Number Of Nucleated Cells Research Articles

Colony-forming potential of murine bone marrow-derived progenitor cells in experimental neurodegenerative pathology and under the impact of melatonin in vivo or in vitro

Cell therapy is a promising direction in the treatment of Parkinson’s disease/parkinsonism and multiple sclerosis, the effectiveness of which can be increased with the help of biologically active factors, in particular melatonin. The purpose of the study: to investigate the colony-forming ability of stromal and hematopoietic cells-progenitors of the bone marrow of mice with experimentally induced neurodegenerative pathology and reveal the possibility to change it under the influence of melatonin in vivo or in vitro. Materials and methods: In adult (6-7 months) male FVB/N (haplotype H-2q) and 129/Sv (haplotype H-2b) mice, the parkinsonism model was reproduced by a single subcutaneous injection of the neurotoxin 1-methyl-4-phenyl -1,2,3,6-tetrahydropyridine (MPTP) at a dose of 30 mg/kg 129/Sv mice also received the neurotoxin cuprizone daily with food (at the rate of 0.2 % of the mass of the pure substance from the daily feed) for three weeks (multiple sclerosis model). Melatonin was administered to the 129/Sv mice intraperitoneally at a dose of 1 mg/kg, daily, at 17.00, from the 8th day of receiving cuprizone, or was added in vitro to the culture of bone marrow cells of mice of both strains with parkinsonism at a dose of 0.1 μg/0.1 mL. In the bone marrow, the absolute number of nucleated cells was counted, the relative content of CD4+ lymphocytes was determined by flow cytometry, and the number of colony-forming precursor cells for fibroblasts (CFU-F) and for granulocyte-macrophages (CFU-GM) in culture in vitro was estimated. Results. In the bone marrow of 129/Sv mice with a model of multiple sclerosis, the number of nucleated cells, CFU-GM and CD4+ cells decrease, while under the influence of exogenous melatonin applied in vivo, the values of these indicators increase to the level of intact animals. After the administration of MPTP in the bone marrow of FVB/N mice, in contrast to 129/Sv mice, the number of nucleated cells and CFU-F decreases. After the incubation with melatonin of bone marrow cells of 129/Sv mice with a model of parkinsonism, an increase in the amount of CFU-F was observed compared to control values, and there were no changes in the indicator values in FVB/N mice. Conclusions. In the bone marrow of mice with the MPTP-model of parkinsonism, a decrease in the absolute amount of CFU-F was found, and in mice with the cuprizone model of multiple sclerosis – CFU-GM. The application of melatonin in vitro increases the colony-forming potential of progenitor cells for fibroblasts in the bone marrow of mice with the MPTP model of parkinsonism. The use of melatonin in vivo increases the colony-forming ability of progenitor cells for granulocyte-macrophages and the relative content of CD4+ cells in the bone marrow of mice with the cuprizone model of multiple sclerosis. Changes in the amount of CFU-F in the bone marrow in parkinsonism and under the influence of melatonin in vitro depend on the H-2 haplotype mice, which can be the basis for the development of personalized cell therapy for this pathology.

Particularities of the bone tissue regenerative properties of the proximal part of the tibia in patients with rheumatoid arthritis with multi-plane knee deformities

Background. Rheumatoid arthritis (RA) has no equal among the musculoskeletal system diseases in terms of the severity of joint damage. Knee joint damage was noted in 65–70 % of RA patients and it is the most common cause of disability. The purpose was to study the indices of osteogenic activity of stromal (mesenchymal) stem cells (MSCs) of the articular bone ends of the knee joint in patients with secondary knee osteoarthritis (KOA) due to RA in the presence of multi-plane deformations. Materials and methods. The studied material was samples of cancellous bone of the anterior and posterior parts of the plateau of the proximal part of the tibia and spongiosa from a depth of 1 cm and 3 cm from the articular surface, namely from the proximal epimetaphysis of the tibia, removed during knee arthroplasty of patients with secon­dary KOA due to RA and in patients with primary KOA in the presence of multi-plane deformations. Using the method of cloning colony-forming units of fibroblasts (CSF) of bone tissues, 38 patients were examined, including 9 patients with RA and 29 patients with primary KOA with damage to the knee joint in the presence of multi-plane deformations. Results. The total number of nucleated cells in 1 cm3 of spongiosa of the front part of the tibial plateau was 2.3 times lower compared to the parameter of this index in the posterior part (p < 0.05). The characteristics of spongy tissue in terms of the number of nucleated cells and the content of CSF in 1 cm3 from a depth of 3 cm from the articular surface of the proximal epimetaphysis of the tibia of patients with RA were almost 3 times higher compared to the indices of patients with primary KOA (p < 0.05). Conclusions. Changes in the renovation processes of bone tissue in patients with secondary KOA due to RA and in patients with primary KOA in the presence of multi-plane deformations are unidirectional, but their expressiveness is greater in patients with RA. At a depth of 3 cm from the articular surface of the proximal epimetaphysis of the tibia, the conditions for secondary fixation of the leg of the knee joint replacement are much better, all other conditions being equal, so the "survival" of the endoprosthesis with an elongated leg will be greater.

