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Background: Safety, tolerability and efficacy of granulocyte colony-stimulating factor (G-CSF) for mobilization of hematopoietic stem and progenitor cells (HSPCs) from healthy donors have been conclusively demonstrated. This explicitly includes, albeit for smaller cohorts and shorter observation periods, biosimilar G-CSFs. HSPC donation is non-remunerated, its sole reward being “warm glow”, hence harm to donors must be avoided with maximal certitude. To ascertain, therefore, long-term physical and mental health effects of HSPC donation, a cohort of G-CSF mobilized donors was followed longitudinally.
Methods: We enrolled 245 healthy volunteers in this bi-centric long-term surveillance study. 244 healthy volunteers began mobilization with twice-daily Sandoz biosimilar filgrastim and 242 underwent apheresis after G-CSF mobilization. Physical and mental health were followed up over a period of 5-years using the validated SF-12 health questionnaire.
Results: Baseline physical and mental health of HSPC donors was markedly better than in a healthy reference population matched for ethnicity, sex and age. Physical, but not mental health was sharply diminished at the time of apheresis, likely due to side effects of biosimilar G-CSF, however had returned to pre-apheresis values by the next follow-up appointment after 6 months. Physical and mental health slightly deteriorated over time with kinetics reflecting the known effects of aging. Hence, superior physical and mental health compared to the general healthy non-donor population was maintained over time.
Conclusions: HSPC donors are of better overall physical and mental health than the average healthy non-donor. Superior well-being is maintained over time, supporting the favorable risk–benefit assessment of volunteer HSPC donation.
Trial registration National Clinical Trial NCT01766934
Background: Blood donation saves lives. Provided they are in good health, male volunteers can donate as often as six times per year from the age of 18 into their late sixties. The burden of blood donation is very unevenly distributed, with a small minority of altruistic individuals providing this critical resource. While the consequences of persistent iron depletion in blood donors have been studied in the context of cancer and coronary heart disease, potential effects of the erythropoietic stress from repetitive large-volume phlebotomy remain unexplored. We sought to investigate if and how repeated blood donations affect the clonal composition of the hematopoietic stem and progenitor cell (HSPC) compartment.
Methods: 105 healthy, male individuals with an extensive blood donation history (median of 120 donations per donor; median age of 66 yrs.) were screened for the presence of clonal hematopoiesis (CH) using a sequencing panel covering 141 genes commonly mutated in human myeloid neoplasms. The control cohort consisted of 103 healthy, male donors with a median of 5 donations per donor and a median age of 63. Donors positive for CH were subsequently studied longitudinally. The pathogenicity of detected variants was compared using established scoring systems. Finally, to assess the functional consequences of blood-donation induced CH, selected CH mutations were introduced by CRISPR-mediated editing into HSPCs from human cord blood (CB) or bone marrow (BM). The effect of these mutations was tested under different stress stimuli using functional ex vivo long-term culture initiating cells (LTC-IC) assays.
Results: Compared to the control cohort, frequent donors were significantly more likely to have mutations in genes encoding for epigenetic modifiers (44.7 vs. 22.3 %), most specifically in the two genes most commonly mutated in CH, DNMT3A and TET2 (35.2 vs. 20.3 %). However, no difference in the variant allele frequency (VAF) of detected mutations was found between the groups. Longitudinal analysis revealed that the majority of the mutations remained at a stable VAF over an observation period of approximately one year. Three DNMT3A variants from the frequent donor cohort were introduced into healthy HSPCs and functionally analyzed: All expanded in response to EPO, but none responded to LPS or IFNγ stimulation. This contrasted with the leukemogenic DNMT3A R882H mutation, which did not expand in the presence of EPO but instead responded strongly to inflammatory stimuli.
Conclusions: Frequent whole blood donation is associated with a higher prevalence of CH driven by mutations in genes encoding for epigenetic modifiers, with DNMT3A and TET2 being the most common. This increased CH prevalence is not associated with a higher pathogenicity of the associated variants and is likely a result of the selection of clones with improved responsiveness to EPO under the condition of bleeding stress. Our data show that even highly frequent blood donations over many years is not increasing the risk for malignant clones further underscoring the safety of repetitive blood donations. To our knowledge, this is the first CH study analyzing a cohort of individuals known for their superior health and survival, able to donate blood until advanced age. Thus, our analysis possibly identified mutations associated with beneficial outcomes, rather than a disease condition, such as mutations in DNMT3A that mediated the improved expansion of HSPCs in EPO enriched environments. Our data support the notion of ongoing Darwinian evolution in humans at the somatic stem cell level and present EPO as one of the environmental factors to which HSPCs with specific mutations may respond with superior fitness.
