Apheresis Unit Research Articles

Establishing a robust chimeric antigen receptor T-cell therapy program in Australia: the Royal Prince Alfred Hospital experience

>himeric antigen receptor (CAR) T-cell therapy is a novel approved cancer treatment that has shown remarkable efficacy in the treatment of patients with relapsed leukemia and lymphoma. Implementation of CAR T-cell therapy in a hospital setting requires careful and detailed planning because of the complexities in delivering this specialist service. A multi-disciplinary approach with dedicated funding is required to meet clinical, scientific, logistic and regulatory requirements. Tisagenlecleucel was the first approved CAR T-cell therapy in Australia. The treatment has been made available to Australian patients in specialist public hospitals through federal and state funding. Royal Prince Alfred Hospital (RPAH) is one of Australia's oldest tertiary referral public health care institutions and was approved for the provision of CAR T-cell therapy service in 2019. A multi-disciplinary clinical program has been established for the collection and cryopreservation of donor cells shipped for manufacturing as well as for the receipt, storage and administration of CAR T-cell therapy and patient management. The program encompasses a Therapeutic Goods Administration-accredited apheresis unit and a state-of-the-art facility for cell processing, cryopreservation and storage. The program's clinical expertise extends to hematology, oncology, intensive care, pharmacy, neurology and radiology services with direct experience in managing patients receiving CAR T-cell therapies. The introduction of CAR T-cell therapies at RPAH was a complex undertaking facilitated by the existing infrastructure and clinical expertise.

Assessing the performance of a plateletpheresis unit at a tertiary-care academic medical center in Mexico: A 6-year experience.

Optimization of platelet (PLT) apheresis collection is a priority to satisfy the increasing demand of hemato-oncology patients. We assessed the performance of a plateletpheresis unit supporting hematology patients. This descriptive retrospective study included 561 plateletpheresis collections from 2013 to 2018. For data analysis, descriptive statistics and receiver operating characteristic (ROC) curve were used. A 5-item satisfaction questionnaire was analyzed. Ninety percent of the donors were males. The median plateletpheresis time was 89 minutes; its success rate was 92.5%; median donor PLT count was 232 × 109 /L, women median PLT count was 247 × 109 /L vs 231x109 /L in men (P=.017). Seventy-seven percent donors were candidates for a double product and 24.5% were processed; 20.8% of these donors had a weight ≤75 and 79.2% >75 kg, P=.003, and 6.6% were women and 93.4% men, P=.161. Thirty-six of donors had ≥250 × 109 /L and 16.8% was processed as a triple product. ROC analysis showed that with donor PLT counts ≥200 × 109 /L the sensitivity for obtaining double products was 0.981 and specificity 0.714, with an area under the curve (AUC)=0.877. The adverse effect rate was 4.3%. Of the potential donors, 6.3% were rejected. The cost of processing single or double products was 430 USD. Comfort and time spent during plateletpheresis were areas for improvement. Platelet count and donor weight predicted PLT yield and obtaining double products. Women had higher PLT counts, but no significant difference was found between donor gender and processed products. Assessment of the apheresis unit can help to improve its performance.

Donor Selection and Deferral Pattern in Convalescent Plasma Donor in Plasmapheresis Unit of a Tertiary Care Hospital

Context: The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in Wuhan, China, in December 2019. The management plan is supportive care with oxygen supplementation and mechanical ventilation. US FDA approved convalescent plasma (CP) for COVID-19 for clinical trials and as an emergency investigational new drug. Although numerous trials are currently investigating the safety and efficacy of CP in COVID-19 patients, there is a paucity of ongoing and published studies evaluating the CP donors' side. This retrospective study reports the CP donors' selection and deferrals. Aim: To evaluate and analyze the donor deferral pattern and its causes among CP donors in a tertiary care hospital blood bank apheresis unit. Settings and Design: Hospital-based retrospective analysis. Subjects and Methods: Donors aged 18–65 years who had recovered from COVID-19 at least 4 weeks previously coming for plasma donation were retrospectively analyzed from July 25, 2020 to January 24, 2021 for a period of 6 months at blood bank apheresis unit, Assam Medical College and Hospital. Results: A total of 396 potential plasma donors were screened during the study period. The donor deferral rate was 39.1%. The permanent deferral was 36.8%, and the temporary deferral was 63.2%. The maximum number of donors deferred because of low antibody (18.7%) followed by low hemoglobin (14.8%). Associated comorbidities, low platelet count, repeat reverse transcription-polymerase chain reaction positivity, transfusion transmitted infection reactivity, intake of medicines, infections, vaccination, and underweight were other causes identified. Conclusion: The pattern of donor deferral is an important tool for blood safety and also provides key areas to focus on a demographic region or policy formulation for donor selection as well as to ensure donor safety.

Therapeutic plasma exchange in neurological diseases: Eleven years experience at a tertiary care center in Turkey.