A prospective study of nanopore-targeted sequencing in the diagnosis of central nervous system infections.

This study aims to prospectively evaluate the ability of nanopore-targeted sequencing (NTS) in the detection of pathogens in cerebrospinal fluid (CSF). It was the first time combining mNGS and microbial culture to verify the NTS-positive results also using 16S rDNA amplification with Sanger sequencing. Although microbial culture was thought to be the gold standard for pathogens detection and diagnosis of infectious diseases, this study suggested that microbial culture of CSF is not the most appropriate way for diagnosing central nervous system (CNS) infection. NTS should be recommended to be used in CSF for diagnosing CNS infection. When evaluating the value of NTS for diagnosis of CNS infections, the results of CSF TNC should be combined, and NTS-positive result is observed to be more reliable in patients with CSF TNC level >10 cells/μL.

Histological changes in pulp-dentin complex in tooth subjected to traumatic occlusion and subluxation

BackgroundThis study evaluated the influence of traumatic occlusion in the dentin-pulp complex a molar teeth submitted to subluxation. Material and methodsNinety Wistar rats were divided into groups Naïve (N), Subluxation (S) and Subluxation with traumatic occlusion (STO) and submitted to histological analysis after 7 and 21 days. A quantitative analysis was submitted to one-way ANOVA and Tukey's post-hoc test, and Chi-square and Bonferronís post-hoc test. ResultsS and STO showed a significant increase in blood vessels area (p < 0.0005), amorphous fundamental substance (p < 0.0005) and reactionary dentin formation (p < 0.0005), as well as a decrease in the nuclear profile (p < 0.0005), odontoblast layer (p = 0.013 and p < 0.0005) by day 7 when compared with N. These changes normalized by day 21, except for the reactionary dentin (p < 0.0005) in both S and STO groups. Interestingly, the STO group exhibited significant changes in the increase of pulp calcification (p < 0.0005), presence of tubules with nuclei (p < 0.0005), and inflammatory infiltrate (p < 0.0005), as well reduction of nuclear profile (p < 0.0005), odontoblast layer (p < 0.0005) compared with N and S at day 21. ConclusionsSTO impaired the defence response and decreased pulp regeneration capacity by increasing the inflammatory infiltrate and pulp calcification, and decreasing the nucleated cell number in the odontoblast layer and central pulp.

The Outcomes of Cord Blood Transplantations Infused with Higher Total Number of Nucleated Cells Were Equivalent to Unrelated Bone Marrow Transplantations

