Transparency on Platelet Transfusion in Routine Cancer Care: The Key for Optimal Blood Usage?

INTRODUCTION: In Germany, up to 75% of platelet concentrates (PCs) are administered to haematological and oncological patients. Only limited transparency exists on the characteristics of haematological/oncological patients receiving PC transfusions, treatment patterns, and guideline adherence in daily clinical routine care. This information would be key for managing platelet supply and optimal platelet usage strategies. This study aimed to analyse data from clinical routine transfusions to fill the aforementioned information gaps and to create an inventory as a blueprint for electronic data capturing systems that allow simplified, recurring analyses. METHODS: Prospective open-label, single-centre, observational study in a German tertiary teaching haematological/oncological setting. All inpatients who received any transfusion of PCs (pathogen-inactivated or conventional) in routine use over a period of 3 months (March 2015-May 2015) were consecutively included. Except for age (≥18 years), no exclusion criteria were applied. For guideline adherence, the Cross-Sectional Guidelines for Therapy with Blood Components and Plasma Derivatives - amended edition 2020 were used. An inventory blueprint was created through a narrative literature review and the data collected in this study. RESULTS: Ninety-four patients received 942 PCs. The mean (±SD) age was 54.6 (±13.9) years, 68% were male and 86% were diagnosed with a haematological disease. Thirteen patients received 42% of all transfused PCs. The mean ± SD number of transfused PC per patient was 10.81 ± 9.24. Five (0.5% per transfusion) minor adverse events were documented. Approximately 19% of PCs were not administered according to existing guidelines. The mean transfusion interval was 1.71 ± 1.1 days, and the mean increment was 12.62 ± 14.7 G/L. The inventory showed which platelet transfusion-specific data should be documented for answering questions in terms of quality, effectiveness, and management of PC transfusions. CONCLUSIONS: Platelet transfusions in a haematological/oncological setting are highly individual in terms of the total number of transfusions and transfusion intervals. The majority of all PC transfusions were given to only a small group of patients. Continuous, structured real-world data collection/evaluation and benchmarking with data from more centres seems essential in determining specific needs in this vulnerable patient group, assessing the quality of transfusion practices, determining effectiveness, and anticipating future demand for platelets and a sustainable blood supply. So far, not all relevant data are collected routinely. The advancing digitalization of health systems offers opportunities to collect and link data and thus make them more accessible and evaluable.

On the trail of blood doping-microRNA fingerprints to monitor autologous blood transfusions in vivo.

Autologous blood doping refers to the illegal re-transfusion of any quantities of blood or blood components with blood donor and recipient being the same person. The re-transfusion of stored erythrocyte concentrates is particularly attractive to high-performance athletes as this practice improves their oxygen capacity excessively. However, there is still no reliable detection method available. Analyzing circulating microRNA profiles of human subjects that underwent monitored autologous blood transfusions seems to be a highly promising approach to develop novel biomarkers for autologous blood doping. In this exploratory study, we randomly divided 30 healthy males into two different treatment groups and one control group and sampled whole blood at several time points at baseline, after whole blood donation and after transfusion of erythrocyte concentrates. Hematological variables were recorded and analyzed following the adaptive model of the Athlete Biological Passport. microRNA profiles were examined by small RNA sequencing and comprehensive multivariate data analyses, revealing microRNA fingerprints that reflect the sampling time point and transfusion volume. Neither individual microRNAs nor a signature of transfusion-dependent microRNAs reached superior sensitivity at 100% specificity compared to the Athlete Biological Passport (≤11% 6 h after transfusion versus ≤44% 2 days after transfusion). However, the window of autologous blood doping detection was different. Due to the heterogenous nature of doping, with athletes frequently combining multiple medications in order to both gain a competitive advantage and interfere with known testing methods, the true applicability of the molecular signature remains to be validated in real anti-doping testings.

Toll-like receptor 7/8-matured RNA-transduced dendritic cells as post-remission therapy in acute myeloid leukaemia: results of a phase I trial.

