Aberrant GPA expression and regulatory function of red blood cells in sickle cell disease.

ABSTRACT: Glycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid-binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), the GPA of RBCs undergo structural changes that affect this interaction. Peripheral blood samples from patients with SCD before and after RBC transfusions were probed for neutrophil and monocyte activation markers and analyzed by fluorescence-activated cell sorting (FACS). RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were coincubated with healthy RBCs (RBCH), GPA-altered RBCs, or GPA-overexpressing (OE) cells and analyzed using FACS. Monocyte CD63 and neutrophil CD66b from patients with SCD at baseline were increased 47% and 27%, respectively, as compared with HS (P = .0017, P = .0162). After transfusion, these markers were suppressed by 22% and 17% (P = .0084, P = .0633). GPA expression in RBCSCD was 38% higher (P = .0291) with decreased Siglec-9 binding compared with RBCH (0.0266). Monocyte CD63 and neutrophil CD66b were suppressed after incubation with RBCH and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction after lipopolysaccharide challenge was restored fully with exposure to RBCH, but not with RBCSCD, from patients in pain crisis, or with RBCH with altered GPA. Pretransfusion RBCSCD do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion, likely by reestablished GPA-Siglec-9 interactions.

Local manufacturing processes contribute to variability in human mesenchymal stromal cell expansion while growth media supplements contribute to variability in gene expression and cell function: a Biomedical Excellence for Safer Transfusion (BEST) collaborative study.

BACKGROUND AIMS: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow-MSC aliquots derived from the same donor source material yet with different growth media. METHODS: Eight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements. The expanded cells were taken through two passages then assessed for cell count, viability, doubling time, immunophenotype, cell function, immunosuppression and gene expression. Results were analyzed by growth media and by center. RESULTS: Center methodologies varied by their local seeding density, feeding regimen, inoculation density, base media and other growth media features (antibiotics, glutamine, serum). Doubling times were more dependent on center than on media supplements. Two centers had appropriate immunophenotyping showing all MSC cultures were positive for CD105, CD73, CD90 and negative for CD34, CD45, CD14, HLA-DR. MSCs cultured in media supplemented with FBS compared with HPL featured greater T-cell inhibition potential. Gene expression analysis showed greater impact of the type of media supplement (HPL versus FBS) than the manufacturing center. Specifically, nine genes were decreased in expression and six increased when combining the four HPL-grown MSCs versus FBS (false discovery rate [FDR] <0.01), however, without significant difference between different sources of HPL (FDR <0.01). CONCLUSIONS: Local manufacturing process plays a critical role in MSC expansion while growth media may influence function and gene expression. All HPL and FBS products supported cell growth.

Assessing deviations for HPCs obtained during COVID-19 (ADHOC): Evaluating impact of the COVID-19 pandemic on cellular therapy products and processes, the BEST collaborative study.

BACKGROUND: The success of allogeneic hematopoietic stem cell transplantation is dependent on a world-wide network of collection centers providing donations that predominantly have been infused as fresh cells. The logistics chain that supports the just-in-time delivery model for stem cell and immunotherapy products was severely stressed by the COVID pandemic, and in early 2020 a number of national and international bodies recommended that cells should be cryopreserved at the collection or transplant center to avoid interruptions in their acquisition or delivery to patients who had started conditioning. STUDY DESIGN: To assess the potential consequences of such pandemic-related deviations to normal practice, we surveyed nine international laboratories to determine if the characteristics or transplant outcomes of allogeneic stem cell donations differed in the immediate periods before and after the switch to routine cryopreservation. RESULTS: Nine centers on two continents provided data for 72 HSC donations just before, and 71 just after, switching to cryopreservation for allogeneic HSC products. No statistically significant differences between the period before and after cryopreservation were noted for time from product collection to receipt, product temperature at receipt, or CD34+ cell viability at receipt. There was an indication of slower absolute neutrophil count recovery after cryopreservation was required (mean time of 15 vs. 17.6 days). DISCUSSION: While there were no apparent changes to most parameters studied, there was an indication of slower neutrophil engraftment that will need to be examined in larger, longer term studies.