Therapeutic plasma exchange (TPE) is an apheresis procedure in which plasma is separated from the blood cellular components ex vivo, allocated, and replaced with another plasma or a plasma-replacing fluid. This study aimed to define the rate of complications and determine TPE distribution in various neurological diseases. Our study is a retrospective analysis of neurologic diseases requiring TPE between 2008 and 2019 that were selected using the medical records of neurology departments and apheresis units database. We performed 1459 TPE procedures on 207 patients between 2008 and 2019. TPE Procedure is most frequently applied in patients with Myasthenia-Gravis syndrome (34.7%). The complication ratio was 1.6% from a total of 1459 TPE procedures. The most commonly specified adverse event was allergic reactions 11 (5.3%), followed by hypotension 6 (2.9%). TPE was safe and tolerable, with manageable complications in experienced hands.

A Novel Process for the Expansion of Functional Human ILC2 Cells for GCHD Prevention

Recently, we have shown that type 2 mouse innate lymphoid cells (ILC2) ameliorate acute GVHD in a mouse model (Bruce et al, 2017). Moreover, it has been shown by us and others that not only is the recovery of ILC2 cells slow in both mouse and humans allogeneic recipients but also the presence of active human ILC2 cells in correlates with lower incidence of acute GVHD. Thus, we sought out to determine if human ILC2 cells could be enriched and expanded in vitro for potential use as a therapy to prevent or treat acute GVHD in humans. Toward this goal, we utilized human peripheral blood cells from apheresis units. We compared three methods to enrich ILC2 cells: RosetteSep kit, EasySep kit and EasySep NK56 kit. In the apheresis unit, ILC2 cells ranged from 0.023-0.062% of the total cells. In comparing the three enrichment methods, the percentage of enriched ILC2 cells varied from 1.1-6.9% with the EasySep ILC2 kit giving the best yields (6.9% and 4.7e4 total ILC2 cells). Interestingly, although the RosetteSep had the lowest percentage enriched, it had a higher yield than the EasySep NK Kit due to the higher yield of total cells (3.4 e6 compared to 4.3 e5 with 3.7 e4 and 2.2e4 ILC2 cells respectively). We next investigated optimal culture conditions. Our initial culture conditions were with α-MEM media with a cytokine cocktail of IL-2, IL-33 and IL-25. Using these conditions, the range of expansion was 500-1500 fold from the initial number of ILC2 at the end of the culture on Day 10. We tested a number of different interventions for optimization: addition of OP9-DL1 as a stromal cell component, addition of IL-7, and addition of TSLP. These results showed that OP9DL1, IL-7 or TSLP did not improve expansion or change the phenotype of the ILC2 cells at the end of the 10 day culture. We also tested different medias (α-MEM and IMDM) with different concentrations of the cytokines (based on the results by Camelo et al, 2017). We found that cells that were enriched using the RosetteSep and then cultured in α-MEM media with 50 ng/ml of IL-33, IL-25 and 20 IU/Ml IL-2 resulted in the best ILC2 expansion (>1000-fold) within 10 days. The final product was >90% pure ILC2 cells (lineage negative, CD161 and Gata-3 expression). These cells produced IL-13 and IL-5 in culture upon stimulation with PMA/ionomycin but did not produce IFN-gamma. Notably, between 50-80% of the ILC2 cells lost expression of CRTH2 (CD294) while in culture, similar to results found by Camelo et al. (2017). A representative experiment is shown in Figure 1.We tested whether injection of human ILC2 cells ameliorated acute GVHD in a humanized mouse model. In this model, PBMCs are used as a positive control for GVHD. Two different approaches were taken. In the first approach we used third party hILC2 cells. On Day 0, irradiated female NSG mice received 15e6 PBMCs +/- 15e6 ILC2 cells. Mice were monitored for GVHD scores and survival. Recipients that received human ILC2 cells had significantly higher percentage mean initial body weight ratios at each time point beginning at Day 8 (P<0.05). At one month, 32% of controls (n=19) had succumbed to GVHD versus 12.5% (n=8) who had received third party human ILC2 cells. In the second approach we used donor-specific hILC2 cells, Irradiated (Day -1) male NSG mice were given 8e6 PBMC with or without donor matched ILC2 cells in 1:1 ratio on day 0. We found a single injection of matched ILC2 cells increased median survival by 10 days increasing survival from 19 days to 29 days for ILC2 treated animals (p<0.01). Taken together, we have discovered a novel method to rapidly expand human ILC2 cells in vitro. We have found that a single dose of hILC2 cells is active to decrease the severity of xenogeneic acute GVHD. These findings provide a rationale basis for the development of hILC2 cells to treat/prevent GI tract GvHD.Figure 1. Apheresis cells were enriched using the ILC2 RosetteSep kit and were cultured in α-MEM media with 50 ng/ml of IL-33, IL-25 and 20 IU/Ml IL-2 for 10 days. The predominant population was ILC2 cells (A) that expressed GATA3 (B; ILC2 shown in red, negative control in blue) and upon stimulation with PMA and ionomycin expressed IL-13 but not IFN-gamma (C). The cells expanded >1000 fold in 10 days (D).Figure 2. Survival of NSG mice following xeno-transplantation with PBMCs and donor matched expanded ILC2. Kaplan-Meier plot of survival following allo-SCT (n=4 per group) Log-rank (Mantel-cox) test used. [Display omitted] DisclosuresSerody:Merck: Research Funding.

