Source Of Hematopoietic Progenitor Cells Research Articles

TNFα induces Caspase-3 activity in hematopoietic progenitor cells CD34+, CD33+, and CD41 + of myelodysplastic syndromes

BackgroundCytopenia is the primary feature of Myelodysplastic Syndrome, even in the presence of hypercellular bone marrow. TNFα is recognized as both a proinflammatory, and proapoptotic cytokine with a well established role in promoting apoptosis in MDS. Therefore, TNFα has the potential to be a valuable biomarker for predicting the progression of cytopenia in MDS. This study aims to establish the role of TNFα exposure in triggering apoptosis through caspase-3 activity in CD34+, CD33+, and CD41 + cells in MDS.MethodsThis study is an in vitro comparative experimental research. Bone marrow mononuclear cells were isolated as the source of hematopoietic progenitor cells. Subsequently, CD34+, CD33+, and CD41 + cells were exposed to rhTNFα, and the caspase-3 activity was measured using flowcytometry.ResultsIn MDS CD33 + and CD41 + caspase-3 activity of rhTNFα exposed cells was significantly higher than without exposed cells. The opposite result was found in CD34 + cells, where the caspase-3 activity without rhTNFα exposed cells was significantly higher than rhTNFα exposed cells.ConclusionrhTNFα exposure led to an elevation in caspase-3 activity in MDS progenitor cells, especially in those that had differentiated into myeloid cell CD33 + and megakaryocyte cell CD41+, as opposed to the early progenitor cells CD34+.

Hematological Parameters: Comparative Evaluation of Performance between Dxh 500 and XN-10 Equipments

Introduction The development of hematopoietic stem cell transplantation (HSCT) was an extremely important milestone for treating certain malignant and non-malignant hematological diseases. The HSCT process contemplates collecting hematopoietic progenitor cells (HPC) from the donor, conditioning chemotherapy, infusion of HPC, and engraftment. Mobilized peripheral blood is the most commonly used HPC source for HSCT due to its ease of obtaining through an apheresis procedure and high progenitor cell count yields compared to bone marrow source, which requires a bone marrow aspiration procedure, usually performed in a surgical center under general anesthesia, and requires the collection of higher volumes to reach the amount of HPC desired. The total nucleated cell count (TNC) corresponds to the sum of all nucleated cells in the sample and is used as a reference for the target dose for both sources of HPC. The TNC provides essential information on the quality of the sample obtained, since an infusion of insufficient number of cells affects the ability to reconstitute the recipient's hematopoietic system. An insufficient TNC can result in poor engraftment, leading to a slow recovery of blood cell production and increasing the risk of complications such as infections and bleeding. Determination of the number of nucleated cells in bone marrow and peripheral blood samples can be performed automatically by hematological analyzers such as the Beckman Coulter DxH 500 and Sysmex XN-10. This study aims to compare the performance of those equipments in determining hematological parameters in bone marrow and apheresis material. Materials and methods We compared the results of 40 HPC samples (20 bone marrow samples and 20 peripheral blood samples collected by apheresis) analyzed in the Sysmex XN-10 and Beckman Coulter DxH 500 equipments. All these specimens were analyzed without dilution and diluted 1:10. The evaluated parameters were: TNC, hematocrit, platelets, lymphocytes, monocytes, and granulocytes. Results The analysis of the results showed a positive Pearson correlation coefficient (above 0.9) between XN-10 and DxH 500 equipment for the evaluation of TNC in diluted samples obtained by apheresis (figure 1) and by bone marrow (figure 2). In pure samples obtained by apheresis the DxH 500 equipment showed non-linearity for TNC but revealed a positive correlation with the XN-10 when evaluating pure bone marrow samples (figure 2). On the other hand, XN-10 equipment showed linearity when correlating TCN between pure and diluted samples obtained by apheresis (figure 1) and bone marrow aspiration (figure 2). Hematocrit analysis in samples obtained by apheresis showed 90% and 100% of agreement for diluted samples between the equipment and for diluted versus pure samples, respectively, when using the cutoff value of 12%. Hematocrit validation using the Beckman Coulter equipment was impaired due to the sample's viscosity and limited linearity. Also, there was a satisfactory hematocrit correlation for pure bone marrow samples and comparing diluted versus pure bone marrow samples with both equipment. The results also showed a good correlation when analyzing the platelet count in pure and diluted samples (bone marrow and apheresis) with XN-10 or DxH 500, as well as comparing the diluted samples versus the pure samples. The use of differential count of nucleated cells (lymphocytes, monocytes and granulocytes) realized by the two evaluated devices was not approved for bone marrow or apheresis samples. Conclusion The determination of TNC, platelet and hematocrit counts using the Sysmex XN-10 equipment were satisfactory for both pure and diluted samples obtained by apheresis or bone marrow. For pure samples obtained by apheresis analyzed by DxH 500, we could observe absence of linearity for TNC and hematocrit. This situation could be explained by the high number of cells in this sample and the consequent high viscosity.

