identified using MHC tetramers. Relative to platelet transfusion alone, prior exposure to poly(I:C) increased the alloantibody response to allogeneic platelet transfusion whereas prior exposure to LPS diminished responses. Prior exposure to poly(I:C) had equivalent, if not moderately diminished, allo-B cell responses relative to platelet transfusion alone and exhibited more robust allo-B cell memory development. Conversely, prior exposure to LPS resulted in diminished allo-B cell frequency, activation, antigen experience, and germinal center formation and altered memory B cell responses. In conclusion, not all inflammatory environments enhance bystander responses and prior inflammation mediated by LPS on gram-negative bacteria may in fact curtail platelet alloimmunization.

Enhanced alloresponse to platelet transfusion due to immune dysregulation following ablative chemotherapy in mice.

Alloimmunization is common following platelet transfusion and can result in negative outcomes for recipients such as refractoriness to subsequent transfusions and rejection of transplants. Healthy people do not receive blood transfusions, and the diseases and therapies that result in a need to transfuse have significant impacts on the immunological environment to which these alloantigens are introduced. Ablative chemotherapies are common among platelet recipients and have potent immunological effects. In this study, we modeled the impact of chemotherapy on the alloresponse to platelet transfusion. As chemotherapies are generally regarded as immunosuppressive, we hypothesized that that they would result in a diminished alloresponse. Mice were given a combination chemotherapeutic treatment of cytarabine and doxorubicin followed by transfusion of allogeneic platelets, and compared to controls given no treatment, chemotherapy alone, or transfusion alone. Alloantibody responses were measured 2 weeks after transfusion, and cellular responses and growth factors were monitored over time. Contrary to our hypothesis, we found that chemotherapy led to increased alloantibody responses to allogeneic platelet transfusion. This enhanced response was antigen-specific and was associated with increased CD4+ and CD8+ T cell responses. Chemotherapy led to rapid lymphocyte depletion followed by reconstitution, non-specific activation of transitional B cells with the highest levels of activation in the least mature subsets, and increased serum levels of B cell activating factor (BAFF). These data suggest that ablative chemotherapy can increase the risk of alloimmunization and, if confirmed clinically, that additional measures to protect these patient populations may be warranted.

Efficient Production of Platelets from Umbilical Cord Blood Stem Cells Using A Collagen-Tragacanth Scaffold in A Multi-Chamber Bioreactor

Background: To date, platelet (PLT) transfusion is the main medical option for the treatment of thrombocytopenia. Nowadays, allogenic PLTs are commonly used for blood donation. Due to the limitations in the preparation and storage of PLTs, researchers try to develop alternative modalities such as bioreactors for the in-vitro expansion and production of PLTs for therapeutic purposes by promoting the differentiation of different types of stem cells into megakaryocytes (MK) and PLTs.
 Materials and Methods: In this experimental study, umbilical cord blood stem cells (UCB-SC) were differentiated into MK. To this end, a new bioreactor system consisting of six compartments and a two-layer scaffold made of collagen and natural tragacanth gum was developed to mimic a bone marrow-like structure. After MKs were loaded to the top layer of the scaffold, the production rate of PLTs was analyzed under shear stress.
 Results: Based on the estimation, each MK produced about 17.42 PLTs loaded into the scaffold collagen. This value emerged to be 23.46 and 9.44 PLTs in the collagen-tragacanth and the pure tragacanth scaffold groups, respectively, the increase of PLTs in the collagen-tragacanth scaffold was statistically significant (p < 0.001).The generated PLTs had a positive response in aggregometry as well as interaction with normal PLTs.
 Conclusion: Taken together, the designed bioreactor with a collagen-tragacanth substrate can be used for the production of a sufficient number of PLTs for clinical purposes.

Efficacy of allogeneic platelet growth factors in actinic cystitis: The resolution of trouble?

