Marrow Harvest Research Articles

A Comparative Quantification in Cellularity of Bone Marrow Aspirated with two New Harvesting Devices, and The Non-equivalent Difference Between A Centrifugated Bone Marrow Concentrate And A Bone Marrow Aspirate As Biological Injectates, Using A Bi-Lateral Patient Model

The first aim of this study was to examine the cellularity and quality of autologous bone marrow aspirates harvested with two novel FDA-cleared devices, namely the Aspire™ bone marrow aspiration system (AS-BMAS) and the Marrow Cellution bone marrow aspiration device (MC-BMAD). Compared to traditional bone marrow harvesting needle systems, both these devices have a closed distal tip, avoiding preferential marrow collection (peripheral blood aspiration) from deeper cavity regions, whereas the side holes facilitate more horizontal marrow extraction. In all patients, a similar harvesting technique was used. The second aim was to demonstrate the effectiveness of mechanical centrifugation of a large volume of extracted bone marrow to produce a bone marrow concentrate (BMC). Finally, we directly compared bone marrow constituents aspirated with MC-BMAD with a BMC, generated by centrifugation of bone marrow harvested using the AS-BMAS. A bi-lateral patient model was used for all comparisons. All cellular analyses included the measurement of Colony-Forming Units-fibroblasts (CFU/f) levels, CD34+cells/ml, Total Nucleated Cells (TNCs)/ml, platelets/ml, and Red Blood Cells (RBCs)/ml in a single, FDA-approved laboratory, compliant with Good Manufacturing Practice regulations. A total of 12 patients consented to the study. In the direct BMA comparison, the AS-BMAS bone marrow yielded significantly higher CFU/f counts and TNC concentrations than the MC-BMAD (1,060/ml, 33.5 × 106/ml, and 610/ml and 28.6 × 106/ml, respectively), with comparable platelet and RBC concentrations. Data following BMA concentration to produce a BMC revealed highly significant cell yields, fewer RBCs, and lower hematocrit (HCT). A direct cellular comparison between the aspirate of the MCBMAD and centrifugated BMC following AS-BMAS marrow extraction showed highly significant differences in cellularity. The AS-BMAS produced cell concentrations of CFU/f, CD34+ cells, TNCs, platelets, and RBCs that were comparable, or greater than, the predicate device. We believe that concentrating bone marrow is a consistent and safe method to produce a BMC that has the potential to be clinically effective. Furthermore, data indicate a non-equivalent difference in BMC cellularity, when compared to a non-filtered and non-centrifugated BMA for clinical use.

Surgical reconstruction of the sternum following cardiac surgery complicated by mediastinitis—implantation of a custom-made sternal implant: a case report

: Mediastinitis represents an uncommon but potentially life-threatening complications following median sternotomy. There remains a lack of consensus regarding the optimal treatment option(s). Custom tridimensional titanium printing provides an effective method for construction of complex prothesis. We report the case of a 49-year-old female with previous sternal dehiscence and deep wound infection following coronary artery bypass grafts, necessitating sternectomy. A year later, she presented with chronic severe pain and audible crepitations of her ribs/clavicles. Lack of identification of a mechanical cause, poor localisation and suboptimal medical pain management culminated in the consideration for a novel approach for sternal reconstruction. By way of bridging the final placement of the titanium prothesis, the patient underwent placement of a methyl methacrylate spacer. The surgical approach involved the removal of the temporary spacer, with tissue samples taken to guide post-operative antimicrobial therapy. Concurrently, the reaming, irrigation and aspiration technique of bone marrow harvesting, for seeding of the central web structure of the implant, was employed under fluoroscopic guidance. Following adequate dissection under the pectoral muscle, and debridement of the anterior chest wall, final placement of the prothesis was made via bicortical fixation to the ribs. Finally, the mobilised pectoral muscles were sutured medially and fixed to the previously placed rectus abdominis flap. Three-dimensional printing of a custom prosthesis offers a novel and effective approach to full reconstruction; maintaining pulmonary mechanics and stability of the anterior chest wall

Single-Agent Cyclosporine for Graft-versus-Host Disease Prophylaxis in Patients with Acquired Aplastic Anemia Receiving Fludarabine-Based Conditioning.