Hospital red blood cell and platelet supply and utilization from March to December of the first year of the COVID-19 pandemic: The BEST collaborative study.

BACKGROUND: At the start of the coronavirus disease 2019 (COVID-19) pandemic, widespread blood shortages were anticipated. We sought to determine how hospital blood supply and blood utilization were affected by the first wave of COVID-19. STUDY DESIGN AND METHODS: Weekly red blood cell (RBC) and platelet (PLT) inventory, transfusion, and outdate data were collected from 13 institutions in the United States, Brazil, Canada, and Denmark from March 1st to December 31st of 2020 and 2019. Data from the sites were aligned based on each site's local first peak of COVID-19 cases, and data from 2020 (pandemic year) were compared with data from the corresponding period in 2019 (pre-pandemic baseline). RESULTS: RBC inventories were 3% lower in 2020 than in 2019 (680 vs. 704, p < .001) and 5% fewer RBCs were transfused per week compared to 2019 (477 vs. 501, p < .001). However, during the first COVID-19 peak, RBC and PLT inventories were higher than normal, as reflected by deviation from par, days on hand, and percent outdated. At this time, 16% fewer inpatient beds were occupied, and 43% fewer surgeries were performed compared to 2019 (p < .001). In contrast to 2019 when there was no correlation, there was, in 2020, significant negative correlations between RBC and PLT days on hand and both percentage occupancy of inpatient beds and percentage of surgeries performed. CONCLUSION: During the COVID-19 pandemic in 2020, RBC and PLT inventories remained adequate. During the first wave of cases, significant decreases in patient care activities were associated with excess RBC and PLT supplies and increased product outdating.

Multicenter evaluation of the IL-3-pSTAT5 assay to assess the potency of cryopreserved stem cells from cord blood units: The BEST Collaborative study.

BACKGROUND: The IL-3-pSTAT5 assay, a new, rapid, and standardized flow-cytometry-based assay may compensate for several limitations of the colony-forming unit (CFU) assay typically used for stem cell potency assessments of cord blood units (CBU). We performed an inter-laboratory evaluation of the performance of this new assay. STUDY DESIGN AND METHODS: This Biomedical Excellence for Safer Transfusion (BEST) Collaborative multicenter, international study included 15 participants from public cord blood banks (CBBs), CBB-supporting research laboratories, and stem cell laboratories. To perform the IL-3-pSTAT5 assay, participating centers received reagents, instructions, and 10 blind CBU samples, including eight normal samples and two samples exposed to a transient warming event. We measured inter-laboratory agreement qualitatively (proportion of correctly classified samples) and quantitatively (coefficient of variation [CV], correlation coefficients, receiver operating characteristics (ROC) curve, and intraclass correlation coefficient [ICC]). RESULTS: The qualitative agreement was 97.3% (i.e., 107/110; Fleiss' kappa = 0.835). The average CV on a per-sample basis was 11.57% among all samples, 8.99% among normal samples, and on a per-center basis was 9.42% among normal samples. In a correlation matrix that compared results across centers, the mean Pearson's correlation coefficient was 0.88 (standard deviation = 0.04). The ICC was 0.83 (95% confidence interval = 0.68-0.95). The area under the curve (AUC) from the ROC curve was 0.9974. DISCUSSION: Excellent qualitative and quantitative agreement was exhibited across laboratories. The IL-3-pSTAT5 assay may therefore be implemented in flow cytometry laboratories to rapidly and reliably provide standardized measures of stem cell potency in CBUs.

Lowering platelet count threshold to 10,000/µL for peripherally inserted central catheter placement safely conserves blood products.