Background: Mesenchymal stromal cells (MSCs), multipotent progenitors that can be isolated from a variety of different tissues, are becoming increasingly important as cell therapeutics targeting immunopathologies and tissue regeneration. Current protocols for MSC isolation from bone marrow (BM) rely on density gradient centrifugation (DGC), and the production of sufficient MSC doses is a critical factor for conducting clinical MSC trials. Previously, a Good Manufacturing Practice (GMP)–compatible non-woven fabric filter device system to isolate MSCs was developed to increase the MSC yield from the BM. The aim of our study was to compare high-resolution phenotypic and functional characteristics of BM-MSCs isolated with this device and with standard DGC technology.
Methods: Human BM samples from 5 donors were analyzed. Each sample was divided equally, processing by DGC, and with the filter device. Stem cell content was assessed by quantification of colony-forming units fibroblasts (CFU-F). Immunophenotype was analyzed by multicolor flow cytometry. In vitro trilineage differentiation potential, trophic factors, and IDO-1 production were assessed. Functionally, immunomodulatory potential, wound healing, and angiogenesis were assayed in vitro.
Results: The CFU-F yield was 15-fold higher in the MSC preparations isolated with the device compared to those isolated by DGC. Consequently, the MSC yield that could be manufactured at passage 3 per mL collected BM was more than 10 times higher in the device group compared to DGC (1.65 × 109 vs. 1.45 × 108). The immunomodulatory potential and IDO-1 production showed donor-to-donor variabilities without differences between fabric filter-isolated and DGC-isolated MSCs. The results from the wound closure assays, the tube formation assays, and the trilineage differentiation assays were similar between the groups with respect to the isolation method. Sixty-four MSC subpopulations could be quantified with CD140a+CD119+CD146+ as most common phenotype group, and CD140a+CD119+CD146+MSCA-1–CD106–CD271– and CD140a+CD119+CD146–MSCA-1–CD106–CD271– as most frequent MSC subpopulations. As trophic factors hepatocyte growth factor, epidermal growth factor, brain-derived neurotrophic factor, angiopoietin-1, and vascular endothelial growth factor A could be detected in both groups with considerable variability between donors, but independent of the respective MSC isolation technique.
Conclusion: The isolation of MSCs using a GMP-compatible fabric filter system device resulted in higher yield of CFU-F, producing substantially more MSCs with similar subpopulation composition and functional characteristics as MSCs isolated by DGC.
Background: Because of limitations of transportation imposed by the COVID-19 pandemic, current recommendation calls for cryopreservation of allogeneic stem cell transplants before patient conditioning. A single cell therapy laboratory was selected to function as the central cryopreservation hub for all European registry donor transplants intended for the Australian-Pacific region. We examined properties of these transplants to ascertain how quality is maintained.
Methods: We analyzed 100 pandemic-related allogeneic mobilized blood-derived stem cell apheresis products generated at 30 collection sites throughout Europe, shipped to and cryopreserved at our center between April and November of 2020. Products were shipped in the cool, subsequently frozen with DMSO as cryoprotectant. Irrespective of origin, all products were frozen within the prescribed shelf-life of 72 h.
Results: Prior to cryopreservation, viable stem cell and leukocyte count according to the collection site and our reference laboratory were highly concordant (r2 = 0.96 and 0.93, respectively) and viability was > 90% in all instances. Median nominal post-thaw recovery of viable CD34+ cells was 42%. Weakly associated with poorer CD34+ cell recovery was higher leukocyte concentration, but not time lag between apheresis or addition of cryopreservant, respectively, and start of freezing. The correlation between pre- and post-thaw CD34+ cell dose was high (r2 = 0.85), hence predictable. Neutrophil and platelet engraftment were prompt with no evidence of dose dependency within the range of administered cell doses (1.31–15.56 × 106 CD34+ cells/kg).
Conclusions: General cryopreservation of allogeneic stem cell transplants is feasible. While more than half of the CD34+ cell content is lost, the remaining stem cells ensure timely engraftment.
Background: The ability to approximate intra-operative hemoglobin loss with reasonable precision and linearity is prerequisite for determination of a relevant surgical outcome parameter: This information enables comparison of surgical procedures between different techniques, surgeons or hospitals, and supports anticipation of transfusion needs. Different formulas have been proposed, but none of them were validated for accuracy, precision and linearity against a cohort with precisely measured hemoglobin loss and, possibly for that reason, neither has established itself as gold standard. We sought to identify the minimal dataset needed to generate reasonably precise and accurate hemoglobin loss prediction tools and to derive and validate an estimation formula.
Methods: Routinely available clinical and laboratory data from a cohort of 401 healthy individuals with controlled hemoglobin loss between 29 and 233 g were extracted from medical charts. Supervised learning algorithms were applied to identify a minimal data set and to generate and validate a formula for calculation of hemoglobin loss.