Cyclosporine (CsA) combined with short-course methotrexate is considered standard-of-care graft-versus-host disease (GVHD) prophylaxis for patients with severe aplastic anemia (AA) who undergo transplantation using cyclophosphamide (Cy) plus anti-thymocyte globulin (ATG) conditioning. However, there is no consensus on optimal post-transplant GVHD prophylaxis for patients undergoing matched related donor (MRD) transplantation using fludarabine (Flu)-based conditioning. We conducted a single-center retrospective analysis of patients with acquired AA (n=106) undergoing MRD transplantation from July 2007 through January 2019. All patients received Flu-Cy-ATG conditioning and single-agent CsA as GVHD prophylaxis. Median age of the study cohort was 20 years (range, 3 to 52) and male to female ratio was 3.8:1. Median time from diagnosis to transplant was 11.5 months (range, 2.8 to 62). Graft source was bone marrow harvest in 71 (68%), combined bone marrow and peripheral blood stem cells in 34 (31%), and peripheral blood alone in 1 (1%) patient. Cumulative incidence of neutrophil engraftment at day 28 was 93.4% (95% confidence interval [CI], 87.3% to 97.1%) while that of platelet engraftment at day 100 was 90.5% (95% CI, 84% to 96%). Cumulative incidence of primary graft failure at day 28 was 6.6% (95% CI, 4% to 8%) while secondary graft failure occurred at a median of 190 days (range, 90 to 415) at a cumulative incidence of 3.7% (95% CI, 2% to 5%). Cumulative incidence of grade II to IV acute GVHD at day 100 was 3.8% (95% CI, 1.4% to 9.9%), while a 1-year probability of chronic GVHD was calculated as 7.5% (95% CI, 2.6% to 15%). Median follow-up post-transplant was 61 months (range, 6 to 144). Overall survival was 84.9%, disease-free survival was 80.2%, and GVHD-free relapse-free survival was 76.3%. This study indicates that single-agent cyclosporine is a feasible option for GVHD prophylaxis in MRD hematopoietic stem cell transplantation using Flu-Cy-ATG conditioning and is associated with very low rates of acute and chronic GVHD.

Introduction of principles of blood management to healthy donor bone marrow harvesting.

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. Observational data from 489 bone marrow (BM) donors were retrospectively analysed, and principles of patient blood management were applied to healthy volunteer BM donations. 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.

Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages—a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in vitro differentiation capacity of isolated MSCs. Bone marrow was obtained from the femoral cavity of twelve donors during total hip arthroplasty in the absence or presence of heparin. No coagulation was observed in the absence of heparin. The number of mononuclear cells was independent of heparin addition. Isolated MSCs were characterized by morphology, population doubling times, expression of cell surface antigens and in vitro differentiation. Results of these analyses were independent of the amount of heparin. Transcriptome analyses of cells from three randomly chosen donors and quantitative realtime PCR (qRT-PCR) analysis from cells of all donors demonstrated no clear effect of heparin on the transcriptome of the cells. This excludes heparin as a potential source of disparate results.

Hematopoietic stem cell donor with IgA nephropathy: Challenges and management algorithm

Donor safety is of prime importance in allogeneic hematopoietic cell transplantation. The Worldwide Network for Blood and Marrow Transplantation (WBMT) standing committee on donor issues has issued a consensus statement regarding suitability criteria for related adult donors. This committee recommends that donors with a history of immune-mediated glomerulonephritis and abnormal urine tests should preferably undergo bone marrow harvest, to avoid the theoretical risk of granulocyte colony-stimulating factor (G-CSF) induced immune flare-up. We discuss here a unique situation where a related donor with a history of IgA nephropathy (IgAN) insisted on a peripheral blood stem cell harvest. We propose a management plan for this situation, which posed challenges about donor suitability.