Despite the low risk of peripherally inserted central catheter (PICC) insertion-related bleeding, the practice of administering prophylactic platelets varies greatly. Limiting unnecessary blood product transfusions reduces transfusion-related adverse events, financial cost, and delays in care. We assessed the impact of lowering prophylactic platelet administration threshold on blood product utilization patterns and bleeding events. This quasi-experimental study was conducted in an urban academic tertiary medical center. The study population included patients with platelet counts ≥ 10,000/µL and < 50,000/µL undergoing PICC placement in 2018 and 2019 when the minimum platelet thresholds were 50,000/µL and 10,000/µL, respectively. The primary outcome was blood product utilization and the secondary outcome was PICC insertion-related bleeding complications. Thirty-five patients using the 10,000/µL (10 K) platelet threshold and 46 patients using the 50,000/µL (50 K) platelet threshold were enrolled. The 50 K group received more platelets before PICC insertion (0.870 ± 0.885 and 0.143 ± 0.430 pools of platelets-per-person, p < 0.001). No patients experienced clinically significant bleeding. Immediately following PICC insertion, minor bleeding occurred in five patients (two [4.3%] and three [8.6%] in the 50 K and 10 K groups, respectively). Bleeding rates between the two cohorts did not differ (p = 0.647). Lowering the minimum platelet threshold from 50,000/µL to 10,000/µL resulted in less prophylactic platelet and total blood product administration with no appreciable difference in PICC insertion-related bleeding.

Kinetics of SARS-CoV-2 antibody responses pre-COVID-19 and post-COVID-19 convalescent plasma transfusion in patients with severe respiratory failure: an observational case-control study.

AIMS: While the SARS-CoV-2 pandemic may be contained through vaccination, transfusion of convalescent plasma (CCP) from individuals who recovered from COVID-19 (CCP) is considered an alternative treatment. We investigate if CCP transfusion in patients with severe respiratory failure increases plasma titres of SARS-CoV-2 antibodies and improves clinical outcomes. METHODS: Patients with COVID-19 (n=34) were consented for CCP transfusion and serial blood draws pretransfusion and post-transfusion. Plasma SARS-CoV-2 antireceptor binding domain (RBD) IgG and IgM titres were measured by ELISA serially, and compared with serial plasma titre levels from control patients (n=68). The primary outcome was survival at 30 days, and secondary outcomes were length of ventilator and/or extracorporeal membrane oxygenation (ECMO) support, length of stay (LOS) in the hospital and in the intensive care unit (ICU). Outcomes were compared with matched control patients (n=34). Kinetics of antibodies and clinical outcomes were compared using LOess regression and ORs, respectively. RESULTS: Prior to CCP transfusion, 74% of patients were anti-RBD seropositive for IgG (median 1:3200), and 81% were anti-RBD IgM seropositive (median 1:320), while 16% were seronegative. The kinetics of antibody titres in CCP recipients were similar to controls. CCP recipients presented with similar survival, duration on ventilatory and/or ECMO support, as well as ICU and hospital LOS compared with controls. CONCLUSIONS: CCP transfusion did not increase the kinetics of SARS-CoV2 antibodies and did not result in improved clinical outcomes in patients with COVID-19 with severe respiratory failure, suggesting that CCP may not be indicated in this category of patients.

Autoantibodies to red blood cell surface Glycophorin A impact the activation poise of circulating leukocytes.