Results: Of the classical supervised learning algorithms applied, the linear and Ridge regression models performed at least as well as the more complex models. Most straightforward to analyze and check for robustness, we proceeded with linear regression. Weight, height, sex and hemoglobin concentration before and on the morning after the intervention were sufficient to generate a formula for estimation of hemoglobin loss. The resulting model yields an outstanding R2 of 53.2% with similar precision throughout the entire range of volumes or donor sizes, thereby meaningfully outperforming previously proposed medical models.
Conclusions: The resulting formula will allow objective benchmarking of surgical blood loss, enabling informed decision making as to the need for pre-operative type-and-cross only vs. reservation of packed red cell units, depending on a patient’s anemia tolerance, and thus contributing to resource management.
Background: The INTERCEPT™ Blood System for Red Blood Cells (RBCs) utilizes amustaline (S‐303) and glutathione (GSH) to inactivate pathogens and leukocytes in transfused RBCs. Treatment‐emergent low titer non‐hemolytic antibodies to amustaline/GSH RBC were detected in clinical trials using a prior version of the process. The amustaline/GSH process was re‐formulated to decrease S‐303 RBC adduct formation.
Study Design and Methods: A standard three‐cell antibody screening panel was modified to include reagent red cells (RRC) with high (S‐303H) or low (S‐303L) S‐303 adduct density as assessed by flow cytometry, representative of the original and current amustaline/GSH treatment processes, respectively. General hospital and RBC transfusion‐dependent patients never exposed, and clinical trial subjects exposed to amustaline/GSH RBC were screened for antibodies to amustaline/GSH RBC using a standardized agglutination assay.
Results: Twelve (0.1%) of 10,721 general hospital and 5 (0.5%) of 998 repeatedly‐transfused patients not previously exposed to amustaline/GSH RBCs expressed natural, low titer (2‐32) IgM and/or IgG (non‐IgG1 or IgG3 isotype) antibodies with acridine (a structural element of amustaline) (n = 14) or non‐acridine (n = 3) specificity. 11 of 17 sera reacted with S‐303L panel RRCs. In clinical studies 81 thalassemia and 25 cardiac surgery patients were transfused with a total of 1085 amustaline/GSH RBCs and no natural or treatment‐emergent S‐303 antibodies were detected.
Conclusion: Standardized RRC screening panels are sensitive for the detection of natural and acquired S‐303‐specific antibodies. Natural low titer antibodies to amustaline/GSH RBC are present in 0.15% of naïve patients. The clinical relevance of these antibodies appears minimal but is under further investigation.
Background and Objectives: Patient blood (more accurately: haemoglobin, Hb) management (PBM) aims to optimize endogenous Hb production and to minimize iatrogenic Hb loss while maintaining patient safety and optimal effectiveness of medical interventions. PBM was adopted as policy for patients by the World Health Organization (WHO), and, all the more, should be applied to healthy donors. Materials and Methods: Observational data from 489 bone marrow (BM) donors were retrospectively analysed, and principles of patient blood management were applied to healthy volunteer BM donations. Results and Conclusion: We managed to render BM aspiration safe for donors, notably completely avoiding the collection of autologous blood units and blood transfusions through iron management, establishment and curation of high-yield aspiration technique, limitation of collection volume to 1·5% of donor body weight and development of volume prediction algorithms for the requested cell dose.
Background and Objectives: Red blood cell (RBC) transfusions are needed by almost every acute myeloid leukaemia (AML) patient undergoing induction chemotherapy and constitute a cornerstone in supportive measures for cancer patients in general. Randomized controlled trials have shown non‐inferiority or even superiority of restrictive transfusion guidelines over liberal transfusion guidelines in specific clinical situations outside of medical oncology. In this study, we analysed whether more restrictive RBC transfusion reduces blood use without affecting hard outcomes.
Materials and Methods: A total of 352 AML patients diagnosed between 2007 and 2018 and undergoing intensive induction chemotherapy were included in this retrospective analysis. In the less restrictive transfusion group, patients received RBC transfusion for haemoglobin levels below 8 g/dl (2007–2014). In the restrictive transfusion group, patients received RBC transfusion for haemoglobin levels below 7 g/dl (2016–2018). Liberal transfusion triggers were never endorsed.
Results: A total of 268 (76·1%) and 84 (23·9%) AML patients fell into the less restrictive and restrictive transfusion groups, respectively. The less restrictive transfusion group had 1 g/dl higher mean haemoglobin levels, received their first RBC transfusions earlier and needed 1·5 more units of RBC during the hospital stay of induction chemotherapy. Febrile episodes, C‐reactive protein levels, admission to the intensive care unit, length of hospital stay as well as response and survival rates did not differ between the two cohorts.