OBJECTIVES: Innovative post-remission therapies are needed to eliminate residual AML cells. DC vaccination is a promising strategy to induce anti-leukaemic immune responses. METHODS: We conducted a first-in-human phase I study using TLR7/8-matured DCs transfected with RNA encoding the two AML-associated antigens WT1 and PRAME as well as CMVpp65. AML patients in CR at high risk of relapse were vaccinated 10× over 26 weeks. RESULTS: Despite heavy pretreatment, DCs of sufficient number and quality were generated from a single leukapheresis in 11/12 cases, and 10 patients were vaccinated. Administration was safe and resulted in local inflammatory responses with dense T-cell infiltration. In peripheral blood, increased antigen-specific CD8+ T cells were seen for WT1 (2/10), PRAME (4/10) and CMVpp65 (9/10). For CMVpp65, increased CD4+ T cells were detected in 4/7 patients, and an antibody response was induced in 3/7 initially seronegative patients. Median OS was not reached after 1057 days; median RFS was 1084 days. A positive correlation was observed between clinical benefit and younger age as well as mounting of antigen-specific immune responses. CONCLUSIONS: Administration of TLR7/8-matured DCs to AML patients in CR at high risk of relapse was feasible and safe and resulted in induction of antigen-specific immune responses. Clinical benefit appeared to occur more likely in patients <65 and in patients mounting an immune response. Our observations need to be validated in a larger patient cohort. We hypothesise that TLR7/8 DC vaccination strategies should be combined with hypomethylating agents or checkpoint inhibition to augment immune responses. TRIAL REGISTRATION: The study was registered at https://clinicaltrials.gov on 17 October 2012 (NCT01734304) and at https://www.clinicaltrialsregister.eu (EudraCT-Number 2010-022446-24) on 10 October 2013.

SERINC5 Is an Unconventional HIV Restriction Factor That Is Upregulated during Myeloid Cell Differentiation.

Classical antiviral restriction factors promote cellular immunity by their ability to interfere with virus replication and induction of their expression by proinflammatory cytokines such as interferons. The serine incorporator proteins SERINC3 and SERINC5 potently reduce the infectivity of HIV-1 particles when overexpressed, and RNA interference or knockout approaches in T cells have indicated antiviral activity also of the endogenous proteins. Due to lack of reagents for detection of endogenous SERINC proteins, it is still unclear whether SERINC3/5 are expressed to functionally relevant levels in different primary target cells of HIV infection and how the expression levels of these innate immunity factors are regulated. In the current study, analysis of SERINC3/5 mRNA steady-state levels in primary lymphoid and monocyte-derived cells revealed selective induction of their expression upon differentiation of myeloid cells. Contrary to classical antiviral restriction factors, various antiviral α-interferon subtypes and proinflammatory interleukins had no effect on SERINC levels, which were also not dysregulated in CD4+ T cells and monocytes isolated from patients with chronic HIV-1 infection. Notably, HIV-1 particles produced by terminally differentiated monocyte-derived macrophages with high SERINC5 expression, but not by low-expressing monocytes, showed a Nef-dependent infectivity defect. Overall, these findings suggest endogenous expression of SERINC5 to antivirally active levels in macrophages. Our results classify SERINC5 as an unconventional HIV-1 restriction factor whose expression is specifically induced upon differentiation of cells towards the myeloid lineage.

Oral Bruton tyrosine kinase inhibitors block activation of the platelet Fc receptor CD32a (FcγRIIA): a new option in HIT?

Activation of the platelet Fc-receptor CD32a (FcγRIIA) is an early and crucial step in the pathogenesis of heparin-induced thrombocytopenia type II (HIT) that has not been therapeutically targeted. Downstream FcγRIIA Bruton tyrosine kinase (BTK) is activated; however, its role in Fc receptor-induced platelet activation is unknown. We explored the potential to prevent FcγRIIA-induced platelet activation by BTK inhibitors (BTKi's) approved (ibrutinib, acalabrutinib) or in clinical trials (zanubrutinib [BGB-3111] and tirabrutinib [ONO/GS-4059]) for B-cell malignancies, or in trials for autoimmune diseases (evobrutinib, fenebrutinib [GDC-0853]). We found that all BTKi's blocked platelet activation in blood after FcγRIIA stimulation by antibody-mediated cross-linking (inducing platelet aggregation and secretion) or anti-CD9 antibody (inducing platelet aggregation only). The concentrations that inhibit 50% (IC50) of FcγRIIA cross-linking-induced platelet aggregation were for the irreversible BTKi's ibrutinib 0.08 µM, zanubrutinib 0.11 µM, acalabrutinib 0.38 µM, tirabrutinib 0.42 µM, evobrutinib 1.13 µM, and for the reversible BTKi fenebrutinib 0.011 µM. IC50 values for ibrutinib and acalabrutinib were four- to fivefold lower than the drug plasma concentrations in patients treated for B-cell malignancies. The BTKi's also suppressed adenosine triphosphate secretion, P-selectin expression, and platelet-neutrophil complex formation after FcγRIIA cross-linking. Moreover, platelet aggregation in donor blood stimulated by sera from HIT patients was blocked by BTKi's. A single oral intake of ibrutinib (280 mg) was sufficient for a rapid and sustained suppression of platelet FcγRIIA activation. Platelet aggregation by adenosine 5'-diphosphate, arachidonic acid, or thrombin receptor-activating peptide was not inhibited. Thus, irreversible and reversible BTKi's potently inhibit platelet activation by FcγRIIA in blood. This new rationale deserves testing in patients with HIT.

Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing-Enabling robust and non-invasive biomarker research.

Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study.

The DNA Inflammasome in Human Myeloid Cells Is Initiated by a STING-Cell Death Program Upstream of NLRP3.

Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during viral and bacterial infections in human myeloid cells. We conclude that targeting the cGAS-STING-LCD-NLRP3 pathway will ameliorate pathology in inflammatory conditions that are associated with cytosolic DNA sensing.

Dramatic impact of blood transfusion on cancer-specific survival after radical cystectomy irrespective of tumor stage.

OBJECTIVE: The aim of the present study was to determine the influence of intraoperative and postoperative blood transfusion on cancer-specific outcome. MATERIALS AND METHODS: Follow-up data were collected from 722 patients undergoing radical cystectomy for urothelial carcinoma of the bladder (UCB) between 2004 and 2014. Median follow-up was 26 months (interquartile range 12-61 months). Outcome was analyzed in relation to the amount of intraoperative and postoperative blood transfusion and different tumor stages. The primary endpoint was cancer-specific survival (CSS) after cystectomy. Kaplan-Meier analysis with log-rank test and Cox regression models were used. RESULTS: Intraoperative blood transfusion was given in 36% (263/722) and postoperative blood transfusion in 18% (132/722). In patients with and without intraoperative blood transfusion, 5 year CSS was 48% and 67%, respectively (p < .001). In patients with and without postoperative blood transfusion, 5 year CSS was 48% and 63%, respectively (p < .001). The number of transfused red blood cell (RBC) units [intraoperatively: hazard ratio (HR) = 1.08, 95% confidence interval (CI) 1.01-1.15, p = .023; postoperatively: HR = 1.14, 95% CI 1.07-1.21, p < .001] was an independent prognostic factor for CSS. The dose-dependent negative effect of transfusions was also found in favorable subgroups (pT1 tumor, hemoglobin ≥13 mg/dl, p = .004) and in a high-volume surgeon subgroup (n = 244, p < .001). CONCLUSIONS: Blood transfusions during and after radical cystectomy were independent prognostic factors for CSS in this retrospective study. Therefore, efforts should be made to reduce the necessity of intraoperative and postoperative blood transfusion in cystectomy patients.

Blood Product Supply in Germany: The Impact of Apheresis and Pooled Platelet Concentrates.

BACKGROUND: In Germany, about 60% of all produced platelet concentrates (PCs) are apheresis PCs (APCs). Ongoing discussions on APC reimbursement and costs might lead to a potential shift in pooled PC (PPC)/APC production. Objective of this analysis was to build a comprehensive model from the societal perspective to evaluate consequences associated with shifts in platelet supply and demand. METHODS: Literature search, desktop researches on platelet supply and demand. Model calculations, time horizon one year: model input from the Paul-Ehrlich-Institute, data 2013. Base case: 19.2% of annual whole blood donations (WBDs) were used for production of 38.5% PPCs, decay of 46,218 PCs (8.0%). Scenarios calculated: variation in PPC proportion of 10-100%. RESULTS: Base case: during PPC production 41,957-83,913 red blood cell concentrates (RBCCs) are estimated to be lost, which corresponds to 1-2% of annual RBCCs in Germany. Scenarios were calculated for a production of 60-100% PPCs: loss is estimated to be 1.5-5.0% of annual RBCCs (65,430-218,099), decay 54,189-69,022 PCs (9.4-12.0%). CONCLUSION: Production of different blood components is interlinked and sensitive to unidimensional decisions. Increasing PPC proportion has negative impact on the RBCC production and on the antigen-matched APC donor pool. Completion of the model calculations to predict the optimal PPC/APC proportion would require evidence on the number of refractory patients, donor pool sizes, and incidences of diseases requiring platelet transfusions.