Donor risk factors and recipient clinical impact of positive microbial contamination after bone marrow harvests - a large academic medical center experience

Background & Aim Background Microbial contamination (+MC) is a complication of bone marrow harvestS. However, risk factors for +MC and implications for recipients are not well-defined. Clinical interventions after +MC range from observation to antimicrobial therapy. Methods, Results & Conclusion Methods We report a retrospective review of 91 pediatric and 101 adult related-donor marrow harvests from Jan 2015 to Mar 2019. Harvests were performed at Boston Children's Hospital (pedi) or Brigham & Women's Hospital (adult) under general anesthesia. Donors received no systemic antimicrobials, just topical sterilization. Samples were cultured in BacT systems and incubated for 14 days at 35°C. Results 19.4% of harvested products from pedi and 24.8% from adult donors demonstrated ≥ 1 +MC result. Mean donor age, weight, gender, and volume harvested differed between pedi and adult donors: 12 vs 40yr, 45.2 vs 79.4kg, male 47% vs 65%, and 622 vs 1200mL. 37% and 39% of products underwent manipulation, respectively (Table 1). Mean time to detection was 4.7 and 6.1 days after pedi and adult harvests. 7.5% and 5.0% of harvests, respectively, yielded +MC ≤ 2 days; 11.8% and 5.9% by 5 days after harvest. Organisms isolated were P. acnes (n=29), Coag-negative Staphylococcus (n=13) and one other Propionibacterium species (n=1). We examined potential risk factors for + MC such as length of harvest (invasive harvesting time); donor age, weight and gender; manipulation; and cell yield (Table 2). In pediatric harvests, +MC correlated with donor age (19 vs 10yr, p Response to positive cultures was at attending physician discretion. Only 9.3% of recipients of a +MC marrow had antimicrobial therapy added, with no product-transmitted infection seen. Neutrophil and platelet engraftment did not differ between +MC vs No Growth marrow recipients. Rates of infusion reactions were within historical norms (19% adult and 11% pedi) and not impacted by +MC. Analyses of donor AEs and +MC unrelated donor harvests are ongoing. Conclusions +MCs are common after pediatric and adult donor harvests. Risk factors may include longer harvest time and higher donor weight but are not clearly modifiable. Despite this +MC incidence, medical implications for recipients and donors after +MC with routine skin flora are few.

Impact of autologous blood transfusion after bone marrow harvest on unrelated donor's health and outcome: a CIBMTR analysis.

Pre-harvest autologous blood collection from bone marrow (BM) donors is performed to meet potential post-operative transfusion needs. This study examines the impact of autologous blood transfusion on BM donor's health and safety. The study included first-time unrelated BM donors from the United States whose BM harvest was facilitated by the National Marrow Donor Program (NMDP) centers between 2006 and 2017. Examination of 7024 BM donors revealed that 60% received at least one unit of autologous blood. The donors who received autologous blood were older, had lower hemoglobin pre-harvest, underwent longer duration of anesthesia, and higher volume BM harvest. Only donors who underwent high-volume BM harvest, defined as a BM harvest volume >27% of donor's blood volume, benefited from autologous transfusion. After a high-volume BM harvest, autologous blood transfusion was shown to decrease grade 2 to 4 collection-associated toxicities within 48 h of BM donation (p = 0.010) and shorten the time to donor-reported "complete" recovery from donation-associated symptoms (p < 0.001). Therefore, autologous transfusion could be avoided as support of marrow donation in the majority of unrelated BM donors and should be limited to cases where the planned BM harvest volume is expected to exceed 27% of donor's blood volume.

Clinical significance of autologous blood transfusions in bone marrow harvest from unrelated donors.

In the Japan Marrow Donor Program (JMDP), autologous blood is collected from most unrelated bone marrow (BM) donors. We retrospectively evaluated 5772 donors who underwent BM harvest between 2010 and 2015 through the JMDP. Autologous blood was collected in 96.8% of the donors; the wastage rate was 0.6%. Allogeneic blood transfusion was not required. The mean hemoglobin (Hb) levels were 12.1g/dL after the BM harvest (mean 891mL) together with autologous blood transfusion (mean 596mL). Propensity-score matching was used to adjust the backgrounds. Among donors with harvested BM of 100-400mL, autologous blood transfusion had no impact on Hb levels or complications after BM harvest. Among donors with harvested BM of > 400mL, more autologous blood transfusion followed by a bleeding volume of ≤ 100mL did not confer clinical benefit to donors compared with less autologous blood transfusion followed by a bleeding volume of > 300mL. The findings of the present study suggest that autologous blood transfusion to BM donors is excessive in terms of Hb changes and post-harvest outcomes.