Conclusion: From our retrospective analysis, we conclude that a more restrictive transfusion trigger does not affect important outcomes of AML patients. The opportunity to test possible effects of the more severe anaemia in the restrictive transfusion group on quality of life was missed.
Background: Healthy volunteer registry donors have become the backbone of stem cell transplantation programs. While most registrants will never become actual donors, a small minority are called upon twice, most commonly for the same patient because of poor graft function. Anecdotal evidence provides no hard reasons to disallow second-time mobilized apheresis, but few centers have treated enough two-time donors for definitive conclusions. Moreover, for reasons unknown, the efficiency of G-CSF varies greatly between donations.
Methods: Comparison of outcomes of first vs. second donations can formally confirm G-CSF responsiveness as intrinsically, likely genetically, determined. In our database, we identified 60 donors (1.3%) who received two cycles of G-CSF 24 days to 4 years apart and systematically compared mobilization outcomes.
Results: First and second mobilization and collection proceeded without severe or unusual adverse effects. First-time mobilization efficiency was highly predictive of second-time mobilization. Neither mobilization efficiency nor time lag between donations affected the similarity of first- and second-time mobilization outcomes.
Conclusions: With the caveat that only donors with an unremarkable first donation were cleared for a second, our data indicate that a second donation is feasible, equally tolerable as a first donation, and efficient. Moreover, the data strongly support the notion of donor-intrinsic variables dictating mobilization response and argue against relevant damage to the stem cell compartment during mobilization with rhG-CSF.
Previous studies reported on the safety and applicability of mesenchymal stem/stromal cells (MSCs) to ameliorate pulmonary inflammation in acute respiratory distress syndrome (ARDS). Thus, multiple clinical trials assessing the potential of MSCs for COVID-19 treatment are underway. Yet, as SARS-inducing coronaviruses infect stem/progenitor cells, it is unclear whether MSCs could be infected by SARS-CoV-2 upon transplantation to COVID-19 patients. We found that MSCs from bone marrow, amniotic fluid, and adipose tissue carry angiotensin-converting enzyme 2 and transmembrane protease serine subtype 2 at low levels on the cell surface under steady-state and inflammatory conditions. We did not observe SARS-CoV-2 infection or replication in MSCs at steady state under inflammatory conditions, or in direct contact with SARS-CoV-2-infected Caco-2 cells. Further, indoleamine 2,3-dioxygenase 1 production in MSCs was not impaired in the presence of SARS-CoV-2. We show that MSCs are resistant to SARS-CoV-2 infection and retain their immunomodulation potential, supporting their potential applicability for COVID-19 treatment.
Background In the pandemic, testing for SARS-CoV-2 by RT-PCR in one of the pillars on which countermeasures are based. Factors limiting the output of laboratories interfere with the effectiveness of public health measures. Conserving reagents by pooling samples in low-probability settings is proposed, but may cause dilution and loss of sensitivity.
Methods We tested an alternate approach (FACT) by simultaneously incubating multiple respiratory swabs in a single tube. This protocol was evaluated by serial incubation of a respiratory swab in up to 10 tubes. The analytics validity of this concept was demonstrated in a five-sample mini pool set-up. It was consequently applied in the testing of 50 symptomatic patients (five-sample pools) as well as 100 asymptomatic residents of a nursing home (ten-sample pools).
Results Serial incubation of a respiratory swab in up to 10 tubes did not lead to a significant decline in viral concentration. The novel FACT-protocol did not cause a false negative result in a five-sample mini-pool setup, with non-significantly differing Ct values between single sample and mini-pool NAT. In two routine applications, all mini pools containing positive patient samples were correctly identified.
Conclusions Our proposed FACT-protocol did not cause a significant loss in analytic or diagnostic sensitivity compared to single sample testing in multiple setups. It reduced the amount of reagents needed by up to 40%, and also reduced hands-on time. This method could enhance testing efficiency, especially in groups with a low pretest-probability, such as systemically relevant professional groups.
Background: In the pandemic, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time polymerase chain reaction is one of the pillars on which countermeasures are based. Factors limiting the output of laboratories interfere with the effectiveness of public health measures. Conserving reagents by pooling samples in low-probability settings is proposed but may cause dilution and loss of sensitivity. Blood transfusion services had experience in performance of high throughput nucleic acid testing (NAT) analysis and can support the national health system by screening of the inhabitants for SARS-COV-2.