Introduction: Choosing a suitable donor is essential to achieve a good response for allogeneic hematopoietic cell transplantation (allo-HCT). Several previous studies have compared unrelated bone marrow transplantation (UR-BMT) and cord blood transplantation (CBT), but the results differed among each study, and their superiority has been controversial. The total number of nucleated cells (TNC) in cord blood unit is crucial for CBT to avoid early complications, including engraftment failure. The outcome of CBT may improve by choosing cord blood unit with a higher TNC. Methods: We retrospectively compared the outcomes of CBT with higher TNC (hCBT) and UR-BMT conducted in our institute from January 1st, 2013, to May 31st, 2023. Cut off point of TNC was the median value. The primary endpoint was overall survival (OS), and the secondary endpoints were non-relapse mortality (NRM), cumulative incidence of relapse, and cumulative incidence of complications. Results: We analyzed a total of 316 patients, including 139 patients who underwent CBT and 177 patients who received UR-BMT. The median infused TNC was 2.71 (range, 0.027-6.42) ×10^7/kg. hCBT group included 69 patients. Significantly more patients in the hCBT group had prior allo-HCTs (hCBT: 18.8%, UR-BMT: 5.6%, p=0.003), and hCBT procedures were more frequently conducted since 2018 (hCBT: 66.7%, UR-BMT: 36.7%, p&amp;lt;0.001). For each group, most patients received tacrolimus and short-term methotrexate as graft-versus-host disease prophylaxis (hCBT: 94.2%, UR-BMT: 95.5%, p=0.277). ABO matched donors were more commonly chosen for UR-BMT (hCBT: 24.6%, UR-BMT: 46.3%, p=0.006). Patients with myelodysplastic syndrome underwent UR-BMT more frequently (hCBT: 7.2%, UR-BMT: 20.9%). Patients with a high or very high disease risk index were more frequently included in the hCBT group (hCBT: 37.5%, UR-BMT: 27.9%, p=0.060). Patient sex, age, days from diagnosis to transplantation, and hematopoietic cell transplantation comorbidity index, conditioning intensity were well balanced between each group. The two-year OS of hCBT was equivalent to UR-BMT (hCBT: 53.6%, UR-BMT: 58.9%, p=0.56) (Figure 1). There was no significant difference in the two-year NRM between the two groups (hCBT: 24.9%, UR-BMT: 25.4%, p=0.87). The hCBT group had a lower neutrophil engraftment rate achieved at day 28 (hCBT: 83.0%, UR-BMT: 92.3%, p&amp;lt;0.001). The two-year relapse rate was similar between the hCBT and UR-BMT groups (hCBT: 26.1%, UR-BMT: 18.7%, p=0.34). The cumulative incidence of acute GVHD tended to be lower in the hCBT group (hCBT: 18.2%, UR-BMT: 33.6% at 100 days, p=0.067), and the same results were observed when analyzing grade 2-4 or grade 3-4 acute GVHD. The cumulative incidence of chronic GVHD was significantly lower in the hCBT group (hCBT: 4.7%, UR-BMT: 48.3%, p&amp;lt;0.001), and the cumulative incidence of severe chronic GVHD was also lower in hCBT group (hCBT: 0.0%, UR-BMT: 13.9%, p&amp;lt;0.001). To reduce the impact of patients' characteristics, propensity score matching was conducted. The covariates included prior allo-HCTs, disease risk index, and allo-HCT conducted before 2018 or not. After propensity score matching, a total of 116 patients were included in the analysis with 58 patients for each group. The two-year OS of hCBT was also equivalent to UR-BMT (hCBT: 61.8%, UR-BMT: 55.2%, p=0.43) (Figure 2). The two-year NRM was slightly lower in hCBT group, but it is not significant (hCBT: 19.1%, UR-BMT: 30.1%, p=0.21). The hCBT group had a late neutrophil engraftment (hCBT: 87.0%, UR-BMT: 89.1% at day 28, p&amp;lt;0.001). The two-year relapse rate was similar between the hCBT and UR-BMT groups (hCBT: 25.0%, UR-BMT: 20.0%, p=0.54). The cumulative incidence of acute GVHD was tended to be lower in hCBT group (hCBT: 19.8%, UR-BMT: 38.7% at 100 days, p=0.057) and the cumulative incidence of chronic GVHD was significantly lower in hCBT group (hCBT: 5.3%, UR-BMT: 56.3%, p&amp;lt;0.001). Discussion: This was the first study to validate that the outcomes of CBT with higher TNC were equivalent to UR-BMT. Although the hCBT group experienced later neutrophil engraftment, the rate of neutrophil engraftment achieved at day 28 was comparable to UR-BMT, and a lower incidence rate of chronic GVHD might be attributed to lower NRM. From these findings, hCBT may be a worth considering option for patients requiring allo-HCT.

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.

The effect of caffeine intake and passive smoking on umbilical cord blood unit's quality parameters.

