Long-Term Cryopreservation of Peripheral Blood Stem Cell Harvest Using Low Concentration (4.35%) Dimethyl Sulfoxide with Methyl Cellulose and Uncontrolled Rate Freezing at -80 °C: An Effective Option in Resource-Limited Settings.

Long-term cryopreservation of peripheral blood stem cells (PBSCs) is highly useful in the setting of tandem/multiple transplantations or treatment of relapse in the autologous hematopoietic stem cell transplantation (HSCT) setting. Even in allogeneic HSCT, donor lymphocyte infusions may be stored for months to years if excess stem cells are collected from donors. Cryopreservation is a delicate, complex, and costly procedure, and higher concentrations of dimethyl sulfoxide (DMSO), a commonly used cryoprotectant, can be toxic to cells and cause adverse effects in the recipient during infusions. In this study, we examined the effect of long-term cryopreservation using 4.35% DMSO (as final concentration) with methyl cellulose and uncontrolled rate freezing in a mechanical freezer (-80 °C) on the viability and colony-forming ability of CD34+ human PBSCs. For patients undergoing autologous HSCT, PBSCs were cryopreserved using DMSO (final concentration of 4.35%) with methyl cellulose. The post-thaw viability of PBSCs was determined using Trypan blue exclusion and flow cytometry-based 7-amino-actinomycin-D (FC-7AAD) methods. Concentrations of CD34+ stem cells and immune cell subsets in post-thaw PBSC harvest samples were assessed using multicolor flow cytometry, and the clonogenic potential of post-thaw stem cells was studied using a colony-forming unit (CFU) assay. CD34+ stem cell levels were correlated with the prestorage CD34 levels using the Pearson correlation test. The viability results in the Trypan blue dye exclusion method and the flow cytometry-based method were compared using Bland-Altman plots. We studied 26 PBSC harvest samples with a median cryopreservation duration of 6.6 years (range, 3.8 to 11.5 years). The median viability of post-thaw PBSCs was >80% using both methods, with a weak agreement between them (r = .03; P = .5). The median CD34+ stem cell count in the post-thaw samples was 9.13 × 106/kg (range, .44 to 26.27 × 106/kg). The CFU assay yielded a good proliferation and differentiation potential in post-thaw PBSCs, with a weak correlation between granulocyte macrophage CFU and CD34+ stem cell levels (r = .4; P = .05). Two samples that had been cryopreserved for >8 years showed low viability. Cryopreservation of PBSCs using 4.35% DMSO with methyl cellulose and uncontrolled freezing in a mechanical freezer at -80 °C allows the maintenance of long-term viability of PBSC for up to 8 years.

The determinants of donor safety and product quality in optimization of apheresis granulocyte harvest: An experience from a tertiary care oncology centre in India.

Allogeneic granulocyte transfusions play a substantial role in treatment of lifethreatening neutropenia-associated infections in patients undergoing intensive chemotherapy and hematopoietic stem cell transplant. Granulocyte harvest by apheresis is considered a safe and effective method to obtain adequate therapeutic granulocyte dosage for clinical effectiveness. This study described the experiences associated with apheresis granulocyte harvest procedures in our tertiary care haemato-oncology centre. We have analysed the incidence of adverse events (AEs) with associated potential risk factors contributing to donor safety and improvement in product quality. Retrospective data of 131 healthy allogeneic donors who underwent granulocyte harvest from May 2016 to July 2020 were analyzed. AEs were observed in overall 29 procedures (22.13%), including 14.50% citrate reactions, 7.6% venous access-related reactions, and 1.52% vasovagal reactions. Older age (p = 0.012) and higher body mass index (p = 0.015) in donors were significant variables found associated with a higher incidence of AEs. There was no significant impact of AEs on granulocyte product yield (p = 0.41) with a median collection yield of 1.73 × 10 10 cells/ unit. In multivariate analysis, post-mobilization parameters like total leukocyte counts (p = 0.036), absolute neutrophil counts (p = 0.042), and platelet counts (p = 0.006) showed a positive correlation with higher product yield. All the donors successfully donated and tolerated granulocyte colony stimulating factor plus dexamethasone mobilization and granulocyte apheresis harvest without any serious AEs. Our study shows that optimal technical and procedural modifications during apheresis granulocyte harvest procedures can overcome the associated potential risks by providing donor safety and improving product quality.