Methods: We evaluated a new approach of a multiple-swab method by simultaneously incubating multiple respiratory swabs in a single tube. Analytical sensitivity was constant up to a total number of 50 swabs. It was consequently applied in the testing of 50 symptomatic patients (5-sample pools) as well as 100 asymptomatic residents of a nursing home (10-sample pools).
Results: The novel method did not cause false-negative results with nonsignificantly differing cycle threshold values between single-swab and multiple-swab NAT. In two routine applications, all minipools containing positive patient samples were correctly identified.
Conclusions: The new method enables countries to increase the total number of testing significantly. The multiple-swab method is able to screen system relevant groups of employees frequently. The example in Germany shows that blood transfusion services can support general health systems with their experience in NAT and their high-throughput instruments. Screening of a huge number of inhabitants is currently the only option to prevent a second infection wave and enable exit strategies in many countries.
Background and objectives: Preoperative anaemia is an independent risk factor for a higher morbidity and mortality, a longer hospitalization and increased perioperative transfusion rates. Managing preoperative anaemia is the first of three pillars of Patient Blood Management (PBM), a multidisciplinary concept to improve patient safety. While various studies provide medical information on (successful) anaemia treatment pathways, knowledge of organizational details of diagnosis and management of preoperative anaemia across Europe is scarce.
Materials and methods: To gain information on various aspects of preoperative anaemia management including organization, financing, diagnostics and treatment, we conducted a survey (74 questions) in ten hospitals from seven European nations within the PaBloE (Patient Blood Management in Europe) working group covering the year 2016.
Results: Organization and activity in the field of preoperative anaemia management were heterogeneous in the participating hospitals. Almost all hospitals had pathways for managing preoperative anaemia in place, however, only two nations had national guidelines. In six of the ten participating hospitals, preoperative anaemia management was organized by anaesthetists. Diagnostics and treatment focused on iron deficiency anaemia which, in most hospitals, was corrected with intravenous iron.
Conclusion: Implementation and approaches of preoperative anaemia management vary across Europe with a primary focus on treating iron deficiency anaemia. Findings of this survey motivated the hospitals involved to critically evaluate their practice and may also help other hospitals interested in PBM to develop action plans for diagnosis and management of preoperative anaemia.
Background: The number of Mesenchymal Stem/Stromal Cells (MSCs) in the human bone marrow (BM) is small compared to other cell types. BM aspirate concentration (BMAC) may be used to increase numbers of MSCs, but the composition of MSC subpopulations and growth factors after processing are unknown. The purpose of this study was to assess the enrichment of stem/progenitor cells and growth factors in BM aspirate by two different commercial concentration devices versus standard BM aspiration.
Methods: 120 mL of BM was aspirated from the iliac crest of 10 male donors. Each sample was processed simultaneously by either Emcyte GenesisCS® (Emcyte) or Harvest SmartPReP2 BMAC (Harvest) devices and compared to untreated BM aspirate. Samples were analyzed with multicolor flow cytometry for cellular viability and expression of stem/progenitor cells markers. Stem/progenitor cell content was verified by quantification of colony forming unit-fibroblasts (CFU-F). Platelet, red blood cell and total nucleated cell (TNC) content were determined using an automated hematology analyzer. Growth factors contents were analyzed with protein quantification assays. Statistical analyses were performed by ANOVA analysis of variance followed by Tukey’s multiple comparison test or Wilcoxon matched-pairs signed rank test with p < 0.05 for significance.
Results: Cell viability after processing was approximately 90% in all groups. Compared to control, both devices significantly enriched TNCs and platelets, as well as the CD45−CD73+ and CD45−CD73+CD90+ cell populations. Further, Harvest significantly concentrated CD45−CD10+, CD45−CD29+, CD45−CD90+, CD45−CD105+, CD45−CD119+ cells, and CD45dimCD90+CD271+ MSCs, whereas Emcyte significantly enriched CD45dimCD44+CD271+ MSCs. BM concentration also increased the numbers of CFU-F, platelet-derived growth factor, vascular endothelial growth factor, macrophage colony-stimulating factor, interleukin-1b, VCAM-1 and total protein. Neither system concentrated red blood cells, hematopoietic stem cells or bone morphogenetic proteins.
Conclusion: This data could contribute to the development of BMAC quality control assays as both BMAC systems concentrated platelets, growth factors and non-hematopoietic stem cell subpopulations with distinct phenotypes without loss of cell viability when compared to unprocessed BM.