Economic Analysis of Posaconazole Prophylaxis in Patients with Acute Myeloid Leukemia Undergoing Remission Induction Chemotherapy from a Hospital Management Perspective

▪IntroductionInvasive fungal infections (IFI) are an important and trending causes of mortality in patients with acute leukemia. Especially during the remission induction, as well as stem cell transplantation, the patients bear the highest risk of IFI due to the prolonged severe neutropenia, long term exposure to broad spectrum antibiotics and cytotoxic chemotherapy.In a meta analysis of primary antifungal prophylaxis (PAT) in patients with malignancy receiving cytotoxic treatment and/or stem cell transplantation, antifungal prohylaxis was related with dramatic decrease in infection related mortality and decreased IFI. In another meta analysis focusing on prophylaxis with mould-active agents or fluconazole in hematologic malignancies has reported a significant decrease in IFI proven or probable, IFI related mortality and also with a concern, increased risk of adverse effects in the mould-active receiving group.Patients and Methods225 patients who have been diagnosed with acute myeloid leukemia (AML) and undergoing intensive treatment (idarubicin 12mg/m2 for three days and cytarabin 100mg/m2 24 h infusion for 7 days) for remission induction have been enrolled in the study in a retrospective manner. Direct health expenses like days of hospitalization, number of CT scans for thorax, development of complications, number of platelet apheresis infusions, mortality during remission induction treatment and total inpatient hospital costs. The benefits included cost reductions after adoption of primary antifungal prophylaxis.Results124 patients were male (55,4%) while 100 were female (44,6%). 90 patients received prophylactic treatment during remission induction therapy (40,2%). 43 patients died during remission induction therapy (19,2%). Mean number of thorax CT scans during remission induction was 1,73 (0-6). Mean number of days for hospitalization was 29,36 (14-88) days. Mean number of units of transfused thrombocyte apheresis was 9,9 (0-43) units. Mean cost of whole remission induction treatment was 9.151,6 (2.872,6-20.483,3) US dolars. Within patients who are not on PAT, 116 patients (86,5%) were treated with an intravenous antimould antifungal treatment while 5 of the patients on PAT (5,5%) received parenteral antimould antifungal treatment, 4 of which were related with neutropenic enterocolitis (p<0.005). 32 of the patients who are not on PAT died during remission induction (23,88%) while 11 of the patients who are on PAT died during remission induction (12,22%) (p=0.021). Mean days of hospitalization for remission induction treatment was 22,61 days for patients on PAT and 33,89 days for patients who are not on PAT (p<0.005). In patients on PAT, mean number of transfused platelet units was 6 (0-9) while 12,51 (4-43) units for patients who are not on PAT (p<0.005). When grouped as paients who are and who are not on PAT, there were significant difference between groups regarding total days of hospitalization, number of transfused platelet apheresis units, number of CT scans for thorax, death during remission induction and total cost of remission induction treatment in favor of patients on PAT. All significant data were analyzed with logistic regression analysis and observed as statistically significant. Results were summarized in Table.DiscussionIn Turkey, social security covers all the treatment for malignancies and complications, with rules and statements that lead the clinicians for decisions. Health Application Communiqué regulates the practice for reimbursement and since 2010, antimould antifungal agents are approved for prophylaxis in patients with AML who are undergoing remission induction therapy or patients undergoing hematopoietic stem cell transplantation. According to the physicians' or center's practices, PAT may be used and reimbursed.In our study, oral suspension form of posaconazole was effective to prevent IFI with a significant decrease in mortality during remission induction treatment. Likewise, PAT with posaconazole was observed to be cost-beneficial regarding the number of CT scans for thorax, number of platelet transfusion and over all costs of hospitalization for remission induction treatment. With its limitations regarding adherence as oral suspension form, tablet form may overcome these limitations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rejuvenated RBCs Increase Oxygen Consumption in a Simulated Exchange Transfusion Model of Sickle Cell Disease Patients