BackgroundActinic cystitis is a severe complication after radiotherapy for prostate cancer. It is a chronic inflammatory process that leads to an alteration of bladder mucosa with formation of petechiae and subsequently hematuria. Actinic cystitis responds poorly to medical treatment, with a heavy burden on patients’ quality of life. Patients with refractory hematuria may undergo cystectomy in the attempt to control bleeding. We conducted a prospective study to evaluate the effectiveness of the allogeneic platelet growth factors for actinic cystitis. Methods and MaterialNine patients with actinic cystitis were enrolled in this study. The primary outcome measures were the effects of the platelet growth factors on the injury of the bladder mucosa. The secondary outcome was the change in quality of life ResultsA total of 9 patients, mean age 68 (range 59–81) underwent a therapeutic program of bladder instillation with allogeneic platelets growth factors for 3 months. Of the 9 patients, all (100 %) had complete resolution of hematuria and urinary symptoms. After three months cystoscopy showed regeneration of the normal bladder mucosa. Biopsies allowed histological confirmation of the finding. DiscussionThe instillation of allogeneic platelet growth factors in actinic cystitis is a new treatment that in this setting of patients appears promising in promoting a resolution of urinary symptoms, hematuria and avoiding a disabling surgery such as cystectomy.

Marginal zone B cells are responsible for the production of alloantibodies following platelet transfusion in mice.

Alloimmunization against platelets remains a potentially serious adverse transfusion event. Alloantibodies produced by the recipient, mainly directed against human leukocyte antigen class I donor antigens, can compromise the therapeutic efficacy of subsequent transfusions, and may lead to refractoriness. Because the mechanism of anti-HLA alloantibody formation is poorly understood, this study aimed to identify the cells involved in the platelet immune response by focusing on the spleen, the main organ that orchestrates this alloimmune response. In the spleen, transfused allogeneic platelets are located in the marginal zone and interact with marginal zone B (MZB) cells, a specialized B-cell population implicated in the capture and follicular delivery of blood-borne antigens. To study the involvement of MZB cells in alloantibody production, we used a murine model reproducing major histocompatibility complex incompatibility between a donor (H2b) and recipient (H2d) that occurs during platelet transfusion. Following weekly H2b platelet transfusions, recipient H2d mice produced anti-H2b immunoglobulin G, which induced a refractory state upon subsequent transfusions. Specific immunodepletion of MZB cells or their displacement from the marginal zone to the B-cell follicles by treatment with an S1P1 antagonist before each transfusion prevented significant alloantibody formation. Under these conditions, transfused platelets were still circulating after 24 hours, whereas they were rapidly removed from circulation in alloimmunized mice. The identification of MZB cells as key players in the plateletalloimmune response opens up new perspectives for minimizing plateletalloimmunization and avoiding the associated refractory state in frequently transfused patients.

Allogeneic platelet gel therapy for refractory abdominal wound healing: A preliminary study.

Refractory abdominal wounds are commonly complicated by surgical site infections, which prolong hospital stays and increase medical costs. There is little clinical data on the use of allogeneic platelet gel (PG) therapy for refractory infected wounds. This study aimed to evaluate the efficacy and safety of allogeneic PGs in the treatment of refractory abdominal wounds. A prospective single-center study was performed in a national abdominal trauma referral center between June 2019 and June 2021. A total of 11 patients with refractory abdominal wounds were treated with allogeneic PGs after the failure of standard medical treatments. The PGs were derived from platelets collected from healthy donors using apheresis, and each PG was tested for platelet count, transfusion-related diseases, aerobic and anaerobic bacteria, and growth factor concentration. Clinical efficacy was evaluated by assessing the wound surface and observing the condition of the wound, including wound area and percentage of granulation. The median age of the patients was 37 years (1st quartile, 3rd quartile (Q1, Q3): 31-55 years), median (Q1, Q3) hemoglobin level was 95 g/L (78-120 g/L) and median (Q1, Q3) serum albumin level was 39.9 g/L (34.9-42.7 g/L). The PG platelet count was 976.5 ±174.9×109/L. Results of transfusion-associated contagion tests for aerobic and anaerobic bacteria were negative. Growth factor contents (pg/mL) were: for transforming growth factor beta 1 (TGF-β1); 2542.39 ±430.60, for platelet-derived growth factor BB (PDGF-BB); 23230.03 ±4236.14 and FOR vascular endothelial growth factor (VEGF); 91.41 ±23.31. The rate of wound healing was 100%, and the median (Q1, Q3) healing time was 30 days (18-40 days). The follow-up period was 5-27 months, during which no recurrence of the wounds was found. The present study demonstrated that allogeneic PGs are a safe and effective treatment for refractory abdominal wounds.