BACKGROUND: Both M and N alleles encode antigens on Glycophorin A (GPA), a red blood cell (RBC) surface sialoglycoprotein. Interaction between RBC GPA and leukocyte surface lectins may downregulate their activation. The current study investigates if RBC autoantibodies against GPA, such as auto-anti-M/N, prime an activated phenotype in peripheral blood leukocytes. METHODS: Leukocyte activation was assessed in whole blood from patients with auto-anti-GPA (anti-M/N) and compared to those with allo-anti-M/N and healthy subjects. Control samples from healthy subjects with no antibodies incubated in vitro with either anti-GPA or anti-Rh were analyzed for neutrophil and monocyte surface activation marker expression, reactive oxygen species (ROS) content, and formation of aggregates with RBCs. Samples incubated with an IgG1 isotype antibody served as controls. RESULTS: Ex vivo, neutrophil CD66b and monocyte CD63 surface expression was increased in patients with auto-anti-M/N compared to those with allo anti-M/N (p = .1757; p = .0698) and to healthy subjects (p = .0186; p = .013). In vitro, neutrophil CD66b and monocyte CD63 surface expression was increased following incubation with anti-GPA compared to anti-Rh (p = .0003; p = .0328) and isotype control (p = .000; p = .0062). Intracellular ROS content increased in both neutrophils and monocytes incubated with anti-GPA compared to anti-Rh (p = .0012; p = .0693) and isotype control (p = .001; p = .0021). Percentage of neutrophil-RBC aggregates was decreased when incubated with anti-GPA compared to isotype control (p < .01). CONCLUSIONS: Neutrophils and monocytes in peripheral blood exposed to an antibody directed against GPA on RBC surfaces, such as M or N antigens, may be primed towards an activated phenotype.

Limitations of current practices in detection of bacterially contaminated blood products associated with suspected septic transfusion reactions.

BACKGROUND: In the setting of suspected septic transfusion reactions, bacterial culture of both the transfused patient and the residual blood component is recommended. Primary bacterial contamination can occur at the time of component collection. Clinically insignificant "secondary contamination" can occur during post-transfusion component discard, retrieval for culture, or manipulation of the bag at the time of culture sampling. STUDY DESIGN AND METHODS: This retrospective, multi-center study analyzes positive residual component culture results and companion patient blood cultures from 15 hospitals, 1 blood center, and all cultured transfusion reactions within the province of Quebec, Canada, over a 5-year period. Imputability was assigned as "definite" (concordant growth), "possible" (discordant growth or lack of growth in patient culture), or "unable to assess" (patient not cultured). RESULTS: There were 373 positive component cultures from 360 unique transfusion reactions, with 276 (76.7%) companion patient blood cultures performed, of which 10 (2.8%) yielded the pathogen detected in the positive component. Of these 10 definite pathogens, 7 (2 Staphylococcus aureus, 3 other staphylococci, and 1 Streptococcus pyogenes and 1 Bacillus sp.) were associated with platelet and 3 (Aeromonas veronii, Staphylococcus epidermidis, and Enterococcus faecalis) with RBC transfusions. RBC and plasma components comprised 70% of positive component cultures. DISCUSSION: The process of performing residual component culture is vulnerable to secondary contamination. The significance of microorganisms recovered from component culture cannot be interpreted in isolation. In the context of low prevalence of primary contamination of blood components, the positive predictive value of a positive component culture result is very low.

When O Bites Back: Unrecognized Acute Hemolytic Reaction from Out-of-Group HLA-Compatible Platelet Transfusion.

Managing a platelet blood product inventory in a hospital-based transfusion service (TS) is challenging. Thus, to optimize platelet inventory availability and to prevent excess outdating, most tertiary care center-based TSs do not require ABO-identical platelet (PLT) transfusions. To mitigate the risk of hemolysis associated with the transfusion of high titer ABO antibody-containing PLT, our institutional policy allows the transfusion of PLT containing ABO-incompatible plasma only if PLT is re-suspended in platelet additive solution (PAS). Despite the steps taken to reduce the risk of hemolytic transfusion reactions to PLT transfusions at our institution, our center has observed hemolytic reactions to PLT in PAS. The current case study highlights the importance of recognizing a hemolytic reaction (HTR) from ABO-incompatible PLT transfusions and discusses the current strategies and recommendations to mitigate this risk.

Passive Immunity Trial for Our Nation (PassITON): study protocol for a randomized placebo-control clinical trial evaluating COVID-19 convalescent plasma in hospitalized adults.