Nucleated red blood cell: a feasible quality parameter of cord blood units

IntroductionUmbilical cord blood is an alternative source of hematopoietic progenitor cells for bone marrow transplantation; however, it is associated with a higher graft failure rate. The presence of a high rate of nucleated red blood cells (NRBCs) seems to be related to a greater capacity for engraftment, although is also associated with fetal distress conditions. We analyzed the correlation of the NRBC with quality parameters and its association with the utilization score of a cord blood unit. Study design and methodData of 3346 units collected in a public cord blood bank from May 2010 to December 2017 were analyzed, retrospectively, to identify factors associated with an increased number of nucleated red blood cells and its correlation with the engraftment capacity measured through total nucleated cells (TNCs) and CD34 positive cells. We also evaluated the utilization score of these units and identified an NRBC cutoff associated with a higher score. ResultsThe median volume collected was 104 mL (42–255), the pre-processing TNC count was 144.77 × 107 (95.46–477.18), the post-processing TNC count was 119.44 × 107 (42.7–477.18), the CD34 count was 4.67 × 106 (0.31–48.01), the NRBC count was 5 (0–202) and the utilization score was 0.0228 (0.00143–0.9740). The NRBC showed a correlation with the collected volume, TNC and CD34 positive cells and a higher utilization score and the receiver operating characteristic (ROC) curve analysis identified the five NRBC/100 leukocytes cutoff that correlates better with the probability of use. No association with pathological conditions and the NRBC rate was observed. ConclusionsThe NRBC is a feasible parameter for the screening of the cord blood unit (CBU) and the minimum cutoff of five NRBC/100 leukocytes can be a strategy in conjunction with the TNC to identify better units for cord blood bank sustainability.

Perspectives on establishing a public cord blood inventory in South Africa

The South African population is highly diverse, both ethnically and genetically. This diversity is particularly true for the African ancestry and various mixed ancestry population groups. These groups are under-represented in national and international bone marrow and peripheral blood donor registries, making it challenging to identify HLA-matched and mismatched unrelated donors when patients from these groups require allogeneic hematopoietic stem and progenitor cell transplantation. In most high-income countries, banked cord blood (CB) units provide an attractive source of hematopoietic progenitor cells for genetically diverse populations. SA does not have a public CB inventory, leaving many patients without access to this important treatment modality. Haploidentical transplantation provides an alternative. In recent years, the use of post-transplant cyclophosphamide has significantly reduced the incidence of graft-versus-host disease after haploidentical transplantation and has improved transplantation outcomes. However, it is difficult to identify suitable haploidentical donors in SA because of family disruption and a high prevalence of HIV. Here the authors provide a brief historical overview of the ethnic and genetic diversity of the country and region. The authors provide a southern African perspective on HLA diversity, consider the allogeneic hematopoietic stem and progenitor cell transplantation landscape and explore the need to establish a public CB bank (CBB) in SA. The health policy and regulatory frameworks that will impact on a CBB in the country SA are also explored. Finally, the authors discuss several matters we believe require attention when considering the establishment of a sustainable public CBB in the South African context.

Umbilical cord blood transplantation: a maturing technology.

The use of umbilical cord blood (CB) as a source of hematopoietic progenitor cells for patients with high-risk hematologic disorders receiving allogeneic hematopoietic cell transplantations (HCTs) has increased significantly. Single-institution and registry studies have shown a decreased relapse rate and an increased transplantation-related mortality rate with similar overall survival rates after allogeneic HCT with CB compared with other donor sources. The transplantation of double CB units has overcome the dose limitation inherent in a single CB unit and thus has markedly extended the use of CB to larger children and adults. Similarly, the use of reduced intensity conditioning in the CB transplantation setting has allowed the treatment of older patients who would be unable to tolerate the myeloablative regimens used in the original CB transplantation protocols.

Human Leukocyte Antigen and Red Blood Cells Impact Umbilical Cord Blood CD34+ Cell Viability after Thawing.

Hematopoietic progenitor cell (HPC) transplantation is a treatment option for malignant and nonmalignant diseases. Umbilical cord blood (UCB) is an important HPC source, mainly for pediatric patients. It has been demonstrated that human leukocyte antigen (HLA) matching and cell dose are the most important features impacting clinical outcomes. However, UCB matching is performed using low resolution HLA typing and it has been demonstrated that the unnoticed mismatches negatively impact the transplant. Since we found differences in CD34+ viability after thawing of UCB units matched for two different patients (p = 0.05), we presumed a possible association between CD34+ cell viability and HLA. We performed a multivariate linear model (n = 67), comprising pre-cryopreservation variables and high resolution HLA genotypes separately. We found that pre-cryopreservation red blood cells (RBC), granulocytes, and viable CD34+ cell count significantly impacted CD34+ viability after thawing, along with HLA-B or -C (R2 = 0.95, p = 0.01; R2 = 0.56, p = 0.007, respectively). Although HLA-B*40:02 may have a negative impact on CD34+ cell viability, RBC depletion significantly improves it.