The effects of several forms of platelet concentrates combined with biomaterial collapan on segmental tibial bone defect regeneration

Objective: the purpose of the study was to evaluate the effects of three forms of allogeneic platelet concentrates on segmental tubular bone defect osseous regeneration.Material and Methods. In 145 laboratory rats tibial shaft segment was resected and stable osteosynthesis of the bone ends was performed. In group 1 animals the bone defect was left graftless, in group 2 animals biomaterial Collapan and shattered orthotopic autologous bone mixture into the bone defect site was placed. In group 3 animals as part of Collapan-autobone mixture defect filling fresh allogeneic platelet rich plasma was added, in group 4 animals allogeneic cryodestructed platelet rich plasma to the Collapan-bone mixture was added and group 5 animals received Collapan-bone mixture with allogeneic platelet rich fibrin added.Results. According to gross dissection data in 23 of the 32 group 3 rats, in 25 of the 37 group 4 rats and in 19 of the 27 group 5 rats complete visual tibial shaft defect healing occurred. In group 2 on visual inspection of tibial bone in 5 of the 10 rats defect healing was noted and in group 1 rats the defect healed in 15 of the 39 animals only. Bone defect healing rates in groups 3, 4 and 5 differed from group 1 healing rate significantly with χ2 = 7,889, χ2 = 6,452 and χ2 = 6,504 respectively, group 2 bone defect healing rate did not differ from that of group 1 (χ2 = 0,439). Histological analysis of specimens from groups 3, 5 and especially from group 4, at 3 weeks after plasty demonstrated active intermediary callus formation at bonesaw-line and neoformed osteoid bone on Collapan-bone chips aggregations surfaces; no intermediary callus formation in specimens from group 2 was found. At 6 to 12 weeks after plasty the most mature osseous tissue in specimens from group 4 was observed, the least mature neoformed osseous tissue amount was common to specimens from group 2.Conclusion. All three allogeneic Collapan-immobilized platelet concentrates studied promoted bone formation and healing of considerable segmental tibial shaft bone defect in rat model. The most evident osteogenesis activating effect was histologically confirmed in cases where cryodestructed platelet rich plasma to Collapan-bone chips mixture was added.

Efficacy of intracorporeal injections of allogenic platelet derived growth factors in the treatment of erectile dysfunction refractory to phosphodiesterase type 5 inhibitors

Efficacy of intracorporeal injections of allogenic platelet derived growth factors in the treatment of erectile dysfunction refractory to phosphodiesterase type 5 inhibitors

Chitosan-dipotassium orthophosphate lyophilizate: a novel in situ thermogel carrier system of allogeneic platelet lysate growth factors

The clinical success of platelet-rich plasma (PRP) is constrained by its limited mechanical strength, rapid disintegration by lytic enzymes, and the consequent short-term release of bioactive growth factors (GFs). Recently, attempts to formulate PRP and other hemoderivatives, such as platelet lysate (PL) have been underway. The current study aimed to formulate allogeneic freeze-dried human platelet lysate (HPL) onto lyophilized chitosan - dipotassium hydrogen orthophosphate (CS/DHO) thermo-sensitive scaffolds. A systemic approach was employed to optimize freeze-drying (FD) procedures targeting predefined critical quality attributes (CQAs). Thermal behavior, vibrational spectroscopy, morphological and moisture content analyses were used to detect possible protein destabilization during formulation and suboptimal cake properties. The effect of CS/DHO concentrations on thermo-responsiveness and release kinetics were investigated. Finally, six-months stability and cytotoxicity studies were carried out. An optimized lyophilizate was attainable with residual moisture of less than 5% and thermoresponsive to 33 °C in less than 3 min. HPL proteins were sustainedly released over five days in a pH-sensitive manner. The stability study indicated plausible physical and biochemical stability. Cell viability testing supported the cytocompatibility of the system. Finally, the lyophilizate variant of CS/DHO thermogel overcomes limited storage stability previously posed as a challenge in freshly prepared thermogels. The developed system overcomes the drawbacks of currently used PRP treatment and provides a novel GF-rich scaffold for wound repair.