BACKGROUND: Convalescent plasma is being used widely as a treatment for coronavirus disease 2019 (COVID-19). However, the clinical efficacy of COVID-19 convalescent plasma is unclear. METHODS: The Passive Immunity Trial for Our Nation (PassITON) is a multicenter, placebo-controlled, blinded, randomized clinical trial being conducted in the USA to provide high-quality evidence on the efficacy of COVID-19 convalescent plasma as a treatment for adults hospitalized with symptomatic disease. Adults hospitalized with COVID-19 with respiratory symptoms for less than 14 days are eligible. Enrolled patients are randomized in a 1:1 ratio to 1 unit (200-399 mL) of COVID-19 convalescent plasma that has demonstrated neutralizing function using a SARS-CoV-2 chimeric virus neutralization assay. Study treatments are administered in a blinded fashion and patients are followed for 28 days. The primary outcome is clinical status 14 days after study treatment as measured on a 7-category ordinal scale assessing mortality, respiratory support, and return to normal activities of daily living. Key secondary outcomes include mortality and oxygen-free days. The trial is projected to enroll 1000 patients and is designed to detect an odds ratio ≤ 0.73 for the primary outcome. DISCUSSION: This trial will provide the most robust data available to date on the efficacy of COVID-19 convalescent plasma for the treatment of adults hospitalized with acute moderate to severe COVID-19. These data will be useful to guide the treatment of COVID-19 patients in the current pandemic and for informing decisions about whether developing a standardized infrastructure for collecting and disseminating convalescent plasma to prepare for future viral pandemics is indicated. TRIAL REGISTRATION: ClinicalTrials.gov NCT04362176 . Registered on 24 April 2020.

Passive Immunity Trial for Our Nation (PassITON): study protocol for a randomized placebo-control clinical trial evaluating COVID-19 convalescent plasma in hospitalized adults.

Background: Convalescent plasma is being used widely as a treatment for coronavirus disease 2019 (COVID-19). However, the clinical efficacy of COVID-19 convalescent plasma is unclear. Methods: The Pass ive I mmunity T rial for O ur N ation (PassITON), is a multicenter, placebo-controlled, blinded, randomized clinical trial being conducted in the United States to provide high-quality evidence on the efficacy of COVID-19 convalescent plasma as a treatment for adults hospitalized with symptomatic disease. Adults hospitalized with COVID-19 with respiratory symptoms for less than 14 days are eligible. Enrolled patients are randomized in a 1:1 ratio to 1 unit (200-399 mL) of COVID-19 convalescent plasma that has demonstrated neutralizing function using a SARS-CoV-2 chimeric virus neutralization assay. Study treatments are administered in a blinded fashion and patients are followed for 28 days. The primary outcome is clinical status 14 days after study treatment as measured on a 7-category ordinal scale assessing mortality, respiratory support, and return to normal activities of daily living. Key secondary outcomes include mortality and oxygen-free days. The trial is projected to enroll 1000 patients and is designed to detect an odds ratio ≤ 0.73 for the primary outcome. Discussion: This trial will provide the most robust data available to date on the efficacy of COVID-19 convalescent plasma for the treatment of adults hospitalized with acute moderate to severe COVID-19. These data will be useful to guide the treatment of COVID-19 patients in the current pandemic and for informing decisions about whether developing a standardized infrastructure for collecting and disseminating convalescent plasma to prepare for future viral pandemics is indicated. Trial Registration: ClinicalTrials.gov: NCT04362176. Date of trial registration: April 24, 2020, https://clinicaltrials.gov/ct2/show/NCT04362176.

Educational and Electronic-Based Tools to Mitigate the Risk of Transfusion Adverse Events.

ABSTRACT: The transfusion of blood products is a widely used practice but comes with the risk of transfusion-associated adverse events and fatalities. The primary aim of this study was to evaluate if strict adherence to transfusion guidelines would lead to a decrease in the rate of transfusion reactions that occurred when blood products were given outside of established indications. Hospital-wide educational programs and dedicated electronic transfusion order sets were used to encourage adherence to guidelines. A secondary aim of this study was to evaluate if a decrease in the incidence of transfusion reactions also lead to a decrease in associated healthcare costs.