Introduction: Exchange transfusion of red blood cells (RBCs) in patients with Sickle Cell Disease (SCD) may require 6 to 8 units to reduce sickle hemoglobin (Hb-S) to less than 30 percent of total hemoglobin (NIH No. 02-2117). Low oxygen affinity (high p50) in patients with SCD is manifested by impaired blood viscosities and reduced hemoglobin concentration. Well characterized RBC metabolic changes during liquid storage of donor RBC include an increase in the affinity of hemoglobin for oxygen (low p50) by depletion of 2,3-diphosphoglycerate (2,3-DPG). Transfusion of stored RBCs (Standard RBC) suggests inefficient and impaired O2 delivery to tissues compared to fresh circulating RBCs (Hasan 1994, Scott 2016) or after exchange transfusion in SCD (Uchida 1998). Extracorporeal manipulation of p50 through RBC rejuvenation increases the level of 2,3-DPG and increases the p50 of stored RBCs by right-shifting the Oxyhemoglobin Dissociation Curve (ODC) (Dennis 1979). Rejuvenation occurs by incubating RBCs at 37 °C for 60 minutes with a rejuvenation solution (Citra Labs). An exchange transfusion simulation model was developed to evaluate the changes in oxygen release capacity of rejuvenated RBCs and the potential impact on estimated oxygen consumption (VO2) compared to Standard RBCs.Methods: Oxyhemoglobin dissociation curves were generated from (5) RBC units (leukocyte-reduced), CPD/AS-1, on Day 0, after 21 days of storage (Standard RBC), and after rejuvenation on day 21 (REJ RBC) which were used to estimate p50 values of the donor/recipient mixtures after each exchange transfusion and to determine VO2 as previously described (Li 2016, Srinivasan 2017). An elevated p50 of 30.4 mm Hg, typical for SCD patients (Uchida 1998), was used to estimate the baseline VO2 (200 mL O2/min). VO2 was determined after each simulated RBC exchange with Standard or REJ RBCs assuming a constant blood volume of 5L, cardiac output of 5 L/min, total hemoglobin concentration of 10 g/dL. The model assumed an exchange rate of one apheresis unit of circulating RBCs to transfusion of one unit of Standard or REJ RBCs.Results: Donor RBC p50 was 26.9 ± 2.0 mm Hg on Day 0 and declined with storage to 17.6 ± 1.5 mm Hg at 21 days (p < 0.001, t-test). Rejuvenation restored p50 to 34.8 ± 2.7 mm Hg which was significantly higher than Day 0 (p < 0.001, t-test). The model predicted a reduction in Hb-S from 70 % to 22%, a decrease in p50 after the 8 unit exchange transfusion to 21.6 mm Hg with Standard RBC, but an increase in p50 to 33.3 mm Hg with REJ RBC (Figures 1a & 1b). Similarly, VO2 increased in the REJ RBC group but decreased sequentially with Standard RBC upon each additional exchange transfusion (Figure 1c). The model predicted a decrease in VO2 to 118 mL O2/min for the Standard RBC group and an increase to 233 mL O2/min with REJ RBC group after the exchange transfusion.Conclusions: The model predicted a baseline VO2 of 200 mL O2/min; however, in fresh whole blood from healthy volunteers with Hb-A, normal VO2 was estimated at 244 mL O2/min (Srinivasan 2017). This simulation suggested that performing an exchange transfusion with REJ RBCs could achieve 95% of normal oxygen consumption assuming a constant cardiac output even though the hemoglobin was maintained at 10 g/dL. While exchange transfusion has been a successful therapy to reduce the Hb-S percentage in chronically transfused SCD patients, improving tissue oxygen exchange may not yet be optimized as evidenced by post transfusion declines in p50 (Miller 1980, Uchida 1998). The model is limited because it does not account for improved rheological properties and blood viscosity following exchange transfusion, which was previously reported to improve exercise capacity even though p50 declined (Miller 1980). The model also did not account for any influence from irreversibly sickled cells. Transfusion practices to correct anemia and improve tissue oxygenation may be less effective than intended due to the diminished oxygen release capacity of standard stored RBC units. Transfusion strategies should consider whether the use of RBCs with increased p50 may be physiologically advantageous.Figure 1: Predicted ODC (a) before and after the simulated exchange transfusion with Standard or REJ RBCs and predicted Hb-A vs Hb-S ratios (b) and VO2 (c) after each unit of the exchange transfusion with Standard or rejuvenated RBCs. [Display omitted] DisclosuresKausch:Zimmer Biomet: Employment, Research Funding. Gray:Zimmer Biomet: Employment, Research Funding. Landrigan:Zimmer Biomet: Employment, Research Funding.

FDA guidance on bacterial contamination risk control strategies to enhance the safety and availability of platelets: advantages and limitations

: Bacterial contamination of platelets was first addressed by primary culture, introduced in US in 2004, which ameliorated but did not solve the problem. Guidance to further decrease bacterial contamination were published by the US Food and Drug Administration in September 2019; recommended implementation is by October 2021. These include three single-step strategies: (I) culture at ≥36 h of ≥16 mL (≥8 mL aerobic, ≥8 mL anaerobic) per split apheresis unit or whole-blood derived pool; 5 day outdate; (II) culture at ≥48 h of ≥16 mL (≥8 mL aerobic, ≥8 mL anaerobic) per split apheresis unit; 7 day outdate; (III) pathogen reduction of apheresis units performed within 24 h of collection (5-day outdate). Several two-step strategies are available: First step has two options: (I) culture ≥36 h of ≥16 mL as in single-step option 1 (5 day outdate); (II) culture at ≥24 h after collection of ≥16 mL (≥8 mL aerobic, ≥8 mL anaerobic) per apheresis collection or split unit, or whole-blood derived pool (3 day outdate). The second step has three options to extend these outdates: (I) secondary culture of each unit of ≥8 mL (aerobic) on ≥ day 3 (extends outdate to 5 days); (II) secondary culture of each unit of ≥16 mL as in step 1 on ≥ day 4 (extends outdate to 7 days); (III) rapid testing of each unit within 24 hours of transfusion on ≥ day 3 (extends outdate to 7 days). This review addresses advantages and limitations of these strategies based on evidence of their efficacy.

Peripheral blood stem cell collection in children with extremely low body weight (≤8kg). What have we learned over the past 25 years and where are the limits?

Hematopoietic progenitor cells-apheresis (HPC-A) collection is now a routine procedure for autologous hematopoietic stem cell transplantation. Here we present our 25 years' experience of HPC-A collection in children weighing 8kg or less, with a focus on the evolution of our standard operating procedures, and the safety limits for these young patients, in the Pediatric Apheresis Unit of Clermont-Ferrand University Hospital (France). Fifteen children weighing 8kg or less underwent 26 HPC-A collections over 25 years. Median CD34+ cell yield by leukapheresis was 4.4 106 /kg. No procedure-related complications were encountered during or after the collection. No patient had profound thrombocytopenia or anemia that needed post-collection transfusions. Our experience in pediatric oncology patients who underwent HPC-A collections shows that this procedure can be performed even in the smallest of children with no increase in toxicity provided all precautions are taken to ensure that the procedure is carried out under the ideal conditions.