Efficacy of Platelet Gel in Children with Stomatitis during Chemotherapy

Abstract Introduction. Stomatitis (oral mucositis) is a very serious adverse effect of anticancer therapy, and it may exacerbate treatment outcomes as well as prognoses. Stomatitis is associated with pain during chewing and swallowing, causing discomfort and interfering with eating. It can also lead to weight loss and a delay in child growth and development. None of the numerous therapies used for stomatitis management can be considered universal or sufficiently effective. Allogeneic platelet gel, a formulation that adheres closely to mucosal lesions and is rich in growth factors, may be an effective treatment for accelerating the erosion-healing process. Methods. A prospective, open-label study on the efficacy of platelet gel used for the treatment of stomatitis in children during chemotherapy. Platelet gel was applied to oral mucosal lesions four times a day. Results. 28 patients with Grade II and Grade III stomatitis were enrolled in the study. The first day after applying platelet gel, 93% of patients reported relief from pain. Within 4 to 5 days, one degree of mucosal lesion regression was reported in 89% of patients, indicating a 3 to 5 day reduction in therapy compared to our previous experience. In only two patients did we observe mild adverse events in the form of a burning sensation. Conclusion. Platelet gel contributed to both pain reduction and acceleration of the oral mucosa healing process. It is a safe and effective therapy for children with stomatitis induced by chemotherapy.

Advances in generating HLA-Universal platelets for transfusion medicine

Objectives‘Universal’ blood typically refers to blood components that can be donated to most individuals. Allogenic platelet refractoriness is often caused by incompatibility of human leukocyte antigens (HLA) and antibodies in the recipient binding donor epitopes. HLA-matched platelets are not always readily available for transfusions, hindered by a general rise in platelet demand, logistical challenges of HLA-selected platelet provisions and the limited HLA repertoire of current stocks. Several groups have therefore progressed towards making in vitro platelets lacking HLA class I molecules, to avoid immunogenic reactions. This review will outline the progress to date in establishing these ‘HLA-universal platelets,’ taking a comparative view of the gene-editing technologies used for their creation while also evaluating the resulting products and the immunogenic properties of HLA-deficient cells. Key findingsUniversal platelets have been differentiated from HLA class I knockdown or knockout progenitors, generated mainly through gene modification techniques targeting B2M, which encodes the β2m-light chain component of HLA class I antigens. B2M-knockdown through RNA interference technology reduces but does not completely deplete HLA class I expression, whereas B2M-knockout via endonucleases (TALENs or CRISPR/Cas9 systems) creates HLA-negative cells. Cultured HLA-silenced or ablated megakaryocytes and platelets are functional and can circulate in mouse models, while evading immune detection. HLA class I proteins serve as ligands for Natural Killer (NK) cell inhibitory receptors. A key concern for patient welfare, using HLA-universal cells, is the triggering of immune reactions including NK cytotoxicity; therefore, a possible approach to circumvent this has been to either retain or co-express alternative inhibitory or immunoregulatory molecules. ConclusionsThe collection of studies brought together and reviewed here show proof-of-concept that functional HLA-universal platelets, inert to immune responses, can be produced using powerful gene-editing techniques. CRISPR/Cas9, in particular, offers versatility to co-engineer multiple specific gene knockouts in combination with options to knock in immunomodulating proteins or platelet factors, to correspondingly further enhance immune tolerance or improve platelet functions. Together these data demonstrate the rapid development and feasibility of moving HLA-universal platelets towards the clinic.

The Role of Platelets in the Induction of Alloimmunization after Platelet Transfusion