Clinical and epidemiological profile of children and adolescents submitted to the hematopoietic cell transplantation

The Hematopoietic stem cell transplantation (HSCT) is used in children as a definitive treatment for various oncological, immune deficiencies, hemoglobinopathy, and malignancies diseases that involve the hematological system, congenital metabolism disorders, among others. To characterize the clinical and epidemiological profile of children and adolescents submitted to HSCT at a referral service in the state of Rio Grande do Norte. This is a quantitative, retrospective, observational, descriptive and analytical quantitative approach approaching the medical records of children and adolescents submitted to HSCT in a referral hospital service for this type of transplantation in the state of Rio Grande do Sul North (RN). The final sample consisted of 35 records patients aged between 2 and 18 years old who underwent HSCT from February 2008 to December 2015 and who presented the data necessary for the study. The records analyzed showed a little majority of male patients (51.42%) and 60.00% of these men were students and 71.42% lived in the state of the Rio Grande do Norte. According to the clinical characteristics, 34.3% of the patients had Acute Lymphoblastic Leukemia and 25.71% had Acute Myeloid Leukemia as the main diagnosis. Gastrointestinal toxicities were the most frequent (97.1%) and all patients received antineoplastic/chemotherapeutic and antiemetic treatment. The allogeneic HSCT was the most frequently performed (57.14%) and the most used source of Hematopoietic progenitor cells (HPC) was the peripheral blood (54.29%) and 5.71% of these patients developed the Graft versus Host Disease (GVHD), of which one was affected by acute GVHD and another by chronic GVHD. Septsis was the most frequent cause of death (60%). The profile of the clinical variables presented by the children and adolescents of this study shows that the most prevalent diagnosis was ALL, the most frequent toxicities were gastrointestinal, cardiac, respiratory and hematological, the most common HSCT was allogeneic peripheral blood and the greatest cause of mortality was sepsis. These data are similar to studies conducted in North America, Europe and Asia.

CXCL12/CXCR4 Signaling Enhances Human PSC-Derived Hematopoietic ProgenitorFunction and Overcomes Early InVivo Transplantation Failure.

SummaryHuman pluripotent stem cells (hPSCs) generate hematopoietic progenitor cells (HPCs) but fail to engraft xenograft models used to detect adult/somatic hematopoietic stem cells (HSCs) from donors. Recent progress to derive hPSC-derived HSCs has relied on cell-autonomous forced expression of transcription factors; however, the relationship of bone marrow to transplanted cells remains unknown. Here, we quantified a failure of hPSC-HPCs to survive even 24 hr post transplantation. Across several hPSC-HPC differentiation methodologies, we identified the lack of CXCR4 expression and function. Ectopic CXCR4 conferred CXCL12 ligand-dependent signaling of hPSC-HPCs in biochemical assays and increased migration/chemotaxis, hematopoietic progenitor capacity, and survival and proliferation following in vivo transplantation. This was accompanied by a transcriptional shift of hPSC-HPCs toward somatic/adult sources, but this approach failed to produce long-term HSC xenograft reconstitution. Our results reveal that networks involving CXCR4 should be targeted to generate putative HSCs with in vivo function from hPSCs.

Review and outlook on unrelated cord blood transplantation for treatment of malignant hematological diseases in adults

There are currently more and more unrelated cord blood transplants (UCBT) performed in adults patients with hematological malignancy. Umbilical cord blood is an important hematopoietic progenitor cell source for patients. Recent studies showed comparable curative effect among patients of acute leukemia receiving UCBT, haplo-identical hematopoietic stem cell transplantation, unrelated bone marrow transplantation and peripheral blood stem cell transplantation. The advantage of single UCBT has low incidence rate of graft versus host disease (GVHD) and is easy to control. Also, single UCBT maintains better graft versus tumor (GVT) effects and better quality of life. However, the disadvantage of UCBT is that the number of umbilical cord blood is so low and that the engraftment of UCBT is slow, especially in UCBT for adults. The immune reconstitution after UCBT is also slow, and results in early infection after transplantation. Improve engraftment and reconstitute immune system can significantly improve clinical outcomes. In this review, we further advance the usage of UCBT in adult patients with malignant hematological disease, and prospect the methods to enhance hematopoietic stem cell expansion, improve engraftment, reconstitute immune system and select the best human leukocyte antigen (HLA) matching. Key words: Umbilical cord blood transplantation; Malignant hematological disease; Graft vs host disease; Graft versus tumor effect; Cord blood bank