Mutualisation des outils de qualité pour les cellules CAR-T : recommandations de la Société francophone de greffe de moelle et thérapie cellulaire (SFGM-TC)

CAR T-cells are anti-cancer immunocellular therapy drugs that involve reprogramming the patient's T-cells using a transgene encoding a chimeric antigen receptor (CAR). Although CAR T-cells are cellular therapies, the organization for manufacturing and delivering these medicinal products is in many ways different from the one for hematopoietic cell grafts or donor lymphocyte infusions. The implementation of this innovative therapy is recent and requires close coordination between clinical teams, the therapeutic apheresis unit, the cell therapy unit, the pharmaceutical laboratory, and pharmacy. Apart from the regulatory texts, which are regularly modified, and the specific requirements of each pharmaceutical laboratory, there is currently no guide to help the centers initiating their activity and there is no specific indicator to assess the quality of the CAR T-cell activity in each center. The purpose of the current harmonization workshop is to clarify the regulatory prerequisites warranted for a center to have a CAR T-cell activity and to propose recommendations for implementing quality tools, in particular indicators, and allowing their sharing.

Extension of platelet shelf life with an improved bacterial testing algorithm.

At Canadian Blood Services, platelet concentrate (PC) shelf life was extended to 7 days with a large-volume, delayed-sampling bacterial screening algorithm. We present the development study and postimplementation results. In the development study, PCs inoculated with five bacteria (various concentrations) were incubated for 7 days with daily sampling for BacT/ALERT cultures and bacterial quantification. After implementation, from August 2017 to December 2019, a total of 223 156 pools and 39 725 apheresis units and 5310 outdated PCs were screened. Since March 2018, cocomponents associated to false-positive results have been released to inventory. In the development study, Klebsiella pneumoniae, Serratia marcescens, and Staphylococcus aureus were detected at concentrations of at least 0.01 colony-forming units (CFUs)/mL at 24 hours postinoculation. However, Staphylococcus epidermidis was detected at concentrations of less than 0.16 CFUs/mL only more than 48 hours postinoculation. After implementation, 776 (0.35%) and 303 (0.77%) initial-positive results and 201 (0.09%) and 16 (0.04%) confirmed-positive results were obtained for pools and apheresis units, respectively, predominantly with Cutibacterium acnes. Other organisms included staphylococci, streptococci, Klebsiella oxytoca and Pseudomonas aeruginosa. One nonfatal reaction involving a 7-day pool contaminated with S. epidermidis occurred. Approximately, 1-in-1000 false-negative screening results were obtained during testing of outdated PCs. Approximately 1000 cocomponents associated with false-positive results were released into inventory. Combined PC outdating at Canadian Blood Services and hospitals was reduced from 18.9% to 13.1%. Screening of 7-day PCs increased bacterial detection mainly of anaerobes and reduced outdating. The incidence of septic transfusion events has decreased approximately threefold. A longer surveillance period is needed to evaluate the value of anaerobic cultures and residual safety risk.

Assessment of Factor V Activity in Apheresis Platelet Units: Implications for Management of FV Deficiency

Abstract Introduction Factor V (FV) deficiency is a rare but significant disorder associated with potentially life-threatening bleeding. Management may involve plasma infusion to increase FV activity. Although the majority (~80%) of FV is present in the plasma, approximately 20% is stored in platelet (PLT) alpha granules. As such, allogeneic PLT infusion is another strategy that has been proposed in the literature to raise FV levels. Although the rationale for this approach is understandable, these recommendations are based largely on anecdotal reports. Since FV is the most labile coagulation factor from an in vitro standpoint and given that allogeneic PLTs are stored at room temperature for 5–7 days after collection, we hypothesized that there would be low FV activity in PLT units and tested our hypothesis by assessing FV levels across PLT products. Material and Methods PLTs were aliquoted from apheresis units only, including those with PLT additive solution (PAS) and from non-PAS units (N=26) on post-collection days 3–6. Supernatant was prepared from PLTS by centrifugation (5000 rpm X 5 min) and was stored at -80oC until analyzed. PLT lysate (PL) was prepared by freezing (-80oC) then thawing (370 C) followed by centrifugation (10,000 rpm X 10 min). FV activity was tested in the PLT unit supernatant and lysate using a clinical-grade assay based on prothrombin time correction of FV deficient plasma and expressed as percent activity; reference range for normal plasma FV levels at our facility is 50–150% activity. Statistical analysis was performed using student t test. The data were expressed as mean ± SD (P-value &amp;lt;0.05 considered significant). Results FV activity was markedly low in PAS PLT units, in both supernatant (3.70% ± 1.02%) as well as PL (3.26% ± 1.02%); N=16. FV activity in non-PAS PLT units (N= 10) was also somewhat low relative to reference range plasma levels in both supernatant (44.55% ± 6.46%) and PL (39.67% ± 6.33%); however, it was significantly higher (P &amp;lt; .001) when compared to PAS PLT units. FV activity from aliquots extracted from the same PAS PLT units and examined serially over storage time (N=3) showed FV levels on day 3 of storage (supernatant 5.03% ± 1.41%; PL: 3.89% ± 1.03%) to be not significantly different than levels measured on day 5 of storage (supernatant: 3.10% ± 0.57%; PL: 2.61% ± 0.41%; P=0.4). Conclusion FV is substantially depleted across PLT components, with particularly low levels observed in PAS PLTs. Given comparable supernatant and PL FV levels, there appears to be little FV reserve available in PLT lysate. Based on these data, plasma should be considered a first-line therapy for most non-complex FV deficiency-associated bleeds. If PLTs are considered for additional use, then non-PAS PLT units should be preferentially employed.