Impact of the novel coronavirus disease and lockdown on the packed red blood cells inventory management: An experience from a tertiary care oncology center in Western India

ABSTRACT Introduction The novel coronavirus disease has disrupted blood transfusion services worldwide. Despite blood transfusion services taking several precautionary measures to minimize the risks of COVID-19 during blood donations, donors became anxious regarding the risk of the COVID-19 infection during the donation and the blood transfusion services was facing the inevitable hazard of blood shortage. Methods The study was conducted at a tertiary care oncology hospital-based blood transfusion services and included analysis of blood donations, packed red blood cell units requirements, and packed red blood cell inventory in the pre lockdown and lockdown phase. New COVID-19 standard operating procedures with enhanced safety guidelines and donor confidence-building measures were implemented at the blood transfusion services. Results The total number of average monthly blood donations decreased in lockdown but the decrease was not statistically significant (238.5 vs. 197.8, P = 0.391). The requisitions for the packed red blood cell cross-matches (722.5 vs. 329.0, P = 0.001) and the packed red blood cell utilization (176.5 vs. 103.3, P = 0.028) for the hospital patients also decreased significantly due to the lockdown. In the lockdown phase, an expressive number of packed red blood cell units were outdated due to the unprecedented fall in the number of patients. In the post-lockdown phase, the packed red blood cell inventory was optimized with decreased outdating via a comprehensive approach. A special emphasis was given to the in-house donations. A second partial lockdown also decreased the blood donations. Conclusion Confidence-building in blood donors and the resolution of logistical issues were crucial for the efficient packed red blood cell inventory management in the lockdown. Implementation of COVID-19 preventive measures helped in the blood donor and blood transfusion services staff safety.

Robust Antitumor Activity and Low Cytokine Production by Novel Humanized Anti-CD19 CAR T Cells.

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBζ T cells produced lower levels of cytokines (IFNγ, TNFα) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBζ T cells. There was a comparable proliferation of h1CAR19-8BBζ T cells and mCAR19-8BBζ T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.

Impact of the novel coronavirus disease and lockdown on the packed red blood cells inventory management: An experience from a tertiary care oncology center in Western India.

INTRODUCTION: The novel coronavirus disease has disrupted blood transfusion services worldwide. Despite blood transfusion services taking several precautionary measures to minimize the risks of COVID-19 during blood donations, donors became anxious regarding the risk of the COVID-19 infection during the donation and the blood transfusion services was facing the inevitable hazard of blood shortage. METHODS: The study was conducted at a tertiary care oncology hospital-based blood transfusion services and included analysis of blood donations, packed red blood cell units requirements, and packed red blood cell inventory in the pre lockdown and lockdown phase. New COVID-19 standard operating procedures with enhanced safety guidelines and donor confidence-building measures were implemented at the blood transfusion services. RESULTS: The total number of average monthly blood donations decreased in lockdown but the decrease was not statistically significant (238.5 vs. 197.8, P = 0.391). The requisitions for the packed red blood cell cross-matches (722.5 vs. 329.0, P = 0.001) and the packed red blood cell utilization (176.5 vs. 103.3, P = 0.028) for the hospital patients also decreased significantly due to the lockdown. In the lockdown phase, an expressive number of packed red blood cell units were outdated due to the unprecedented fall in the number of patients. In the post-lockdown phase, the packed red blood cell inventory was optimized with decreased outdating via a comprehensive approach. A special emphasis was given to the in-house donations. A second partial lockdown also decreased the blood donations. CONCLUSION: Confidence-building in blood donors and the resolution of logistical issues were crucial for the efficient packed red blood cell inventory management in the lockdown. Implementation of COVID-19 preventive measures helped in the blood donor and blood transfusion services staff safety.

Comparative analysis of platelet concentrates prepared after two hours and overnight storage of buffy coat at room temperature.