Human Mesenchymal Stromal Cell (MSC) Characteristics Vary Among Laboratories When Manufactured From the Same Source Material: A Report by the Cellular Therapy Team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

BACKGROUND: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods and different source materials. The purpose of this study was to assess the impact on MSC characteristics when different laboratories propagated MSCs from cultures initiated with BM aliquots derived from the same donor source material. METHODS AND METHODS: Five aliquots from each of three different BM donors were distributed to five independent laboratories. Three laboratories plated whole BM and two laboratories a mononuclear BM cell fraction. Four laboratories cultured in media supplemented with fetal bovine serum (FBS) and one laboratory used human platelet lysate (hPL). Initial cell seeding densities (i.e., P0) ranged from 19.7 × 103/cm2-282 × 103/cm2 and for second seeding (i.e., P1) 0.05 × 103-5.1 × 103 cells/cm2. Post-thawed MSCs from each laboratory were analyzed for cell viability, immunophenotype, tri-lineage differentiation, fibroblast colony-forming units (CFU-F), gene expression, and immunosuppressive activity. RESULTS: Transit times from BM collection to receipt by laboratories located in the United States ranged from 16.0-30.0 h and from 41.5-71.5 h for a laboratory in Asia. Post-thaw culture derived MSCs rom BM #1, #2, and #3 exhibited viabilities that ranged from 74-92%, 61-96%, and 23-90%, respectively. CFU activity from BM #1, #2, and #3 per 200 MSCs plated averaged 45.1 ± 21.4, 49.3 ± 26.8 and 14.9 ± 13.3, respectively. No substantial differences were observed in immunophenotype, and immunosuppressive activities. Global gene expression profiles of MSCs revealed transcriptome differences due to different inter-laboratory methods and to donor source material with the center effects showing greater molecular differences than source material. CONCLUSION: Functional and molecular differences exist among MSCs produced by different centers even when the same BM starting material is used to initiate cultures. These results indicated that manufacturing of MSCs by five independent centers contributed more to MSC variability than did the source material of the BM used in this study. Thus, emphasizing the importance of establishing worldwide standards to propagate MSCs for clinical use.

A multicenter evaluation of heterogeneity in cellular therapy processing laboratory procedure times to assess workload capacity.

BACKGROUND: Growth in size and complexity of clinical hematopoietic progenitor cell (HPC) transplant programs necessitates parallel increases in cellular therapy laboratory (CTL) workload. Typically individually developed, HPC product processing is labor and time intensive. Variation in procedure type and numbers across CTLs complicates direct comparisons, and benchmark data are not readily available. STUDY DESIGN AND METHODS: Studies were undertaken at seven CTLs. Transplant volume and staff numbers were determined. Staff recorded time performing tasks broken down into steps: paperwork, product acceptance, transport/infusion, processing, and cryopreservation. Times were added to obtain total times for 15 common CTL procedures. RESULTS: Annual transplant volume ranged from 53.4 to 463.2, with products processed by a range of 2 to 10 dedicated CTL staff. Paperwork time constituted 23.7% to 62.3% total time; product processing time accounted for 1.8 (for National Marrow Donor Program product receipt) to 62.6% (for red blood cell reduction of allogeneic HPC products from bone marrow) of total processing time. Mean time for 15 procedures ranged from 1.27 to 8.28 hours (standard deviation range, 0.35-2.71 hr). Mean time for products from bone marrow versus peripheral blood was 6.6 ± 2.0 versus 5.5 ± 1.1 hours (p = 0.02). Cryopreservation (6.5 ± 1.6 vs. 4.4 ± 0.85 hr; p < 0.01) and manipulation (6.4 ± 1.5 vs. 4.4 ± 0.85 hr; p < 0.01) added time. CONCLUSION: CTL procedures are time intensive, with wide intra- and inter-CTL variation. Paperwork accounted for substantial portion of total time across procedures. Bone marrow source, cryopreservation, and manipulation contributed to longer times. These findings provide concrete data on which to build regarding CTL workload capacity.