The role of therapeutic leukapheresis in hyperleukocytotic AML.

PURPOSE: Hyperleukocytosis in AML with leukostasis is a serious life-threatening condition leading to a high early mortality which requires immediate cytoreductive therapy. Therapeutic leukapheresis is currently recommended by the American Society of Apheresis in patients with a WBC>100 G/l with signs of leukostasis, but the role of prophylactic leukapheresis before clinical signs of leukostasis occur is unclear. PATIENTS: We retrospectively analyzed the role of leukapheresis in 52 patients (median age 60 years) with hyperleukocytotic AML with and without clinical signs of leukostasis. Since leukapheresis was performed more frequently in patients with signs of leukostasis due to the therapeutic policy in our hospital, we developed a risk score for early death within seven days after start of therapy (EDd7) to account for this selection bias and to independently measure the effect of leukapheresis on EDd7. RESULTS: 20 patients received leukapheresis in combination to chemotherapy compared to 32 patients who received chemotherapy only. In a multivariate logistic regression model for the estimation of the probability of EDd7 thromboplastin time and creatinine remained as independent significant parameters and were combined to create an EDd7 risk score. The effect of leukapheresis on EDd7 was evaluated in a bivariate logistic regression together with the risk score. Leukapheresis did not significantly change early mortality in all patients with a WBC≥100 G/l. DISCUSSION: Prophylactic leukapheresis in hyperleukocytotic patients with and without leukostasis did not improve early mortality in our retrospective study. Larger and prospective clinical trials are needed to validate the risk score and to further explore the role of leukapheresis in AML with hyperleukocytosis.

Coding of tissue preparations with eurocode in Germany.

SUMMARY: A safe look back of products requires their unique identification. Blood products are encoded in Germany with Eurocode since 1987. EU Directives 2004/23/EC und 2006/86/EC demanded unique identification and safe look back procedure also for tissues and cells. Eurocode IBLS e.V. and the DGTI working parties 'Tissue Preparations' and 'Automation and Data Processing' supplemented the already available Eurocode nomenclature for blood products with further data structures for tissue preparations and deliberated the federal authorities during the EU hearings. In result all EU member states can administer the coding system oneself, but have to take care about the 'key code' structure as defined and the common part at the begin of the ID number of the preparations. Eurocode today offers an EU-conform coding system considering various aspects of blood, tissue and cell preparations in an ISO-standardized form.

"Eurocode International Blood Labeling System" enables unique identification of all biological products from human origin in accordance with the European Directive 2004/23/EC.

Due to their limited availability and compatibility, biological products must be exchanged between medical institutions. In addition to a number of national systems and agreements which strive to implement a unique identification and classification of blood products, the ISBT 128 was developed in 1994, followed by the Eurocode in 1998. In contrast to other coding systems, these both make use of primary identifiers as stipulated by the document ISO/IEC 15418 of the International Organization for Standardization (ISO), and thus provide a unique international code. Due to their flexible data structures, which make use of secondary identifiers, both systems are able to integrate additional biological products and their producers. Tissue and cells also constitute a comparable risk to the recipient as that of blood products in terms of false labeling and the danger of infection. However, in contrast to blood products, the exchange of tissue and cells is much more intensively pursued at the international level. This fact is recognised by Directives 2004/23/EC and 2006/86/EC of the European Union (EU), which demand a standardized coding system for cells and tissue throughout the EU. The 2008 workshop agreement of the European Committee for Standardization (CEN) was unique identification by means of a Key Code consisting of country code corresponding to ISO 3166-1, as well as competent authority and tissue establishment. As agreed at the meeting of the Working Group on the European Coding System for Human Tissues and Cells of the Health and Consumers Directorate-General of the European Commission (DG SANCO) held on 19 May 2010 in Brussels, this Key Code could also be used with existing coding systems to provide unique identification and allow EU traceability of all materials from one donation event. Today Eurocode already uses country codes according to ISO 3166-1, and thus the proposed Key Code can be integrated into the current Eurocode data structure and does not need to be introduced separately. The Eurocode product classification for all products is based on its own unique coding system, which can be accessed over the internet by all users who are not themselves members of Eurocode. In summary, it can be said that the standardized single coding system for tissues and cells requires only unique sections in the data structure such the Key Code to fulfil the requirements of the EU Directive. Thus, various systems currently in place in different EU member states can continue to operate if the Key Code as suggested by the EU is integrated into them. The classification and description of each product characteristic is currently being discussed by the DG SANCO Working Group on the European Coding System for Human Tissues and Cells. Following intensive scrutiny in light of the stipulations laid out in EU Directives 2004/23/EC and 2006/86/EC as well as the CEN/ISSS workshop agreements, the Germany Federal Ministry for Health and organisations representing German tissue establishments under the responsibility of the German Society of Transfusion Medicine and Immunohematology, Working Party "Tissue preparations" proposed in 2009 that Eurocode be adopted for the donor identification and product coding of tissue and cells in Germany. The technical details for implementation have already been completed and are presented in the current article.