During erythropoiesis, haematopoietic stem cells (HSCs) differentiate in successive steps of commitment and specification to mature erythrocytes. This differentiation process is controlled by transcription factors that establish stage- and cell type-specific gene expression. In this study, we demonstrate that FUSE binding protein 1 (FUBP1), a transcriptional regulator important for HSC self-renewal and survival, is regulated by T-cell acute lymphocytic leukaemia 1 (TAL1) in erythroid progenitor cells. TAL1 directly activates the FUBP1 promoter, leading to increased FUBP1 expression during erythroid differentiation. The binding of TAL1 to the FUBP1 promoter is highly dependent on an intact GATA sequence in a combined E-box/GATA motif. We found that FUBP1 expression is required for efficient erythropoiesis, as FUBP1-deficient progenitor cells were limited in their potential of erythroid differentiation. Thus, the finding of an interconnection between GATA1/TAL1 and FUBP1 reveals a molecular mechanism that is part of the switch from progenitor- to erythrocyte-specific gene expression. In summary, we identified a TAL1/FUBP1 transcriptional relationship, whose physiological function in haematopoiesis is connected to proper erythropoiesis.
Background: Perioperative anaemia leads to impaired oxygen supply with a risk of vital organ ischaemia. In healthy and fit individuals, anaemia can be compensated by several mechanisms. Elderly patients, however, have less compensatory mechanisms because of multiple co-morbidities and age-related decline of functional reserves. The purpose of the study is to evaluate whether elderly surgical patients may benefit from a liberal red blood cell (RBC) transfusion strategy compared to a restrictive transfusion strategy.
Methods: The LIBERAL Trial is a prospective, randomized, multicentre, controlled clinical phase IV trial randomising 2470 elderly (≥ 70 years) patients undergoing intermediate- or high-risk non-cardiac surgery. Registered patients will be randomised only if Haemoglobin (Hb) reaches ≤9 g/dl during surgery or within 3 days after surgery either to the LIBERAL group (transfusion of a single RBC unit when Hb ≤ 9 g/dl with a target range for the post-transfusion Hb level of 9–10.5 g/dl) or the RESTRICTIVE group (transfusion of a single RBC unit when Hb ≤ 7.5 g/dl with a target range for the post-transfusion Hb level of 7.5–9 g/dl). The intervention per patient will be followed until hospital discharge or up to 30 days after surgery, whichever occurs first. The primary efficacy outcome is defined as a composite of all-cause mortality, acute myocardial infarction, acute ischaemic stroke, acute kidney injury (stage III), acute mesenteric ischaemia and acute peripheral vascular ischaemia within 90 days after surgery. Infections requiring iv antibiotics with re-hospitalisation are assessed as important secondary endpoint. The primary endpoint will be analysed by logistic regression adjusting for age, cancer surgery (y/n), type of surgery (intermediate- or high-risk), and incorporating centres as random effect.
Discussion: The LIBERAL-Trial will evaluate whether a liberal transfusion strategy reduces the occurrence of major adverse events after non-cardiac surgery in the geriatric population compared to a restrictive strategy within 90 days after surgery.
Trial registration: ClinicalTrials.gov (identifier: NCT03369210).
(1) Background: Refractory acute graft-versus-host disease (R-aGvHD) remains a leading cause of death after allogeneic stem cell transplantation. Survival rates of 15% after four years are currently achieved; deaths are only in part due to aGvHD itself, but mostly due to adverse effects of R-aGvHD treatment with immunosuppressive agents as these predispose patients to opportunistic infections and loss of graft-versus-leukemia surveillance resulting in relapse. Mesenchymal stromal cells (MSC) from different tissues and those generated by various protocols have been proposed as a remedy for R-aGvHD but the enthusiasm raised by initial reports has not been ubiquitously reproduced.
(2) Methods: We previously reported on a unique MSC product, which was generated from pooled bone marrow mononuclear cells of multiple third-party donors. The products showed dose-to-dose equipotency and greater immunosuppressive capacity than individually expanded MSCs from the same donors. This product, MSC-FFM, has entered clinical routine in Germany where it is licensed with a national hospital exemption authorization. We previously reported satisfying initial clinical outcomes, which we are now updating. The data were collected in our post-approval pharmacovigilance program, i.e., this is not a clinical study and the data is high-level and non-monitored.
(3) Results: Follow-up for 92 recipients of MSC-FFM was reported, 88 with GvHD ≥°III, one-third only steroid-refractory and two-thirds therapy resistant (refractory to steroids plus ≥2 additional lines of treatment). A median of three doses of MSC-FFM was administered without apparent toxicity. Overall response rates were 82% and 81% at the first and last evaluation, respectively. At six months, the estimated overall survival was 64%, while the cumulative incidence of death from underlying disease was 3%.
(4) Conclusions: MSC-FFM promises to be a safe and efficient treatment for severe R-aGvHD.