Today cord blood (CB) is a valuable source of hematopoietic stem cells to treat many hematological disorders. One of the limitations of CB utilization is the reduced number of nucleated cells including stem cells. Therefore, CB banks around the world have developed strategies in an attempt to improve donor selection and the quality of the CB inventory. This study aimed to determine the impact of passive smoking and caffeine consumption on CB quality. CBs were obtained from mothers who gave birth at King Abdulaziz Medical City. All mothers gave their informed consent. Personal interviews about the mother's demographics, smoking status and exposure, and caffeine consumption executed, followed by a chart review to analyze maternal and neonatal factors. Laboratory testing was performed on all collected CB units. Using descriptive statistics, maternal and newborn factors were analyzed. T-test or Mann-Whitney U Test, as appropriate, for continuous variables analysis to study the effect of second hand smoking and coffee consumption for the primary outcome. Our study demonstrated a reduction in CB MNC, including lymphocytes, in caffeine consumers among pregnant donors, as well as a reduction in cell potency activities, including total CFU and BFU-E. The effect of passive cigarette smoking on the same cohort was insignificant. Outcome of this study will help in optimizing the quality and quantity of stem cell harvesting from CB to get the maximum benefit and such knowledge will raise the awareness among pregnant women.

Trolox Antioxidant as a Factor in Stabilization of Human Cord Blood Nucleated Cells During Cryopreservation

The paper presents experimental data on the determination of preservation rate of human cord blood nucleated cells and their apoptosis/necrosis stages to determine the number of viable functionally active cells after cryopreservation in solutions with different concentrations of DMSO and the water-soluble analogue of vitamin E, the antioxidant trolox. Counting the number of human cord blood nucleated cells after freezing in media with the addition of trolox revealed their maximum preservation in the samples with 7.5% DMSO and 50, 70 or 200 μM of the antioxidant. Using the flow cytometry with the addition of the Annexin V FITC reagent, which specifically binds to phospholipids, and the DNA dye 7-amino-actinomycin D (7-AAD), it was established that trolox in concentrations of 50–70 μM provided an increase in the number of viable cells with intact membrane (AnnexinV─/7AAD─) by 12–16% compared to the control, which involved the use of only DMSO in the cryoprotective solution. The obtained results indicate the effectiveness of using the antioxidant trolox and the prospects of developing trolox-containing cryoprotective mixtures for freezing and long-term storage of nucleated cord blood cells, including hematopoietic progenitor cells.

5599228 PITFALLS IN DIAGNOSING THROMBOTIC THROMBOCYTOPENIC PURPURA IN SICKLE CELL DISEASE

Background: Thrombotic microangiopathies are diagnosed by thrombocytopenia, microangiopathic haemolytic anaemia and evidence of end organ damage. In TTP this includes neurological symptoms, renal impairment and cardiac microthrombi. Confirmation of the diagnosis is severe deficiency (<10%) of the metalloproteinase ADAMTS13 and/or the presence of an inhibitor. The diagnosis of TTP in sickle cell disease (SCD) can be challenging as SCD is characterised by a chronic state of haemolysis often with very abnormal peripheral blood morphology so clinical features suggestive of TTP can be due to complications of SCD. More specifically, the coexistence of neurological impairment and thrombocytopenia seen in fat embolism syndrome (FES) complicating SCD can lead to an erroneous diagnosis of TTP. FES typically affects non-homozygous patients and those with a previously mild course of their illness manifesting with respiratory failure, neurological signs, thrombocytopenia and potential involvement of any system leading to high morbidity and mortality. Peripheral blood morphology typically shows very high number of nucleated red blood cells (nRBC). Aims: To identify published cases of TTP in patients with SCD and review the diagnostic approach. Methods: A PubMed literature search was performed using the terms “sickle cell” and “thrombotic thrombocytopenic purpura”/”TTP”. Results: Of 19 cases identified, 9 were individual case reports and a case series of 10. All patients had very mild course of their SCD. The median nadir platelet count was 38 x 109/L (7-70). 84% had neurological involvement either at presentation or shortly afterwards; primarily altered sensorium/drop in GCS. 79% patients also had severe type I respiratory failure either preceding or occurring simultaneously with the development of neurological signs and associated with extensive changes on chest imaging. Schistocytes were identified in all cases but in the vast majority at relatively low levels; at the same time, all showed large numbers of nRBCs. 18 of the 19 cases were published after 1998; in 15 there was no mention of ADAMTS13 testing at all. In the 3 cases with ADAMTS13 levels, all were normal; 2 samples were obtained before therapeutic plasma exchange (TPE) and 1 after. 18 patients received TPE and 1 an infusion of donor plasma. However, prior to TPE, all patients had already received red cell transfusion achieving Hb S levels of 30% or less. There were 2 (11%) deaths while the remainder of patients achieved complete recovery. One due to intracranial haemorrhage and the other, sepsis in the context of immunosuppression for prevention of TTP recurrence. The presence of early respiratory failure, the inconclusive morphological findings, the degree of thrombocytopenia, and the complete absence of confirmatory ADAMTS13 results bring the diagnosis of TTP to question in all cases. Given the clinical presentations, we suspect that some of these patients had FES. The positive outcomes may be explained by the fact that all patients received adequate red cell transfusion, but also the possible beneficial effect of TPE in acute complications of SCD with a hyper inflammatory component such as FES. Conclusion: The diagnosis of TTP in patients with SCD is challenging due to many overlapping features. It should not be made without confirmatory ADAMTS13 results and the possibility of alternative diagnoses should be explored.