Pre- and post-bone marrow harvest anaemia is associated with lower CD34+ stem cell collection, high harvest volume and female gender.

Donor safety is paramount when performing bone marrow stem cell harvest. The incidence of full blood count (FBC) abnormalities among donors and variables associated with anaemia after marrow harvest are not well established. To describe the frequency of FBC abnormalities prior to bone marrow stem cell harvest and to identify variables associated with post harvest anaemia. Outcomes of 80 consecutive adult marrow harvests performed at our centre were analysed retrospectively. FBC abnormalities were present in 28% of donors prior to marrow harvest with normocytic anaemia the most common abnormality in 13%. Reduced donor haemoglobin (Hb) was independently correlated with lower CD34+ cell count per kg of recipient body weight. Anaemia (Hb < 100 g/L) was seen in 20% of donors after harvest with median decrease in Hb of 19 g/L. Variables independently associated with anaemia after harvest included donor to recipient weight ratio (P = 0.011), high collection volume (P = 0.044) and female gender (P = 0.023). Total nucleated cell and CD34 concentration in the final collected product were associated with the inverse of harvested marrow volume (P < 0.001). Pre-harvest anaemia should be corrected where possible particularly in female donors. Marrow collection volume should be minimised to reduce post-harvest anaemia, optimise CD34+ cell number and improve nucleated and stem cell concentrations in the harvest product.

Điều trị chấn thương sọ não cấp tính bằng ghép tế bào gốc tự thân tại Bệnh viện Trung ương Huế

TREATING ACUTE TRAUMATIC BRAIN INJURY BY USING AUTOLOGOUS BONE MARROW DERIVED STEM CELLS AT HUE CENTRAL HOSPITAL The number of acute traumatic injuries caused by accidents has been increasing in recent years, leading to death or serious complications in cognitive behavior or social function. Few pre-clinical studies around the world have shown the ablity of stem cells in neuroprotection. Therefore, we apply autologous stem cells transplants in two acute traumatic brain injury patients to evaluate the effectiveness of stem cell therapy. Method: Three male patients aged 23 and 49 years with a postresuscitation Glasgow Coma Scale of 6 and 8 were treated with autologous mononuclear cells delivered intravenously within 2-3 hours after bone marrow harvesting, mesenchymal stem cells were isolated and expanded in culture before the system administrating through vein after 7-10 days. To determine the safety of the procedure, systemic and cerebral hemodynamics were monitored during bone marrow harvest; infusion-related toxicity was determined by hepatic enzymes, and renal function. Result and conclusion: There were no significant changes in liver, kidney and hematological criteria. BI and Glasgow indexes increased significantly compared to the control group. There was no abnormal complication within 4-6 weeks after cell transplantation. Autologous stem cell therapy is safe and effective for patients with acute brain injury. Keywords: Stem cells; Mesenchymal stem cell; Bone marrow; Acute traumatic brain injury

Outcomes of allogenic hematopoietic cell transplantation for childhood chronic myeloid leukemia: Single‐center experience

Despite the apparent efficacy and favorable toxicity profile of TKIs, allogeneic SCT remains the only curative treatment for CML especially in younger patients, but TRM should be considered. We evaluated the clinical outcomes of pediatric CML patients who had SCT in our center. This retrospective study included children with CML, who received an allogeneic SCT at Children Cancer Hospital Egypt, 57357, from 2007 to 2017. All patients received myeloablative conditioning chemotherapy containing busulfan/cyclophosphamide followed by stem cell infusion from MRD. From 121 patients diagnosed with CML, 43 had available MRD and subjected to HSCT while 78 patients continued TKI therapy. The median time to transplant from diagnosis was 13months. At initial diagnosis, there were 39 patients in CP and 4 had blastic crises. Bone marrow harvest was the stem cell source in 32 patients, while 11 cases received mobilized peripheral blood stem cells with average stem cell dose of 4.45×106 /kg. The probabilities of overall survival and event-free survival at 5years were 97.4% and 79.8%, respectively. TRM at 100days and TRM at 1-year post-transplant were 0%. The incidence of chronic GVHD was significantly higher in peripheral blood than bone marrow stem cell source (P=.004). Considering the excellent survival rates and very low TRM, HSCT is still a valid option for pediatric patients with newly diagnosed CML with best using marrow stem cell source to avoid a significant risk of cGVHD and its related complications.