Total leukocyte count-based predictor tool for calculating hematopoietic progenitor cell dose in bone-marrow harvest

Background: Hematopoietic progenitor cell transplantation is increasingly being used as the curative therapy in India for various clinical conditions. There are three main sources of hematopoietic progenitor cells; hematopoietic progenitor cell-apheresis (HPC-A), hematopoietic progenitor cell-bone marrow (HPC-M) and hematopoietic progenitor cell-Umbilical Cord (HPC-C). The number of CD34+ cells in HPC-C is fixed. In HPC-A, the number of CD34+ cells collected is based on the baseline peripheral blood counts and a second harvest can be easily performed. The trickiest calculation of CD34+ cells adequacy is in case of HPC-M harvest, where the end point of harvest cannot be predetermined. Aim: The aim was to study the accuracy of a total leukocyte count (TLC)-based predictor tool in predicting CD34+ dose in comparison to the actual CD34+ cell counts in the harvest. Materials and Methods: This was a prospective study to validate the tool. The data captured included patient and donor demographic data, disease condition, mobilization regimen of the donor, progenitor cell harvest data, dose collected, cryopreservation if any, progenitor cell infusion data, engraftment, and follow-up of the patient including day thirty and day hundred chimerism in a tertiary care hospital. Results: Five patients were included in the study. For each patient, the target dose and volume were collected in a single HPC-M harvest attempt, and no repeat harvests were required. The average volume of HPC-M harvest collected was 195 ml. The average CD-34+ cell dose in HPC-M harvest achieved was 4.3 × 106/kg (Range = 3.39–6.42). This was 85.7% of the targeted dose calculated on the basis of TLC-based predictor tool. Conclusion: This study reiterates the importance of a simple TLC-based predictor tool (formula) for estimation of HPC-M volume to be harvested.

"Tailoring" Hematopoietic Progenitor Cell Collection: Impact of a Data-Driven Prediction Algorithm for Blood Volume Processing in Large Volume Leukapheresis

Background:The predominant source of hematopoietic progenitor cells (HPCs) for hematopoietic stem cell transplantation (HSCT) is peripheral blood (PB). HPCs are collected for HSCT using large volume leukapheresis (LVL) that typically involves processing >4 blood volumes to ensure adequate yield of HPCs. LVL is time-intensive, causes volume overload and hypocalcemia, and in small children, requires prolonged sedation. The purpose of this quality improvement project was to assess whether a collection efficiency-based, data-driven prediction algorithm for HPC collection can meaningfully decease the volume of blood processed in well-mobilized patients.MethodsWe evaluated the CD34+ cellular collection efficiency 2 (CE2; see below) in 240 consecutive adult autologous (A-auto), 59 adult allogeneic (A-allo), and 23 pediatric autologous (P-auto) collections ("training set"). In the training set, mean (1SD) CD34+ CE2s in A-auto, A-allo and P-auto collections were 47% (16%), 51% (14%), and 59% (32%), respectively (Fig. 1).CD34 Collection Efficiency 2 (CE2) was calculated as below:CD34+ CE2= CD34+ yield/[BVP x Precollection PB CD34 count]Blood Volume to be processed (BVP) was calculated as below:BVP= CD34+ cell target/ [CD34+ CE2 x Precollection PB CD34+ count]In order to have a high level of confidence that the targeted dose of CD34+ cells will be collected with implementation of the prediction formula, the CD34+ CE2 was set at the 3rd percentile (30%) in the A-auto group, and lowest attained CD34+ CE2 in the A-allo (30%) and P-auto groups (25%) for the A-auto, A-allo and P-auto prediction formulae, respectively. These CD34+ CE2s were used to calculate the BVP in a large cohort of consecutive patients from Froedtert Memorial Lutheran Hospital and the Children's Hospital Wisconsin which included 461 A-Auto, 90 A-Allo and 16 P-Auto collections over a period of 16 months (2/2015- 5/2016). Data was collected from both prediction-driven truncated and standard non-truncated LVLs and comparisons were made with regards to blood volume processed, duration of LVL, financial impact and time to neutrophil and platelet engraftment.ResultsAfter implementation of the BVP prediction formula, 91 truncations were performed in 42 (9%), 41 (46%) and 8 (50%) of 461 A-auto, 90 A-allo and 16 P-auto collections, respectively, representing an overall truncation rate of 16% (Fig. 2). Of the 91 total procedures truncated, 4 procedures (all A-Auto) failed to reach the CD34+ collection target, however, cell yields were extremely close to targets and were not deemed to be prediction failures (Table 1). In truncated LVL procedures, reduction in volume of blood processed was 34% overall, with average decreased processing of 31%, 36% and 46% (of 4 blood volumes) in A-auto, A-allo and P-auto collections, respectively (Fig. 3). The average time for a randomly selected group of 20 standard 4BV collections was 4 hr 49 mins. A 34% truncation in BVP would result in the procedure concluding 1 hr 38 mins earlier and this prediction formula based intervention is predicted to save on average $132 per patient ($35/hr Nursing time; $25/hr Cell Processing technologist time, and 35% overhead) and total savings of $12,039 over 91 truncated procedures during the 16 month period assessed. Median neutrophil and platelet engraftment were not significantly different in autologous HSCT patients with truncated vs. non-truncated procedures (10 days each for neutrophil engraftment, and 20 days vs. 19 days in truncated vs. non-truncated procedures for platelet engraftment).ConclusionThe use of a data-driven HPC prediction algorithm resulted in the truncation of 16% of all LVL procedures. The volume of truncation was significant in most cases and did not adversely impact the ability to collect the desired number of stem cells and resulted in comparable neutrophil and platelet engraftment time. This approach may be particularly important in mitigating risks of LVL in populations with higher concern for adverse events such as children, and patients with poor tolerance for LVL-induced volume overload. Finally, in our experience, this approach has resulted in enhancing patient satisfaction associated with shorter procedures and promotes optimal use of healthcare resources by limiting costs. [Display omitted] DisclosuresPadmanabhan:Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591; Terumo BCT: Consultancy, Honoraria; Schlesinger & Associates: Consultancy, Honoraria; LEK Consulting: Consultancy, Honoraria; Mallinckrodt Pharmaceuticals: Consultancy, Honoraria; Fenwal (Fresenius Kabi): Research Funding.