Blood type incompatible cardiac transplantation in young infants.

OBJECTIVE: Donor organ shortage in pediatric heart transplantation (HTx) is causing mortality rates of 30-50% on the waiting list. Due to immaturity of the immune system of newborns and infants, ABO-incompatible HTx may be an option to increase donor availability. We present our experience with ABO-incompatible HTx. METHODS: Three infants were transplanted ABO-incompatible since 12/2004: (1) hypoplastic left heart complex, (2) restrictive hypertrophic cardiomyopathy, (3) dilative cardiomyopathy. Age at HTx was 7, 5, and 3.5 months. All recipients had blood type O, donors were A, A, and B. Informed consent was given by parents, the ethics committee, and Eurotransplant. RESULTS: Preoperative isohemagglutinin titers were low (Patient 1: 1:4 for anti-A1, A2, B, Patient 2: 1:4, 1:1, 1:4 for anti-A1, A2, B, respectively, and Patient 3: 0 for all, but quick spin 1+ for all). Intraoperatively, plasma was separated from red blood cells and discarded up to six times until antibodies were eliminated. Immunosuppressive induction with ATG was started for 5 days. Basic immunosuppression consisted of tacrolimus, mycophenolate mofetil, and prednisone. Extubation was performed on days 15, 2, and 1, respectively. After a follow-up of 17, 16, and 12 months all patients are well, ventricular function is excellent without any acute rejection periods; Patient 1 is still on dialysis. Isohemagglutinin titers against donor blood type have disappeared in follow-up. CONCLUSIONS: ABO-incompatible cardiac transplantation shows good short-term results in young infants and seems to be a safe procedure to lower the mortality on the waiting list.

Aripiprazole augmentation of clozapine in treatment-resistant schizophrenia: a clinical observation.

BACKGROUND: Therapeutic options for patients with treatment-resistant schizophrenia are limited, and combination treatment with atypical antipsychotic drugs is an often used strategy. We tested the hypothesis that the combination of aripiprazole and clozapine would lead to an improvement in this patient group. METHODS: Eleven patients with treatment-resistant schizophrenia participated in this clinical trial and received a combination of aripiprazole and clozapine. Patients had to have remained on a stable dose of clozapine for at least 6 months in order to ensure a reasonable opportunity to respond to clozapine monotherapy. Clinical status was evaluated at baseline and at 3 months' follow-up using the Brief Psychiatric Rating Scale (BPRS). RESULTS: All patients completed 3 months' combination treatment. There was a significant reduction in the mean BPRS score in seven patients (63.6%) over the 3 months of combination treatment. Augmentation with aripiprazole in clozapine-treated patients did not result in a corresponding increase in adverse effects. Use of the combination allowed a significant reduction in the daily dose of clozapine. CONCLUSIONS: Combined application of clozapine and aripiprazole is in accordance with a neurobiological rationale and appears to be safe and well tolerated without increased risk of adverse effects.

Successful ABO-incompatible heart transplantation in two infants.

In the pediatric age group shortage of donor hearts leads to mortality rates of 30-50% on the waiting list. Because of the immaturity of the immune system of infants, ABO-incompatible heart transplantation may be an option to increase donor availability. We transplanted two infants with blood type O at the age of 7 and 5 months, respectively, with complex congenital heart disease. Intraoperative plasma exchange was performed during cardiopulmonary bypass followed by standard immunosuppression. Both recipients received a blood type A donor organ. Plasma was exchanged up to six times until anti-A antibodies were eliminated. No hyperacute rejection occurred, ventricular function is excellent and there have been no acute rejection episodes up to 4 months after transplantation. Anti-A antibody titers remained low and eventually disappeared. ABO-incompatible cardiac transplantation shows good short-term results in young infants and appears to be a safe procedure to reduce mortality on the waiting list.