Improving Marrow Collections By Use of Five Side Hole Needle

Authors Harvey, E., Abhyankar, S., Merkel, D, Shune, L. Objectives Marrow harvesting is used to treat both malignant and nonmalignant hematologic disorders. We describe here our method of decreasing both anesthesia and collection times and maintaining or improving the total nucleated cell dose of the product. Method We analyzed 227 marrow harvest between October 2010 and August 2018. From the period of October 2010 through July 2018, we utilized the standard aspirate/biopsy needle. Total number of harvests during this time was 167. Starting in August of 2018, we switched to a 5-side hole marrow harvest needle. Total number of harvest utilizing this needle was 60. Counts were checked midway during all harvests and again close to estimated completion to verify stopping point. Results Anesthesia time for donors decreased 27% from an average of 83 minutes to 61 minutes (44 minimum- 96 maximum). Total harvest time was decreased by 32%, from an average of 44 minutes with the single hole needles to 30 minutes with the five side hole needs. (11 minimum – 66 maximum) (32% decrease). Total nucleated cell dose averaged 4.91 × 108/kg with the use of the single hole needs (n=167) and with the 5-side hole needles the average was 5.13 × 10 8 (N=60) Conclusion Changing from the single hole needle to the five (5) side hole needle improved the efficiency of the marrow harvest from both the collection and an anesthesia timepoints. By decreasing the anesthesia and collection times, we minimize the overall effects of the collection procedure on the donor. Traditional single hole beveled needle on left, 5 side hole needle on right. Close-ups of 5 Side Hole Needle

Donor Sex, Age, and Body Mass Index on Bone Marrow Harvest Collection Quality Outcomes

Introduction To evaluate factors of sex, age, and body mass index (BMI) on the bone marrow (BM) harvest collection outcomes of total nucleated cell (TNC) yield, anesthesia time, and sterility; a retrospective review of BM harvest donors at our institution from 2008 to 2017 was conducted. Donors were compared by sex and quartiles based on age and BMI. Materials and Methods Fifty-one donors underwent BM harvest. Thirty-three, 64.7%, of donors were male while 18, 35.3%, were female. Ages ranged from 20 to 72 years, median 33. BMI ranged from 18.7 to 43.4 kg/m2, median 27.7. BMI quartiles approximate WHO nutritional categories: first quartile, Normal weight; second and third quartiles, Overweight; fourth quartile, Obese. As TNC number targets are not donor dependent, outcomes were standardized by concentration: TNC/ml for TNC yield, seconds/ml for anesthesia time. Results There were no significant differences in TNC yield between sexes and between age quartiles. There was a significantly higher TNC yield in the fourth quartile of BMI (>32.5 kg/m2) compared to the first quartile of BMI (≤24.3), p = 0.032. There were trends towards longer median anesthesia times in females, highest BMI quartile, and youngest age quartile. Cultures were positive in 5, 9.8%, products; all occurred in the male donor group, 15.2% of male donors. Conclusion Selection biases might explain the greater number of male donors and donors with a greater BMI. There were no significant differences in TNC yields between sexes and between age quartiles. There was a significantly higher TNC yield in the fourth quartile of BMI compared to the first. Median anesthesia times were longest in female, youngest quartile of age, and Obese donors but did not approach statistical significance. BM products collected from male donors were significantly more likely to have positive cultures. Sample size limits the power of these analyses.

Autologous Whole Blood Donation in Allogeneic Marrow Harvest – Is It Truly Needed?