Hematopoietic differentiation is driven by transcription factors, which orchestrate a finely tuned transcriptional network. At bipotential branching points lineage decisions are made, where key transcription factors initiate cell type-specific gene expression programs. These programs are stabilized by the epigenetic activity of recruited chromatin-modifying cofactors. An example is the association of the transcription factor RUNX1 with protein arginine methyltransferase 6 (PRMT6) at the megakaryocytic/erythroid bifurcation. However, little is known about the specific influence of PRMT6 on this important branching point. Here, we show that PRMT6 inhibits erythroid gene expression during megakaryopoiesis of primary human CD34+ progenitor cells. PRMT6 is recruited to erythroid genes, such as glycophorin A. Consequently, a repressive histone modification pattern with high H3R2me2a and low H3K4me3 is established. Importantly, inhibition of PRMT6 by shRNA or small molecule inhibitors leads to upregulation of erythroid genes and promotes erythropoiesis. Our data reveal that PRMT6 plays a role in the control of erythroid/megakaryocytic differentiation and open up the possibility that manipulation of PRMT6 activity could facilitate enhanced erythropoiesis for therapeutic use.
Hematopoietic differentiation is controlled by key transcription factors, which regulate stem cell functions and differentiation. TAL1 is a central transcription factor for hematopoietic stem cell development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation. Knowledge of the target genes controlled by a given transcription factor is important to understand its contribution to normal development and disease. To uncover direct target genes of TAL1 we used high affinity streptavidin/biotin-based chromatin precipitation (Strep-CP) followed by Strep-CP on ChIP analysis using ChIP promoter arrays. We identified 451 TAL1 target genes in K562 cells. Furthermore, we analysed the regulation of one of these genes, the catalytic subunit beta of protein kinase A (PRKACB), during megakaryopoiesis of K562 and primary human CD34+ stem cell/progenitor cells. We found that TAL1 together with hematopoietic transcription factors RUNX1 and GATA1 binds to the promoter of the isoform 3 of PRKACB (Cβ3). During megakaryocytic differentiation a coactivator complex on the Cβ3 promoter, which includes WDR5 and p300, is replaced with a corepressor complex. In this manner, activating chromatin modifications are removed and expression of the PRKACB-Cβ3 isoform during megakaryocytic differentiation is reduced. Our data uncover a role of the TAL1 complex in controlling differential isoform expression of PRKACB. These results reveal a novel function of TAL1, RUNX1 and GATA1 in the transcriptional control of protein kinase A activity, with implications for cellular signalling control during differentiation and disease.
Background: Red blood cell (RBC) depletion is a standard graft manipulation technique for ABO-incompatible bone marrow (BM) transplants. The BM processing module for Spectra Optia, “BMC”, was previously introduced. We here report the largest series to date of routine quality data after performing 50 clinical-scale RBC-depletions.
Methods: Fifty successive RBC-depletions from autologous (n = 5) and allogeneic (n = 45) BM transplants were performed with the Spectra Optia BMC apheresis suite. Product quality was assessed before and after processing for volume, RBC and leukocyte content; RBC-depletion and stem cell (CD34+ cells) recovery was calculated there from. Clinical engraftment data were collected from 26/45 allogeneic recipients.
Results: Median RBC removal was 98.2% (range 90.8–99.1%), median CD34+ cell recovery was 93.6%, minimum recovery being 72%, total product volume was reduced to 7.5% (range 4.7–23.0%). Products engrafted with expected probability and kinetics. Performance indicators were stable over time.
Discussion: Spectra Optia BMC is a robust and efficient technology for RBC-depletion and volume reduction of BM, providing near-complete RBC removal and excellent CD34+ cell recovery.
MiR144/451 expression is repressed by RUNX1 during megakaryopoiesis and disturbed by RUNX1/ETO
(2016)
Abstract: A network of lineage-specific transcription factors and microRNAs tightly regulates differentiation of hematopoietic stem cells along the distinct lineages. Deregulation of this regulatory network contributes to impaired lineage fidelity and leukemogenesis. We found that the hematopoietic master regulator RUNX1 controls the expression of certain microRNAs, of importance during erythroid/megakaryocytic differentiation. In particular, we show that the erythorid miR144/451 cluster is epigenetically repressed by RUNX1 during megakaryopoiesis. Furthermore, the leukemogenic RUNX1/ETO fusion protein transcriptionally represses the miR144/451 pre-microRNA. Thus RUNX1/ETO contributes to increased expression of miR451 target genes and interferes with normal gene expression during differentiation. Furthermore, we observed that inhibition of RUNX1/ETO in Kasumi1 cells and in RUNX1/ETO positive primary acute myeloid leukemia patient samples leads to up-regulation of miR144/451. RUNX1 thus emerges as a key regulator of a microRNA network, driving differentiation at the megakaryocytic/erythroid branching point. The network is disturbed by the leukemogenic RUNX1/ETO fusion product.