Background Platelet (PLT) transfusion can elicit alloimmune responses leading to alloantibody formation. These alloantibodies can opsonize PLTs which causes rapid clearance of transfused PLTs and thereby render the transfusion ineffective. Contaminating WBCs in PLT concentrates are important for the induction of this alloimmunization which is evident from the fact that leukoreduction of platelet concentrates significantly reduced the frequency of alloimmunization. Despite universal leukoreduction, however, a residual risk remains. Currently, it is unknown if this is due to the residual WBCs (<1*106 / transfusion) in PLT concentrates or if PLTs themselves can also induce primary HLA-alloimmunization. Furthermore, it is not known which factors increase or reduce the likelihood of alloimmunization. Therefore, the aim of our study was, first, to determine the involvement of PLTs in the induction of alloimmunization, and second, to investigate whether storage of PLT concentrates might be a risk factor for alloimmunization.Methods To determine if PLTs are phagocytosed and presented by antigen presenting cells, monocyte derived dendritic cells (moDCs) were incubated at 37°C with allogeneic PLTs. After incubation, moDCs were either fixed and analysed for phagocytosis of PLTs using z-stacks obtained with confocal microscopy, or moDCs were lysed and HLA-DR was immunoprecipitated from this lysate. Thereafter, HLA-DR presented peptides were eluted and measured using mass spectrometry. Furthermore, HLADRB3*01:01+ moDCs were incubated with either HPA1a+ or HPA1b+ PLTs and later exposed for 24 hours to HPA1a-specific HLADRB3*01:01+ CD4 T cells after which production of IFNy by T cells was determined using ELISA. To investigate if storage may be a potential risk factor for alloimmunization, moDCs were incubated with freshly isolated PLTs or PLTs stored 7 days under blood bank conditions. Finally, in some experiments, freshly isolated PLTs were activated using thrombin-receptor agonistic peptide, treated with calcimycin A23187 to induce apoptosis, or incubated with either purified phosphatidylserine (PS) or phosphatidylcholine (PC). Subsequently, phagocytosis of these PLTs by moDCs was quantified using imaging flow cytometry.Results Confocal microscopy clearly showed that PLTs are phagocytosed by moDCs. Mass spectrometry analyses showed presentation of peptides derived from proteins exclusively expressed in PLT by HLA class II of moDCs. In addition, moDCs induced a significant HPA1a+ specific CD4 T cells response (measured as IFNy production) after incubation with HPA1a+ PLTs, which was not observed after incubation with HPA1b+ PLTs. PLTs obtained from stored PLT concentrates were significantly more phagocytosed compared to fresh PLTs. This might be caused by increased presence of apoptotic platelets in stored PLT concentrate, as phagocytosis of apoptotic platelets was significantly increased compared to activated or unstimulated PLTs. This increased phagocytosis was partially caused by PLT surface expression of PS. Furthermore, apoptotic PLTs also induced a stronger CD4 T cell response.Discussion Our studies clearly show that peptides of phagocytosed PLTs are presented by DCs, resulting in a platelet-specific CD4 T cell response. This implicates that not only WBCs, but also PLTs themselves can induce alloimmunization after PLT transfusion. As a result, factors that increase phagocytosis of PLTs could also increase the risk of alloimmunization after PLT transfusion. In this respect, PLTs stored for 7 days showed increased phagocytosis as compared to fresh PLTs. This increase is at least partly due surface expressed PS caused by storage-induced apoptosis and resulted in a stronger platelet-specific CD4 T cell responses compared to DCs incubated with untreated fresh PLTs. Although these in vitro experiments only partly represent the complex processes that are involved in an immune response in vivo , these data suggest that storage of PLT concentrates may be associated with increased alloimmunization. DisclosuresNo relevant conflicts of interest to declare.

Continuous Platelet Transfusion As a Potential Desensitization Regimen in HLA Class I Alloimmune-Mediated Platelet Refractoriness

Background: The development of HLA antibodies is responsible for 4-8% of platelet transfusion refractoriness (PTR). The presence of HLA antibodies is a clinical complication that is generally managed by the selection of products that are negative for the antigens cognate to the patient's antibodies. Often, this selection is facilitated by targeted recruitment of donors with known HLA types. However, for very broadly HLA-alloimmunized patients, compatible products may be exceedingly scarce or completely unavailable, precluding the ability to consistently provide products the patient will likely increment to and benefit from. Here we describe a case of a patient with severe aplastic anemia (platelet count chronically <1 K/uL) and platelet transfusion refractoriness secondary to broad HLA-alloimmunization (98% PRA). HLA compatible products were unavailable due to the patient's rare HLA type and breadth of antibodies, and attempts at procuring compatible products via multiple national agency searches were unsuccessful. Thus, the patient could only be supplied with HLA incompatible products, which yielded insufficient post-transfusion platelet corrected-count increments (CCI). Her course was eventually complicated by multifocal intracranial hemorrhages, necessitating ICU admission with aggressive platelet transfusion support, immune-targeting therapy consisting of IVIg, and aminocaproic acid and recombinant activated factor VIIa.Methods: A continuous platelet infusion, or “platelet drip,” was employed in order to provide platelet transfusion support in the face of her severe thrombocytopenia with profound refractoriness. This consisted of consecutive transfusions of 4-6 equivalent unit apheresis platelet products (“doses”) transfused over 4 hours with a 15 minute post-transfusion CBC obtained from Q8 to Q12 hours to assess platelet increments. This ultimately resulted in 24-30 equivalent unit platelet product infusions/day.Results: Two days into the continuous platelet infusion, the patient began incrementing appropriately to occasional platelet products (CCI>>5) and, consequently, the infusions were able to sustain a platelet count >10K/uL, with a range of 20-70 K/uL, over the subsequent week. The improved counts, along with the patient's improved clinical condition and stable imaging findings showing resolving intracranial hemorrhages, allowed the transfusion support to be goal-directed targeting a prophylactic platelet threshhold of 10K/uL. Continued monitoring of the patient's CCIs enabled the identification of permissive antigens (i.e., allogeneic platelets with HLA antigens predicted to be incompatible with the patient's serum by solid-phase testing but were found to result in satisfactory increments); this knowledge allowed progressive widening of her permissive donor pool and enabled continued prophylactic support as needed. After three weeks the patient was able to be discharged from the ICU.Subsequent HLA class I antibody screens using flow-cytometry based single antigen bead (SAB) technology revealed progressive decrease in the patient's mean fluorescence intensity for most of her antibody specificities, with the majority of her class I A antibodies falling to undetectable or near-undetectable levels (while previously >5000 MFI). Simple regression analysis (RStudio 1.0.153, R version 3.4.1) was used to test to how level of exposure to individual HLA platelet antigens (as measured by the number of doses with a given antigen) affected subsequent MFI levels. The results of the regression indicate that pre MFI and exposure explain 70.6% of the variance (R2=0.706, F(2,78)=93.7, p=<2.2e-16). It was found that exposure is a significant predictor of decreased MFI (b=-107.20, p=0.01422). High initial MFI was strongly associated with high subsequent MFI (b=0.75, p= <2.2e-16).Conclusions: This data raises the possibility that large bolus platelet infusions could not only be of immediate therapeutic benefit in patients with HLA-alloimmune-mediated platelet refractoriness, but may also accelerate the evanescence of the responsible antibodies. Further research is warranted to explore this possibility, and to ascertain the potential magnitude of this effect in comparison to competing factors such as natural evanescence and/or B-cell immunosuppressive or immunomodulatory therapies. DisclosuresNo relevant conflicts of interest to declare.