Authors Harvey, E, Abhyankar, S., Merkel, D., Shune, L. Background The collection of autologous units in allogeneic marrow donors, while no longer required by the National Marrow Donor Program, is still practiced by some collection centers. Collection centers have the option to request collection of autologous unit(s), however, this practice can cause a delay in time to transplant as well as the possibility of putting donors through procedures that are not needed. If a marrow collection is delayed or cancelled, autologous units may expire. Donors may also experience anemia prior to the harvest due to donation of autologous units. Method Our Center analyzed our marrow harvests collected between 2010 and July 2018. We checked a complete blood count (CBC) on the day of the harvest, both prior to and following completion of the marrow collection. Results Of the 227 marrow harvests that were reviewed, there were no donors that required transfusion support post-harvest. The average drop in the hemoglobin was 2.67 g/dL. (N=227 with a range of +1.3 g/dL to -6.8 g/dL). Of the donors whose hemoglobin dropped greater than 5 grams (N=6), 1 received 1 liter of IVF, one 1.3 L, 2 received 3 L and one received 5.5 L of IVF, possibly leading to hemodilution of these samples. Additionally, 2 of these were drawn from the IV line, which could also contribute to dilution of the sample. Average percentage of total blood volume taken from these 6 donors was 25%, which was consistent with the 21% average among all harvests, therefor the lower hemoglobin was felt to be dilutional and not an accurate portrayal of the actual hemoglobin. Conclusion Collection of autologous units is not needed in healthy allogeneic marrow donors. The practice of autologous unit collections can delay time to transplant for the recipient. It can also put the donors through additional and unnecessary procedures. Lastly, the units may be wasted if the harvest is delayed or cancelled.

Establishment of Pediatric Bone Marrow Transplant Unit in Public Sector Hospital, a Dream Accomplished.

<h3>Background</h3> Bone Marrow Transplant (BMT) is a highly sophisticated curative treatment of many Hematological, immunological, metabolic and Oncological conditions. Pakistan is a Lower middle income country. The treatment facilities are very limited as compared to the need of the region. Our Pediatric Hematology/Oncology unit is in one of the busy center of the country. We decided to start the First ever Public sector Pediatric Bone Marrow Transplant. <h3>Objective</h3> This study was done to know & enlighten the hurdles in establishment of BMT unit in Public Sector Hospitals in LMIC. The task to start BMT was taken as a challenge in collaboration with NIBD & BMT, Karachi, Pakistan in 2016. The Physicians and other supportive Team were sent for short term training.On return the Team started renovation of old building. In next step arrangement of instrumentation was done step by step, includes Cell separators, Platelet Aphresis Machines, HEPA Filters, CBC Analyzer, Handy Electrolyte Machine, Fridge (-80C⁰) for Cryopreservations & Plasma Storage, PRBC storage fridge and Blood grouping/Cross matching reagents and Gel Cards arranged. Then two patients with Beta Thalassemia Major were selected who had full matched sibling donors. The standard Conditioning Protocol of BU/Cy/ATG with GVHD prophylaxis Steroids/MTx and Cyclosporine started after saving the Autologus Bone Marrow Harvest. The Bone Marrow Harvest was done on both donors on same day and Infusion was done. The MNC and CD34 count were appropriate. The senior team members of Pediatric Hem/Onco department were involved and senior most staff nurses had taken care of the patients along with mothers of the patients. But unfortunately one patient died of Severe Pneumonia along with Pharyngeal abscess on day +11 and the second on +25 due to Primary engraftment Failure and severe Pulmonary Hemorrhage. The initial two patient's mortality depressed the Hospital administration as well as the Treating Team. We decided to review the supportive care techniques, restricted the entry and limited the Treating staff. After getting these improvements we again decided to start the BMT unit again in March 2018 and selected Aplastic Anemia patient of 8 years who had 6/6 matched sibling donor.The five days conditioning was done with Fludarabine, Cyclophsphamide and GVHD prophylaxis with Cyclosporin, MTX and steroids. PBSC was done on cell separator and stem cell infusion done. Patient engrafted Neutrophil on +10, Platelets on +18 and RBC on +45. Th immnunosupression was tapered off 6 months post BMT and now free of medication. Since then we have done so far total 23 patients including Aplastic Anemia, Thalassemia, Fanconi Anemia.Our success rate is 60%. I know it is far behind the international data but we are thriving to improve it day by day. <h3>Conclusion</h3> The persistent struggle is needed for establishment of BMT units in LMICs. <h3>Recommendation</h3> When there is a will, there is a way.