INTRODUCTION: The overnight storage of the buffy coat (BC) at room temperature has logistic and operational advantages for the blood centre. The present study aimed to evaluate the impact of an overnight hold (stored) of BC at room temperature in comparison with the 2-hour hold (fresh) of buffy coats on the platelet concentrate (PC) characteristics. METHODS: A total of 60 BCs were included in the study, 30 PCs (fresh) were prepared after two hours holding time of the BCs and the other 30 PCs (stored) were prepared after the overnight BC storage at room temperature. The primary endpoint of PCs evaluation was the platelet yield, volume, pH, WBC count, RBC count, and platelet swirling in the PC and the secondary endpoints were glucose concentration, lactate, LDH, and sterility of the PCs. All the tests were performed on the day+1 of the blood collection. RESULTS: There was no difference concerning the volume, RBC count, and swirling between the two groups (P>0.05). The PCs from the fresh BC had higher pH and glucose concentration (P<0.05). On the other hand, the overnight hold of BC produced higher platelet counts, WBC counts, lactate, and LDH levels (P<0.05). All the 60 PCs did not record any bacterial growth on the culture media for the sterility results. CONCLUSION: The overnight hold of BC produces a higher platelet yield with higher storage lesions. This may also allow better supervision, ensuring better quality control.

Bortezomib and cyclophosphamide based chemo-mobilization in multiple myeloma.

Hematopoietic stem and progenitor cell (HSPC) mobilization regimens in multiple myeloma typically use filgrastim (GCSF) alone or combination of GCSF with plerixafor or high-dose cyclophosphamide. Murine model and human studies have shown HSPC mobilization potential of bortezomib. A total of 37 patients underwent mobilization using bortezomib 1.3 mg/m2 on day 1, 4, 8 and 11, cyclophosphamide 1 g/m2 on day 8 and 9, and GCSF 10 µg/kg from day 10 (B-Cy-GCSF). This regimen was compared with our earlier cohort of patients where cyclophosphamide was given at dose of 1 g/m2 on day 1 and day 2 followed by GCSF 10 µg/kg from day 4 (Cy-GCSF). In B-Cy-GCSF group, median CD34 cells collected were 9.21 × 106/kg (range 4.95-17.1) while in the Cy-GCSF cohort, the median CD34 cell yield was 8.2 × 106/kg (0.4-24.2). Target CD34 cells yield of 5 × 106/kg was achieved with single apheresis in 58.6% of patients after B-Cy-GCSF mobilization as compared to 44.3% in Cy-GCSF group (p = 0.07). Three patients failed mobilization after Cy-GCSF, while no patients failed mobilization in bortezomib group. Addition of bortezomib to Cy-GCSF mobilization showed a trend towards increased CD34 collection and reduced need for apheresis sessions.

Challenges in platelet inventory management at a tertiary care oncology center during the novel coronavirus disease (COVID-19) pandemic lockdown in India.

The novel coronavirus disease (COVID-19) has been declared a pandemic by the world health organization and to limit the spread of the disease, many countries in the world, including India, had enforced a lockdown. Despite no restriction over the platelet donation activities, plateletpheresis donors became apprehensive regarding the possible risk of spread of the COVID-19 during the platelet donation and in the hospital premises. Many of them started hesitating for platelet donations. With this, the blood center started having an acute shortage of platelets. Various confidence-building steps were implemented by the blood center to promote voluntary plateletpheresis. The blood center staff and individual donors were educated to prevent the spread of COVID-19. The donor organizations and plateletpheresis donors were informed about the steps to be taken by the blood center during the donation and necessary steps for the prevention of the possible spread of COVID-19. With the help of these measures, the confidence of the individual platelet donors and the donor organizations was restored in the blood center and regular plateletpheresis was continued. These measures may also be useful to other blood centers in the COVID-19 pandemic and this experience may be useful if a similar pandemic lockdown happens in the future.

Organization of the outdoor blood donation drives amid novel coronavirus pandemic and national lockdown: An experience from a tertiary care oncology institution in India.

The conduct of blood donation drives became difficult amid novel coronavirus disease pandemic and national lockdown. Despite no restriction for the outdoor blood donation drives, voluntary blood donor organizations (VBDOs) and individual donors became apprehensive regarding the possible risk of spread of the infection during blood donation. Various confidence-building measures were taken to decrease this hesitation. Numerous preventive measures were taken at the blood bank and at the donation venue to limit the possible risk of the spread of infection. With the help of these measures, the confidence of the individual blood donors and the VBDOs was restored and multiple blood drives were organized.