Validation of long-term handling and storage conditions for hematopoietic stem cell products for autologous transplants.

Hematopoietic stem cells (HPSCs) are multipotent stem cells that can differentiate into lymphoid and myeloid progenitors, giving rise to white blood cells (WBCs), red blood cells (RBCs), and platelets. HPSCs are a widely used treatment for many hematological non-malignant and malignant disorders. HPSCs can be used in the fresh or cryopreserved state for future use. Fresh HPSCs are typically stored at 2-6°C for up to 72 hours and are primarily used for allogeneic transplants or autologous transplants in myeloma and lymphoma patients. However, in some cases of autologous donations, HPSC transplantation is delayed more than three days after collection. In such situations, the cells are thawed after short-term preservation, resulting in a 35% cell viability loss. This study aimed to investigate the quality of HPSCs products after long-term storage exceeding 72 hours. The quality of HPSCs products was assessed by measuring viable CD34+ cell count, the total number of nucleated cells (TNC), and HPSCs recovery after different storage intervals of up to 120 hours in hypothermal storage. The mean total cell viability decreased by 2.18% within 72 hours and 7.4% within 120 hours, while mean CD34+ cell recovery was 92.61 % at 72 hours and 83.83 % at 120 hours in hypothermal storage. The mean TNC recovery was 89.93% at 72 hours and 76.18 % at 120 hours. All products were free from bacterial contamination for up to 120 hours under hypothermal storage conditions.

Evaluation of bone marrow aspirates using the automated hematology analyzer Sysmex XN-3000.

This study evaluated the feasibility of the Sysmex XN-3000 automated hematology analyzer for the assessment of total nucleated cells (TNC) and bone marrow (BM) cell density in routine bone marrow aspiration (BMA) samples. A total of 54 BMA samples from 39 hematological patients were evaluated. The number of megakaryocytes was calculated by a specific gating algorithm using the body fluid mode of the WBC differential (WDF) channel. Lipid contents were calculated through a newly developed algorithm utilizing the WDF channel. The ratio of lipid particles over TNCs by the WNR channel was compared with the BM cellularity assessed by the BM biopsy. The myeloid/erythroid (M/E) ratio was calculated by measuring the number of myeloid cells in the WDF channel and the number of nucleated red blood cells (NRBCs) in the WNR channel. XN-3000 counts and microscopic results showed a linear correlation in TNC (R2 =.98, p < .001), megakaryocytes (R2 =.59, p=.002), NRBC (R2 =.84, p < .001), and M/E ratio (R2 =.59, p < .001). There were significant differences in the lipid/TNC ratios of hypercellular, normocellular, and hypocellular BMs measured by XN-3000 (p < .001). Receiver-operating characteristic analysis detected cut-off values of the lipid/TNC ratio of >0.4054 for hypoplasia and <0.157 for hyperplasia. The sensitivity and specificity for hypoplasia were 100% and 88%, and for hyperplasia were 89% and 86%, respectively. XN-3000 provides a quantitative assessment of BM cellularity, supporting the qualitative assessment by myelogram and BM biopsy.

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.