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, an important number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow (BM), supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPC). To overcome the limit of BM harvest/HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex vivo expansion of HSPC coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1- defective patients were transduced with a clinically-optimized lentiviral vector expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NOD scid gamma common chain (NSG) recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPC coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by BM fibrosis.

Evaluation of the Sysmex XN automated hematopoietic progenitor cell enumeration for timing of peripheral blood stem cell harvest

BackgroundEnumeration of stem cells is essential in the management of peripheral blood stem cell (PBSC) harvest. An alternative to the gold standard flow cytometric CD34+ stem cell count is the fully automated hematopoietic stem cell (HPC) count on the Sysmex XN hematology analyzer. Materials and methodsEighty-nine patients and healthy stem cell donors who underwent PBSC harvest were included in the study. Stem cells were enumerated in pre-harvest peripheral blood and the apheresis yield by both flow cytometric CD34+ stem cell enumeration and by the Sysmex XN HPC count. ResultsThe Sysmex HPC concentration overestimated the CD34+ stem cell concentration by a ratio of 1.3 in average. The agreement between the two methods was poor at concentration <40 stem cells/μL (Bias: 45 %, 95 % limits of agreement: -71 - 160 %). CD34+ stem cell concentration and HPC concentration correlated well in pre-harvest peripheral blood (R=0.73, slope=0.96). We established a positive cut off >43.5 HPC/μL, where PBSC harvest can be initiated. And a negative cut off <16.5 HPC/μL, where harvest should be postponed or other mobilizing regimens or bone marrow harvest should be considered. 33 % of measurements were in between the negative and positive cut-off and would require a supplementary CD34+ cell count. ConclusionAlthough Sysmex HPC count correlates well with CD34+ cell count in peripheral blood, the agreement between the two methods is poor, especially at low concentrations, namely in the clinical decision range. Sysmex HPC count as a surrogate for CD34+ cell count should, therefore, be used with caution.

The effect of a single consecutive volume aspiration on concentrated bone marrow from the proximal humerus for clinical application

BackgroundLow aspiration volumes have been recommended to allow for higher concentrations of progenitor cells during bone marrow harvesting. However, these guidelines then require multiple aspiration attempts in order to maximize cellular yield. The purpose of this study was to investigate the effect of a single, high-volume aspiration with four consecutive aliquots on the number of nucleated cells (NCs) and colony-forming units (CFUs) in concentrated bone marrow aspirate (cBMA) taken from the proximal humerus.MethodscBMA was taken from the proximal humerus of patients undergoing arthroscopic rotator cuff surgery. Four 12-mL double syringes were used consecutively from a single trocar to obtain four 10 cc aliquots. Each then underwent centrifugation to create a fractionated layer rich in nucleated cells. Following cellular separation, NCs were counted and CFUs were evaluated after incubation of 7–10 days. Cellular comparisons between each aliquot were performed along with their interaction with patient age and sex.ResultsTwenty-nine patients (55.9 ± 4.6 years) were included in this study. The number of NCs and CFUs showed significant differences between the four aliquots of aspirate, with the first 10 cc aliquot providing the highest amount (p < 0.001, respectively). No significant differences were found between the sum of the three sequential aliquots compared to the initial 10 cc sample. There were no significant differences between male and female patients (p > 0.05). Increasing age resulted in no significant decrease in the number of NCs and CFUs across the four consecutive aliquots (p > 0.05).ConclusionIn conclusion, while the initial aliquot provided the greatest number of nucleated cells and cultured CFUs, the addition of each sequential volume aspirate yielded similar amounts in total. This demonstrates the potential effectiveness of obtaining of higher volume aspirates from the proximal humerus during rotator cuff repair.