A massive dose of allogeneic platelet gel for wound hemostatic therapy on patients with giant thoracic aortic aneurysm: report of 2 cases

Autologous platelet-rich plasma (PRP) or platelet gel (PG) has been widely used in clinical treatment. Allogeneic PRP or PG may also become a safe and effective alternative method. In this study, two cases with giant thoracic aortic aneurysm were reported where massive doses of allogeneic PG were used to spray the thoracic aortic aneurysm wall suture wrapped in artificial blood vessels, tumors blood vessel wall anastomotic site, and incision site of surgical operation. The volumes of 220 mL and 250 mL PG were applied on two patients respectively, to clot bleeding and decrease the mediastinal and pericardial drainage days after operation. The drainage tubes were pulled out on the 4th day after operation. The patients were transferred from ICU to a cardiothoracic surgery ward on the 4th and 5th day respectively. This study suggests that allogeneic platelet concentrate, as a source of PRP to prepare PG, may be used to promote and help the clotting and wound healing on surgical operation.

Anatomopathological Findings in Deep Second Degree Burns Locally Treated with Allogeneic Platelet Lysate

Anatomopathological Findings in Deep Second Degree Burns Locally Treated with Allogeneic Platelet Lysate

Preoperative autologous platelet pheresis reduces allogeneic platelet use and improves the postoperative PaO2/FiO2 ratio in complex aortic surgery: a retrospective analysis.

An autologous platelet-rich plasma pheresis (aPP) strategy can harvest partial whole blood that is separated into erythrocytes, plasma and platelets, and can reduce blood loss and transfusion during cardiovascular surgery using cardiopulmonary bypass (CPB). However, the blood and organ conservation effects of this technique have not been confirmed in the context of complex aortic surgery. Perioperative records of 147 adult patients who underwent complex aortic surgery were analysed retrospectively. All patients received regular blood conservation treatment, and 57 patients received aPP. Whether or not the participants were propensity matched, decreased platelet and cryoprecipitate transfusions were found in the aPP group (both P < 0.001), but there were non-significant differences in erythrocyte transfusion, Sequential Organ Failure Assessment scores and other outcomes when compared with the same parameters in the non-aPP group. The aPP group had a higher arterial oxygen partial pressure to inhaled oxygen concentration ratio on postoperative days 1, 2 and 7 than the non-aPP group (P < 0.001, P < 0.001 and P = 0.048, respectively). The utilization of aPP was associated with a reduction in allogeneic platelet and cryoprecipitate transfusions as well as minor lung-protective effects during complex aortic surgery using CPB.