Novel Techniques for Ex Vivo Expansion of Cord Blood: Clinical Trials.

Cord blood (CB) provides an excellent alternative source of hematopoietic progenitor cells (HPC) for patients lacking human leukocyte antigen-matched peripheral blood or bone marrow graft for transplantation. However, due to the limited cell dose in CB graft, it is associated with prolonged time to engraftment, risk of graft rejection, infections, and treatment-related mortality. To increase the cell dose, a variety of ex vivo expansion techniques have been developed. Results of traditional methods of CB expansion using cytokines alone were disappointing. Expanding CB cells with mesenchymal progenitor cells led to sizeable increase in graft content and improved engraftment. Other methods used HPC-differentiation blockers, such as nicotinamide analogs, copper chelators, inducing constitutive Notch signaling, or an aryl hydrocarbon receptor antagonist (StemReginin1). Many of these methods lead to substantial expansions of total nucleated cells and CD34+ cells, and significantly improved time to neutrophil or platelet engraftment in patients transplanted with the expanded products compared to the recipients of unmanipulated CBT. These studies differ not only in the expansion method but also with regards to the cytokines used, patient population, conditioning regimens, and transplantation practices, to name a few. Some of these methods employed expansion of a portion of CB unit in the setting of single CBT, while others in the setting of double CBT. Here, we review various procedures used for CB expansion and highlight some of the key differences. Novel methods of improving engraftment that aim at improving bone marrow homing potential of CB cells are not reviewed.

Autologous Hematopoietic Progenitor Cell Infusion Adverse Event Associated with Apheresis Product Granulocyte Content