Impact of COVID-19 pandemic in the activity of a Therapeutic Apheresis unit in Italy

IntroductionThe recent Coronavirus Disease 2019 (COVID-19) outbreak has led to profound and rapid changes in the Italian and Veneto Region Healthcare System. This context also includes the quick reorganization which the Apheresis Unit (AU) of the Padova University Hospital, i.e. the Regional Reference Center for Therapeutic Apheresis (TA), had to face. Material and MethodsThe study retrospectively evaluated the TA activity (procedures performed, patients treated and consultations) during the COVID-19 pandemic, from March to April 2020, comparing the activity in the same time period in 2018 and 2019. ResultsIn the period analyzed, a significant reduction in both the total number of procedures performed and of patients treated, respectively by 17 % and 16 % for the procedures and by 19 % and 20 % for patients treated compared to the same period of 2018 and 2019, respectively, was observed. A concomitant reduction in requests for TA consultation for new patients (both outpatients and inpatients) was observed, equal to 32 % and 21 % compared to 2018 and 2019, respectively. ConclusionMany reasons determined the observed reduction in the TA activity during the recent COVID-19 outbreak. The AU itself was quickly reorganized in terms of location and supplies to allow for the appropriate COVID-19 patients care. Many non urgent cases, after multidisciplinary discussion between Clinicians and Apheresis Specialists, were deferred, maintaining close phone and e-mail contact with patients.

Closed and automated CAR T cell lentiviral transduction by spinoculation

Background & Aim There is a growing need to close and automate chimeric antigen receptor (CAR) T cell therapy manufacturing. CAR T workflows depend on an ex vivogene transfer step for therapeutic efficacy. To streamline this step while keeping flexibility during process development, we introduced a stand-alone protocol software that uses existing Sepax C-Pro technology. SpinOculation C-Pro protocol software automates lentiviral vector (LVV) transduction and maintains a functionally closed system within a single-use disposable kit, and no viral entry enhancers are required. We present different user cases highlighting the flexibility of the SpinOculation C-Pro protocol software parameters to influence process development of LVV transduction efficiencies in combination with application-specific upstream processes and viral source. Methods, Results & Conclusion Methods Case 1 used frozen apheresis units from one healthy donor. Case 2 used fresh apheresis units from three healthy donors. Case 1 included thawing and washing of the apheresis unit with closed, automated processes.In both cases, T cell enrichment and activation were performed using manual, open processes on the first day of the workflow. Activation was continued in T-flask culture for the next 24 hours. Then, on culture day 1 in both cases, cells were split to perform parallel SpinOculation C-Pro process and manual transduction processes in static or shake flask for case 1 and in static flask for case 2. Table 2 shows the SpinOculation C-Pro protocol software parameters used in the two cases. Conclusion As these user cases demonstrate, SpinOculation C-Pro protocol software offers a solution for a closed, automated LVV transduction process step where transduction efficiency can be comparable to a manual, open process. The open, flexible software parameters provide a wide potential for process development and optimization by allowing users to vary multiple parameters . It is important to consider that many factors influence the performance that can be achieved. Notably, transduction efficiency is highly dependent on LVV source and the MOI. Moreover, the upstream preparation of T cells, as well as the method and reagents used to expand transduced T cells, can impact performance.SpinOculation C-Pro protocol software completes our solutions to meet the growing need for fully automated workflow solutions for chimeric antigen receptor T cell therapies to help standardize processes and reduce the impact of donor variability. There is a growing need to close and automate chimeric antigen receptor (CAR) T cell therapy manufacturing. CAR T workflows depend on an ex vivogene transfer step for therapeutic efficacy. To streamline this step while keeping flexibility during process development, we introduced a stand-alone protocol software that uses existing Sepax C-Pro technology. SpinOculation C-Pro protocol software automates lentiviral vector (LVV) transduction and maintains a functionally closed system within a single-use disposable kit, and no viral entry enhancers are required. We present different user cases highlighting the flexibility of the SpinOculation C-Pro protocol software parameters to influence process development of LVV transduction efficiencies in combination with application-specific upstream processes and viral source. Case 1 used frozen apheresis units from one healthy donor. Case 2 used fresh apheresis units from three healthy donors. Case 1 included thawing and washing of the apheresis unit with closed, automated processes.In both cases, T cell enrichment and activation were performed using manual, open processes on the first day of the workflow. Activation was continued in T-flask culture for the next 24 hours. Then, on culture day 1 in both cases, cells were split to perform parallel SpinOculation C-Pro process and manual transduction processes in static or shake flask for case 1 and in static flask for case 2. Table 2 shows the SpinOculation C-Pro protocol software parameters used in the two cases.