Author Summary: The regulatory network between transcription factors, epigenetic cofactors and microRNAs is decisive for normal hematopoiesis. The transcription factor RUNX1 is important for the establishment of a megakaryocytic gene expression program and the concomitant repression of erythroid genes during megakaryocytic differentiation. Gene regulation by RUNX1 is frequently disturbed by mutations and chromosomal translocations, such as the t(8;21) translocation, which gives rise to the leukemogenic RUNX1/ETO fusion protein. We found that RUNX1 regulates microRNAs, which are of importance at the megakaryocytic/erythroid branching point. Specifically, RUNX1 down-regulates expression of the microRNA cluster miR144/451 during megakaryocytic differentiation by changing the epigenetic histone modification pattern at the locus. We could show that miR451, one of the micorRNAs of the miR144/451 cluster, supports erythroid differentiation. We found that expression of miR451 is repressed by the RUNX1/ETO fusion protein, resulting in up regulation of miR451 target genes. Our data support the notion that RUNX1 suppresses the erythroid gene expression program including the erythroid microRNA miR451 and that the RUNX1/ETO fusion protein interferes with normal gene regulation by RUNX1.
Background aims: Immunomagnetic enrichment of CD34+ hematopoietic “stem” cells (HSCs) using paramagnetic nanobead coupled CD34 antibody and immunomagnetic extraction with the CliniMACS plus system is the standard approach to generating T-cell-depleted stem cell grafts. Their clinical beneficence in selected indications is established. Even though CD34+ selected grafts are typically given in the context of a severely immunosuppressive conditioning with anti-thymocyte globulin or similar, the degree of T-cell depletion appears to affect clinical outcomes and thus in addition to CD34 cell recovery, the degree of T-cell depletion critically describes process quality. An automatic immunomagnetic cell processing system, CliniMACS Prodigy, including a protocol for fully automatic CD34+ cell selection from apheresis products, was recently developed. We performed a formal process validation to support submission of the protocol for CE release, a prerequisite for clinical use of Prodigy CD34+ products.
Methods: Granulocyte-colony stimulating factor–mobilized healthy-donor apheresis products were subjected to CD34+ cell selection using Prodigy with clinical reagents and consumables and advanced beta versions of the CD34 selection software. Target and non-target cells were enumerated using sensitive flow cytometry platforms.
Results: Nine successful clinical-scale CD34+ cell selections were performed. Beyond setup, no operator intervention was required. Prodigy recovered 74 ± 13% of target cells with a viability of 99.9 ± 0.05%. Per 5 × 10E6 CD34+ cells, which we consider a per-kilogram dose of HSCs, products contained 17 ± 3 × 10E3 T cells and 78 ± 22 × 10E3 B cells.
Conclusions: The process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.
The genetics responsible for the inter-individually variable G-CSF responsiveness remain elusive. A single nucleotide polymorphism (SNP) in the 3’UTR of CXCL12, rs1801157, was implicated in X4-tropic HiV susceptibility and later, in two small studies, in G-CSR responsiveness in patients and donors. The position of the SNP in the 3’UTR together with in-silico predictions suggested differential binding of micro-RNA941 as an underlying mechanism. In a cohort of 515 healthy stem cell donors we attempted to reproduce the correlation of the CXCL12 3’UTR SNP and mobilization responses and tested the role of miR941 in this context. The SNP was distributed with the expected frequency. Mobilization efficiency for CD34+ cells in WT, heterozygous and homozygous SNP individuals was indistinguishable, even after controlling for gender. miR941 expression in non-hematopoietic bone marrow cells was undetectable and miR941 did not interact with the 3’ UTR of CXCL12. Proposed effects of the SNP rs1801157 on G-CSF responsiveness cannot be confirmed in a larger cohort.
nefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.
The decision in September 2011 in the UK to accept blood donations from non-practicing men who have sex with men (MSM) has received significant public attention. Will this rule change substantially boost the number of blood donations or will it make our blood less safe? Clearly, most European countries have a blood procurement problem. Fewer young people are donating, while the population is aging and more invasive therapies are requiring more blood. Yet if that was the reason for allowing non-practicing MSM to donate, clearly re-admission of some other, much larger populations that are currently deferred from donation should likewise be considered. As far as risks for blood safety are concerned, evidence has been provided that the current quality of infectious disease marker testing significantly mitigates against, although does not completely eradicate, risks associated with admission of donors with a high risk of carrying certain blood-transmissible agents. However, it could be argued that more effective recruitment of the non-donor pool, which is substantially larger than the group of currently ineligible donors, would be a better strategy. Recruitment of this group will benefit the availability of blood without jeopardizing the current excellent safety profile of blood.