Peripheral Blood to Marrow Collection White Cell Count Ratio As an Indicator of Harvest Quality and Transplant Outcomes in G-CSF-Primed Marrow Grafts

Background: G-CSF-primed bone marrow (G-BM) is used in some centers as the product of choice in related donor transplants because of lower rates of GVHD and fast engraftment (Morton 2001; Deotare et al. 2015). Marrow collections substantially diluted with peripheral blood may, however, compromise the GVHD-sparing effect of G-BM. Concerns over the variability in the quality of bone marrow harvest have been reported (Spitzer et al. 1994; Prokopishyn et al. 2019). We explored the role of a simple novel quality indicator, i.e peripheral blood to marrow harvest WBC ratio, as a predictor of GVHD risk. Materials and methods: We analysed 156 successive bone marrow harvests for patients suffering from severe thalassemia using G-BM donated by fully matched (10/10 or 12/12) related donors (143 siblings and 13 mothers) at Sankalp-People Tree Centre for Paediatric BMT - Bangalore (80 patients) and Sankalp-CIMS Centre for Paediatric BMT - Ahmedabad (76 patients). G-CSF priming was done for 5 days before the transplant with 10 mcg/kg/day in 2 divided subcutaneous doses and peripheral counts were obtained within 24 hours of collection. G-BM was collected in bags with 20 ml 50 U/ml heparin for the first 200 ml and further 10ml/100ml of marrow. We defined quality of marrow as follows: Quality of Marrow = WBC count of G-CSF primed BM / Peripheral WBC count of donor Quality of Marrow &lt; 0.9 was categorised as poor as it signifies that the concentration of WBCs in bone marrow is same or lesser than the pre-harvest peripheral counts. EBMT criteria was used for grading GVHD. We calculated the odds of aGVHD based upon the quality of marrow. Beside marrow quality, the sex of the recipient seemed to impact GVHD rates and thus regression analysis was used to study the impact of G-BM quality independent of other factors. Statistics were performed on R software (version 3.5.x). Mann Whitney and Fisher's exact tests were used to compare numeric and categorical data respectively. Results: Of the 156 harvests, 18 had poor marrow quality; 33% (6 of 18) of these developed acute GVHD grade II-IV as opposed to 9% (13 of 138) (see Figure 1). Between those who developed GVHD and those who did not, marrow quality (p=0.012), patient sex (p=0.006) and sex mismatch status (p=0.014) were significantly different. Differences in total cell dose (p=0.65), transplant centre (p=0.62), bone marrow cell count (p=0.14) and peripheral cell count before harvest (p=0.72), donor age at BMT (p=0.42), relationship with the donor (p=1.0) and average volume of marrow per draw of syringe (p=0.23) showed no statistical significance. Variables with significant difference (sex mismatch and bone marrow quality) were entered in the Cox's proportional hazards model for acute GVHD II-IV which showed that poor marrow quality is the most significant independent risk associated with a hazard ratio of 3.6 (p=0.009), followed by male to male transplants which were associated with a hazard ratio of 10.6 (p=0.03) See Table 1. Further, poor quality marrow was associated with lower cell count in marrow (p&lt;0.001), higher peripheral counts (p&lt;0.001), higher collection volume (p=0.035) and was seen more in one centre compared to the other (p&lt;0.001). Donor age (p=0.81), donor's relationship (p=1.0), donor sex (0.62), donor's carrier status (p=0.64), donor weight (p=0.85) and average volume of marrow per draw of syringe (p= 0.76) were not statistically significant. Discussion: The role of tolerance induced by mesenchymal stem cells in reduction (Aggarwal 2005) and even the treatment of GVHD (Le Blanc et al. 2004; Baron and Storb 2012) has been reported elsewhere and supports our findings though this is yet to be established (Fisher et al. 2019). Simple quality control of BM could be highly beneficial in all settings in minimising acute GVHD. This is particularly important in low- and middle-income countries where GVHD can be very challenging (Saleem et al. 2018) and is associated with high costs of management and prolonged hospitalisation (Grubb et al. 2016). Our indicator is accurately measurable, inexpensive, precise and timely; further it does not involve any sophisticated equipment (flow cytometry for CD counts), thus may be widely applicable and usable. Conclusion: Quality of BM harvest, as indicated by PB to BM WBC ratio, is a simple, effective and inexpensive informative indicator in the context of G-BM. Poorer quality of marrow collection may be contributing to increased risk of acute GVHD. Disclosures No relevant conflicts of interest to declare.