Introduction: Autologous hematopoietic progenitor cell (HPC) transplantation after high-dose chemotherapy has been used for several years in the treatment of various diseases. Over the past few years, peripheral blood have become the standard hematopoietic progenitor cell source. This process requires frozen HPC graft storage in mechanical freezers or liquid nitrogen for their subsequent infusion. Addition of a cryoprotectant solution such as dimethylsulfoxide (DMSO) is required in order to prevent ice crystals formation during HPC freezing process. Clinical toxicity has been classically attributed to thawed HPC DMSO content. However, adverse events still have been described after DMSO removal. The amount of granulocytes in the apheresis product has been correlated to adverse event occurrence and severity. In this study, we evaluated correlation between pre leukapheresis peripheral blood granulocytes count and adverse event occurrence during thawed HPC infusion.Patients and Methods: We retrospectively analyzed data from 361 patients submitted to autologous HPC transplantation from January 1999 to December 2013. HPC collection was performed with large volume leukapheresis, in a continuous-flow blood separator (Spectra, TerumoBCT, USA), with the mononuclear cell collection protocol, and ACD-A as anticoagulant solution. The apheresis product was diluted to maintain cell concentration less than 150x109 cells/L or 300x109 cells/L for storage in mechanical freezer or liquid nitrogen, respectively. A 5% DMSO solution was added to the HPC stored in mechanical freezer, whereas 10% DMSO was added for storage in liquid nitrogen. Complete blood count and CD34+ cell determination were performed in pre collection peripheral blood and the apheresis product. CD34+ cell determination was performed in the equipment Coulter Epics XL-MCL Flow Cytometer (Beckman Coulter, USA) according to the ISHAGE protocol. The HPC graft was thawed and infused without further manipulation. Infused volume limit was 10 ml/kg b.w or 40 mL of DMSO/day. Infusion was fractionated whenever required. Antihistamine drug and corticosteroid were given before all infusion. Adverse events included: nausea, vomiting, fever; hemodynamic, neurological, gastrointestinal, renal disorders; cardiac conduction disturbances, and hypersensitivity.Results: The patients´ median age was 50±17 yo (min: 1 max: 86), weight 74±19 kg (10-143). The underlying diseases were: Multiple Myeloma, 105 (29%), NonHodgkin lymphoma, 99 (27%), Acute Myeloid Leukemia, 37 (10%), solid tumor and multiple sclerosis 32 (9%) each and Hodgkin Lymphoma, 30 (8%) patients. CBC median values: hemoglobin, 10.9±1.6 g/dl (9.8-12.0), platelet 74,2±86 x109/L (44-138), white blood cell 20.8±18.3x109/L(9.6-34.4), granulocyte 16.5±15.8x109/L (6.6-28.9), and CD34+ cell, 23±79/mm3 (12-54). Apheresis product median values: total volume 235±47 mL (215-256), white blood cell 175±113x109/L (122-257), granulocyte 43.5±67.6x109/L (13.4-93.9), and CD34+ 0.8±2.4x109/L (0.4-2.0). Total nucleated cell collected was 5.7±4.5x108/kg b.w (3.9-8.5), total CD34+ 2.5±7.3x106/kg b.w (1.2-6.3). A total of 490 (74%) apheresis products were stored in mechanical freezer. A total of 289 thawed HPC infusions occurred in the study period. Adverse event occurred in 28 (10%) cases. The most common manifestations were hypertension and nausea/vomiting. Mild or moderate adverse event occurred in 26 infusions and severe adverse event in 02 cases. There was no difference in the infused HPC variables between patients with and without adverse event. However, significant difference was found in the apheresis product variables: median granulocyte count in the group with adverse reaction 74.7±52.5x109/L (39.2-103.6) vs. 43.1±61.6x109/L (12.6 to 89.2) in the group without adverse reaction, p=0.0158; total nucleated cells 8.1± 8.3x108/kg b.w (5.8-13) vs. 5.8±4.7x108/kg b.w (3.8-8.6), p=0.0033.Conclusion: The apheresis product granulocyte count and total nucleated cells were correlated with adverse event during infusion of HPC cryopreserved without further manipulation after thawing. DisclosuresNo relevant conflicts of interest to declare.

Determination of the Volume of HPC, Apheresis Products Based on Weight in Grams

Hematopoietic progenitor cells (HPC) collected by apheresis [HPC(A)] have become the most common source of HPC used for transplantation. HPC(A) products have a more variable content of CD34+ progenitor cells than marrow or umbilical cord blood, especially when collected from autologous donors. Most often the CD34+ cell content of previous collections is used to decide whether or not additional collections are needed to reach the target CD34+ cell infusion dose. Accurate assessment of CD34+ cell counts are imperitive in order to properly determine the need for additional apheresis procedures. CD34 content is typically measured by flow cytometry, either using a dual platform method that requires determination of the percentage of CD34+ cells within the total nucleated cells or a single platform method that uses counting beads to directly determine the number of CD34+ cells in a given volume of product. Both methods depend on accurate determination of the volume of the product in order to calculate the total number of CD34+ cells that have been collected. Volume estimates of HPC source products are most often performed using the product weight in grams (g) after subtracting the weight of the bag in which the product is collected. The weight is then divided by the product’s specific gravity to obtain the volume. The specific gravity of a substance is the ratio of the density of the substance to the density of a reference substance, such as water (1 g = 1 mL). The specific gravity of whole blood has been defined as approximately 1.058, whereas the specific gravity of platelet products is approximately 1.030. The difference between the two products is a function of the cellular content. Blood has a higher specific gravity since it contains more high-density cells (red blood cells (RBCs) and granulocytes) than platelet concentrates (largely platelets and lymphocytes). The composition of marrow is similar to blood, so a specific gravity of 1.058 is used. However, there are no published data that estimates the specific gravity of HPC(A) products and since these products are enriched for cells that are more dense than platelet products but less dense than whole blood products neither of the two established specific gravities is optimal to use. We sought to determine what the specific gravity of an apheresis product truly is so as to determine the optimal factor to estimate volume from weight for HPC(A) products. To this end our laboratory sampled 54 well-mixed HPC(A) products at receipt and used a reverse pipetting method and a sensitive analytical balance to weigh 1 mL aliquots of product. A hematology analyzer was used to measure the total number of white blood cells (WBC) and RBCs per mL of product. The 54 sampled products contained on average 2.7 ± 1.1 x 10E8 WBCs per mL and 5.5 ± 2.0E8 RBCs per mL, which is similar to the average seen for 823 products collected since 2010 of 2.81 ± 1.33 x 10E8 WBCs per mL. The weight in grams was significantly associated with the total number of RBCs + WBCs in a given product (p=0.026) but not with total WBC alone, indicating that RBC content was a contributing factor. The average weight (i.e. specific gravity) of 1.0 mL HPC(A) aliquots was 1.047 ± 0.009 g, a value between that of whole blood and platelet products. Based on these data we recommend that laboratories use a factor of 1.047 to convert the weight of HPC(A) products in grams to volume to more accurately determine the number of CD34+ cells contained in a given product. DisclosuresNo relevant conflicts of interest to declare.