A Case of Smoldering Multiple Myeloma with Simultaneous Increase of a Polyclonal Immunoglobulin

Anwarul Islam1* and Joanne Becker2 Author Affiliations 1Clinical Associate Professor of Medicine, Division of Hematology/Oncology, Department of Medicine, Buffalo General Hospital, Buffalo, New York 14203, USA 2Associate Professor and Clinical Chief, Division of Laboratory Medicine, Department of Pathology and Laboratory Medicine, Medical Director, Blood Bank and Therapeutic Apheresis Unit, Roswell Park Cancer Center, Buffalo, New York 14203, USA Received: April 08, 2020 | Published: April 23, 2020 Corresponding author: Anwarul Islam, Clinical Associate Professor of Medicine, Division of Hematology/Oncology, Department of Medicine, Buffalo General Hospital, Buffalo, New York 14203, USA DOI: 10.26717/BJSTR.2020.27.004468

A Randomized Trial Examining Plasma Exchange Using the Becton Dickinson (BD) Catheter In an Outpatient Apheresis Unit

A Randomized Trial Examining Plasma Exchange Using the Becton Dickinson (BD) Catheter In an Outpatient Apheresis Unit

Blood Supply and Demand in Korea: What is in Store for the Future?

PurposePresently, Korea is facing new challenges associated with an imbalance in blood supply and demand. The purpose of this study was to examine trends in blood supply and demand in Korea over the past 10 years through 2018 and to propose what to prepare in the future.Materials and MethodsAge demographics in Korea were analyzed using data from the Statistics Korea. Blood donation and blood supply data were analyzed using Blood Services Statistics 2018 by the Korean Red Cross. Blood transfusion data from hospitals in 2018 were obtained from the Health Insurance Review and Assessment Service.ResultsIn 2018, 2883270 whole blood and apheresis units were collected in Korea. The Korean Red Cross supplied 4277762 blood components to 2491 hospitals. The overall blood donation rate was 5.6%, and the most frequent donors were young male donors. Leukoreduced red blood cells (RBCs) constituted 25% of all RBCs used, and 40% of all platelets were supplied by single-donor platelets. The self-sufficiency rate of domestic plasma with which to produce plasma-derived medicinal products was 68.7% in 2018. Blood use was the most frequent among patients aged 70–79 years.ConclusionBlood management in Korea is changing rapidly due to a low birth rate, rapid aging, and an increase in severely ill patients who require most of the blood supply. Therefore, future plans to promote donation at a national level and optimal use of blood in hospitals is necessary.

Fibrinogen-Mediated Platelet Microaggregate Formation in Stored Whole Blood

Introduction: Whole blood is regaining popularity in resuscitation after hemorrhage. The platelet storage lesion that has been observed in apheresis platelets also exists in whole blood, where platelet count and aggregation response decline rapidly after collection and during storage in the blood bank. Platelet microaggregates form in unfiltered whole blood, resulting in relatively large but unstable thrombus formation in a flow model over collagen. We hypothesized that the presence of fibrinogen activates platelets within the whole blood unit and predisposes them toward premature aggregate formation. Methods: Fresh whole blood (WB) was collected in citrate tubes (4.3%, 10.9 mM) from healthy donors (n=4) according to an institutionally approved standard operating procedure. Blood was centrifuged at 200-g to collect the platelet-rich plasma (PRP). The remaining red blood cell (RBC) fraction was centrifuged at 2000-g to pellet RBCs which were washed with HEPES-buffered saline and repelleted; platelet-poor plasma (PPP) was saved. The PRP fraction was centrifuged with 1 µM prostacyclin at 700-g, and the platelet pellet was washed with Tyrode's buffer, repelleted, and resuspended in either fibrinogen-deficient plasma (Fg-DP) or PPP. Platelets were combined with washed RBCs to generate a reassembled whole blood sample containing 150,000 platelets/µl and a hematocrit of 40%. Samples were added to minibags and stored at 4 °C for 14 days. On days of testing (0, 3, 7, 14), 1 ml aliquots were stained with 1 µM calcein-AM for 30 min at 37 °C and run on the BioFlux 1000 microfluidics platform with arterial flow rates generating a shear rate of 980 s-1 over a collagen surface. Fluorescence microscopy images were captured and quantified over 10 min to observe platelet adhesion to the surface. Complete blood count (CBC) was measured on each aliquot. In a follow-up study, unmanipulated, stored whole blood was passed through a standard transfusion filter (200 µm pores) to determine the effects of removing microaggregates prior to microfluidics flow testing at each time point. Results: CBC results are in Figure 1, illustrating the decline of platelet count over storage as previously observed. Figure 2 shows representative images captured at the end of the 10 min flow period. In samples containing normal plasma, thrombus formation is impaired by day 3 of storage with aggregate structures appearing immediately in the microscopy images. These aggregates attach and release periodically over the course of flow, an effect that persisted through day 14. In samples reconstructed with Fg-DP, no microaggregates were observed during storage, although the density and size of thrombi diminished over time. In filtered whole blood samples, thrombus formation on days 3 and 7 more closely resembled the Day 0 fresh samples, but aggregates appeared on day 14 despite filtration. Discussion: WB contains all of the elements required for resuscitation after hemorrhage, however the platelet storage lesion may affect its function. The interaction of platelets and fibrinogen in a relatively static environment results in activation and aggregation of platelets. In apheresis units, studies have shown that using a platelet additive solution to dilute fibrinogen results in better platelet performance for longer periods of storage. Further study is required to determine the clinical significance of aggregate formation and whether mitigation strategies should be explored. Disclosures No relevant conflicts of interest to declare.

In Vitro Functionality of hiPSC Derived PLT+: The Utility of Microfluidic Assays in Determining Potency

Introduction: There is a growing shortage of platelets, the principal blood cells responsible for clot formation and blood vessel repair at sites of active bleeding. Platelet BioGenesis (PBG) is developing a commercial-scale, donor-free platelet (PLT+) production process using a cGMP-grade human induced pluripotent stem cell line (hiPSC) to address this issue. The PLT+ activity is being characterized extensively through multiple approaches that measure hemostatic function both in vitro and in vivo, with the ultimate goal of determining a clinical dose in humans. Alongside tests such as the current gold standard thrombin generation assay (TGA), we have developed an in-house microfluidic model to measure the ability of PLT+ to adhere to extracellular proteins under flow. The assay captures the surface receptor dynamics, as well as the ability of PLT+ to initiate coagulation. Materials and Methods: The thrombin generation assays are performed according to manufacturer's instructions. For the in-house microfluidic assay (MFA), fibrillar collagen, fibrinogen, or von Willebrand Factor is patterned in a commercial microfluidic device (Ibidi Slide VI 0.1). PLT+ (DNA-, calcein +, CD61+, CD42a+ cells from our bioreactor) are concentrated in normal pooled plasma, platelet additive solution, or platelet wash buffer to a normalized concentration of 1e8/mL, and are labeled with the mitochondrial dye DiOC6. Calcium chloride (7.5 mM) is added and the solution is perfused with a syringe pump at 50 1/s over the bioactive surface for 10 min. Images are captured every 10 seconds and quantified with ImageJ. After perfusion, the samples are fixed with 4% paraformaldehyde and can be stained for further characterization. Results and Discussion: PLTs+ are functionally non-inferior to human donor (blood) platelets and appear more active than Day 4/5 stored apheresis unit platelets. The TGA of PLT+ shows a more rapid generation of thrombin but similar total potential to donor platelets (Figure A). In the microfluidic assay, PLT+ (green DiOC6, red PAC1, figure B) adhere readily to the collagen surface under low shear flow, but do not adhere to non-functionalized surfaces, indicating GPVI functionality. As measured by surface coverage, the PLT+ resuspended in PAS adhere faster to the surface than either freshly washed donor platelets or apheresis platelets (p=0.01). When the PLT+ are preincubated in normal pooled plasma prior to perfusion, we observe fibrin formation and the aggregates show clot retraction, which supports the TGA results. Adhesion to surface bound fibrinogen and VWF is also comparable to donor platelets (GP IIb/IIa and GPIb function,). Combined, the TGA and MFA data reflect results of traditional more resource intensive assays, suggesting their viability as a rapid and flexible platform to study platelet function and function as release assays for in vitro generated PLTs+ Conclusion: Current work demonstrates that PLT+ are functional and a future alternative to donor derived platelet units. The MFA is a valuable tool for the characterization of this new therapeutic because of its specific control of surface protein-receptor interactions, shear forces, and flexibility to look at multiple markers. Alongside the TGA, the MFA will be a release assay for the PLT+. Figure Disclosures Lehmann: Platelet Biogenesis: Employment. Burton:Platelet Biogenesis: Employment. Szemethy:Platelet Biogenesis: Employment. Valdez:Platelet BioGenesis: Employment. Thon:Platelet BioGenesis: Employment.

Leukocytapheresis in nonmobilized donors for cellular therapy protocols: Evaluation of factors affecting collection efficiency of cells

Collection efficiency (CE1) of cells refers to the number of cells that are collected from the total number of cells processed by the apheresis device. Limited data are available about the CE1 of cells when performing leukocytapheresis in nonmobilized donors for cellular therapy purposes. The aim of our study was to evaluate donor- and procedure-related characteristics that might influence the CE1 of cells. Variables that predicted the CE1 of cells were analyzed by longitudinal linear regression in a series of 1071 leukocytapheresis procedures on 249 nonmobilized donors. Donor-related characteristics considered were gender, age, total blood volume, and complete blood count (CBC) data. Procedure-related characteristics considered were vascular access and device used. Older donor age was associated with a decrease in the CE1 of leukocytes, lymphocytes, monocytes, and mononuclear cells (MNCs; sum of leukocytes and monocytes) and with an increase in the CE1 of platelets (P < .05). Preprocedure CBC data (leukocytes, lymphocytes, monocytes, and platelets) were associated with either a statistically significant increase or decrease in the CE1 of cells. Central line used as vascular access was associated with a statistically significant decrease in the CE1 of MNCs (P = .02). Donor's age, preprocedure CBC data as well as central line used as vascular access were factors associated with CE1 of cells. Knowing these characteristics is helpful in the apheresis unit when designing cellular therapy protocols in order to maximize the CE1 of the desired cell and to personalize collection variables for each donor.