Application of a Modified Immunomagnetic Positive Selection Method for Isolation of Human CD34+ Stem/Progenitor from Cord Blood

Umbilical cord blood (UCB) and isolated umbilical cord blood stem cells (UCBSCs) have become an alternative source of hematopoietic progenitor cells for transplantation. The aim of this study was to test the effectiveness of some modifications of human hematopoietic stem cells isolation protocols with the intention of improving the output and viability of CD34+ cells and progenitor subpopulations progeny that can be obtained from a sample of human umbilical cord blood. By that, we contribute to current studies on the human hematopoietic stem cells (HSCs) in order to bank UCB units suitable for basic research of very long- term hematopoietic as well as for transplantation. Cord blood samples were transformed to buffy coat prior to the isolation of HSCs which was performed by two steps involving CD34 pre-enrichment using human cord blood CD34 positive selection kit and an Immunomagnetic cell separation, targeting CD34 surface antigen. CD34+ cells were immunophenotyped by four-color fluorescence, using a large panel of monoclonal antibodies (CD34/PE, CD45/FITC, CD38/APC, CD33/Per-Cy, HLA-DR/PE, CD117/APC, CD123/Per-Cy, CD105-FITC, CD56/ PE, CD14/Per-Cy, CD19/Per-Cy and CD3/APC) recognizing different lineage or activation antigens. Our results showed that the percentage of CD34+ cells in whole human cord blood samples was 0.02% of total cells. After isolation by two-step, combining CD34 pre-enrichment and Immunomagnetic isolation, the frequency of CD34+ stem cells represented 0.65% among total MNCs and 83.53% among total isolated cells. This isolation leaded to a purity of over 95% and viability of 98.60%. In addition, we found that the percentage of CD34+ cells which are CD45+ was 83.53%, whereas CD34+CD38- cells comprised 21.70%. About 70.85% of isolated CD34+ cells were characterized by the absence of human leukocyte antigen-DR (HLA-DR). Concerning the CD117, CD33, CD123 and CD105 antigens which characterize true stem cells, we found a high expression percentage among isolated HUCB CD34+ cells (81.26%, 57.14% 47.45%, 58.52% for CD117, CD33, CD123 and CD105, respectively), while a very small number displayed markers of advanced myeloid commitment, such as CD14 (Myeloid lineage, 0.7%) and CD56 (NK-cell lineage, 4.48%), or those of lymphoid differentiation: CD3 (T-cell lineage, 5.22%), and CD19 (B-cell lineage, 1.76%). After testing 12 samples of cord blood using modified positive magnetic isolation technique, no variations in subpopulations were observed from sample to sample. We conclude that our modified technique enabled us to obtain an important proportion of primitive hematopoietic progenitors, as suggested by the absence of HLA-DR and CD38, as well as the presence of CD117, CD33, CD123, and CD105 on their surface. These cells are recognized as having long term reconstitution capacity within the human CD34+cell population.

Mutation of the NPM1 gene contributes to the development of donor cell–derived acute myeloid leukemia after unrelated cord blood transplantation for acute lymphoblastic leukemia

Donor cell leukemia (DCL) is a rare but severe complication after allogeneic stem cell transplantation. Its true incidence is unknown because of a lack of correct recognition and reporting, although improvements in molecular analysis of donor-host chimerism are contributing to a better diagnosis of this complication. The mechanisms of leukemogenesis are unclear, and multiple factors can contribute to the development of DCL. In recent years, cord blood has emerged as an alternative source of hematopoietic progenitor cells, and at least 12 cases of DCL have been reported after unrelated cord blood transplantation. We report a new case of DCL after unrelated cord blood transplantation in a 44-year-old woman diagnosed as having acute lymphoblastic leukemia with t(1;19) that developed acute myeloid leukemia with normal karyotype and nucleophosmin (NPM1) mutation in donor cells. To our knowledge, this is the first report of NPM1 mutation contributing to DCL development.

Expression of early hematopoietic markers in cord and mobilized blood

G-CSF mobilized peripheral blood and cord blood are major sources of hematopoietic progenitor cells. These cells are characterized by the expression of "early" antigens. We have evaluated the coexpression of hematopoietic cell markers CD34, CD133, CD90, CDCP1, CD117 and activation antigen CD38 using multicolor flow cytometry. We show that (1) cells being positive for every single antigen form a separate population. (2) Percentage of cells expressing each "early" antigen are twice more in the cord blood than in the mobilized blood. The content of cells with complex progenitor phenotype (CD34+/CD38-/CD117, CD133+/CD34+/CD38-, CDCP1+/CD34+/CD38- etc.) is equal in mobilized and cord blood. (3) There are strong positive correlations between the expression of CD34, CD133, CD117 and CDCP1 in both groups. Positive correlation exists for CD90 with CD34, CD133, CDCP1 and CD117 only in cord blood and is not significant in mobilized blood. The analyses of early antigens coexpression with activation marker CD38 revealed that hypothesis on sequential activation and loss of expression of the aforementioned antigens is not confirmed. We assume that there is global regulation of the expression of CD34, CD133, CDCP1 and CD117. Yet expression of CD38 could be reversibly abolished during maturation of the hemapoetic cells and CD117 could be expressed not only on myeloid cells.

Avoiding room temperature storage and delayed cryopreservation provide better postthaw potency in hematopoietic progenitor cell grafts

Conditions for maintaining hematopoietic progenitor cells (HPCs) before cryopreservation remain controversial. An understanding of the impact of time and temperature during nonfrozen storage can contribute to the maintenance of the quality of products, improving transplantation outcomes. The objective of this study was to determine the influence on cell potency of thawed products from three sources of HPCs after prolonged storage at different temperatures before cryopreservation. Viable cell counts by flow cytometry and colony-forming unit (CFU) recoveries were assessed on cord blood (CB), mobilized peripheral blood stem cell (PBSC), and bone marrow (BM) samples over 72 hours using two different storage conditions, refrigerated (4-8°C) or room temperature (19-22°C). To determine the effects of delayed freezing on progenitor recoveries, paired samples were evaluated before and after cryopreservation. All samples maintained at refrigerated temperatures resulted in higher recoveries than those at room temperature in all variables assessed. Specifically, when assessing for CFU yields after thawing, the impact of time on BM resulted in a significant loss as soon as 24 hours (n = 10, 36.4 ± 28.0%, p = 0.003). This decrease was also observed for PBSCs and CB but at 48 hours of fresh storage (PBSCs n = 11, 32.7 ± 26.2%, p = 0.006; CB n = 10, 39.6 ± 26.4%, p = 0.001). Our data suggest that HPC products are better maintained at refrigerated temperatures before cryopreservation. Delaying cryopreservation should be minimized to avoid significant losses in cell potency.

Umbilical cord blood transplantation: a maturing technology

Abstract The use of umbilical cord blood (CB) as a source of hematopoietic progenitor cells for patients with high-risk hematologic disorders receiving allogeneic hematopoietic cell transplantations (HCTs) has increased significantly. Single-institution and registry studies have shown a decreased relapse rate and an increased transplantation-related mortality rate with similar overall survival rates after allogeneic HCT with CB compared with other donor sources. The transplantation of double CB units has overcome the dose limitation inherent in a single CB unit and thus has markedly extended the use of CB to larger children and adults. Similarly, the use of reduced intensity conditioning in the CB transplantation setting has allowed the treatment of older patients who would be unable to tolerate the myeloablative regimens used in the original CB transplantation protocols.

Does Umbilical Cord Blood-derived CD34+ Cell Concentration Depend on the Weight and Sex of a Full-term Infant?

Umbilical cord blood has recently been considered as an alternative source of hematopoietic progenitor cells for clinical application. Patient survival in allogenic cord blood transplantation is critically dependent on the cord blood-derived total nucleated cell count and the total CD34 cell count/kg of the body weight. A number of factors such as maternal age, gestational age, newborn's sex and weight, umbilical cord length, and placental weight can influence the volume, amount of mononuclear cells, and the CD34 cell concentration. Cord blood was collected from normal vaginal and cesarean deliveries. It was immediately processed and assessed for the total nucleated cell count and CD34 cell concentration. Assessment of maternal and neonatal parameters such as gestational age, baby's birth weight, and sex was carried out with the CD34 cell concentration. The mean CD34 cell concentration was 0.21±0.24% for the group with <2500 g (low birth weight) birth weight of the baby (n=104). The mean CD34 cell concentration was 1.84±1.12% for the group with ≥2500 g (normal birth weight) birth weight of the baby (n=396). A strong positive correlation was found between birth weight of the baby with cord blood-derived CD34 cell concentration (*P<0.0001, r=0.81). A positive correlation was found between gestational age and cord blood-derived CD34 cell concentration (*P<0.0001, r=0.31). No significant correlation was found between the baby's sex with the CD34 cell concentration and the total nucleated cell count. This study concludes that the higher the birth weight of the baby, the better the yield of the CD34 cell concentration, and hence these should be the preferred samples for infusion.