Effect of Hypoxia-Supported Umbilical Cord Mesenchymal Stem Cells on the Expansion of Cord Blood Mononuclear Cells in vitro

To explore the effect of hypoxia-supported umbilical cord mesenchymal stem cell (UC-MSC) on the expansion of cord blood mononuclear cell （MNC） in vitro. The isolated cord blood mononuclear cells were inoculated on the preestablished umbilical cord mesenchymal stem cell layer and cultured under hypoxic conditions (3％ O2) and the experimental groups were normoxia (MNCs were cultured under normoxic conditions), hypoxia (MNCs were cultured under hypoxic conditions), UC-MSC (MNCs were cultured with UC-MSC under normoxic conditions), and UC-MSC+hypoxia (MNCs were cultured with UC-MSC under hypoxic conditions). To further investigate the combinational effect of 3 factors of SCF+FL+TPO (SFT) on expansion of cord blood MNCs in vitro in hypoxia-supported UC-MSC culture system, the experiments were further divided into group A (MNCs were cultured with UC-MSC and SFT under normoxic conditions), group B (MNCs were cultured with UC-MSC under hypoxic conditions), group C (MNCs were cultured with UC-MSC and SFT under hypoxic conditions). The number of nucleated cells (TNC), CD34+ cell, CFU and CD34+CXCR4+, CD34+CD49d+, CD34+CD62L+ cells of each groups were detected at 0, 7, 10 and 14 days, respectively. Compared with group hypoxia and UC-MSC, group UC-MSC+hypoxia effectively promoted the expansion of TNC, CD34+ cell and CFU, and upregulated the expression level of adhesion molecule and CxCR4 of the cord blood CD34+ cell（P<0.05）. After culturing for 14 days, compared with group A and group B, group C effectively promoted the expansion of cord blood MNC at different time points（P<0.05）, and the effect of group A was better than that of group B at 7 and 10 days（P<0.05）. Hypoxia-supported UC-MSC efficiently promoted the expansion and expression of adhesion molecule and CXCR4 of cord blood CD34+ cell, and the effect of expansion could be enhanced when SFT 3 factors were added.

Cord Blood Plasma and Placental Mesenchymal Stem Cells-Derived Exosomes Increase Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells While Maintaining Their Stemness.

Mesenchymal stem cells (MSCs) have been used for ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) to maintain their primitive characters and long-term reconstitution abilities during transplantation. Therapeutic effects of MSCs mainly rely on paracrine mechanisms, including secretion of exosomes (Exos). The objective of this study was to examine the effect of cord blood plasma (CBP)-derived Exos (CBP Exos) and Placental MSCs-derived Exos (MSCs Exos) on the expansion of UCB HSCs to increase their numbers and keep their primitive characteristics. CD34+ cells were isolated from UCB, cultured for 10 days, and the expanded HSCs were sub-cultured in semisolid methylcellulose media for primitive colony forming units (CFUs) assay. MSCs were cultured from placental chorionic plates. CBP Exos and MSCs Exos compared with the control group significantly increased the number of total nucleated cells (TNCs), invitro expansion of CD34+ cells, primitive subpopulations of CD34+38+ and CD34+38-Lin- cells (p &lt; 0.001). The expanded cells showed a significantly higher number of total CFUs in the Exos groups (p &lt; 0.01). CBP- and placental-derived exosomes are associated with significant ex vivo expansion of UCB HSCs, while maintaining their primitive characters and may eliminate the need for transplantation of an additional unit of UCB.

Effect of wheat-grain moxibustion on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition

To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition, and to explore the possible mechanism of wheat-grain moxibustion in treating bone marrow inhibition. Forty-five SPF male CD1(ICR) mice were randomly divided into a blank group, a model group and a wheat-grain moxibustion group, 15 mice in each group. The bone marrow inhibition model was established by intraperitoneal injection of 80 mg/kg of cyclophosphamide (CTX). The mice in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), 3 moxa cones per acupoint, 30 s per moxa cone, once a day, for 7 consecutive days. The white blood cell count (WBC) was measured before modeling, before intervention and 3, 5 d and 7 d into intervention. After intervention, the general situation of mice was observed; the number of nucleated cells in bone marrow was detected; the serum levels of interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were measured by ELISA; the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc in bone marrow cells was measured by Western blot and real-time PCR method. Compared with the blank group, the mice in the model group showed sluggish reaction, unstable gait, decreased body weight, and the WBC, number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were decreased (P<0.01), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was decreased (P<0.01). Compared with the model group, the mice in the wheat-grain moxibustion group showed better general condition, and WBC, the number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were increased (P<0.01, P<0.05), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was increased (P<0.05). Wheat-grain moxibustion shows therapeutic effect on bone marrow inhibition, and its mechanism may be related to activating Wnt/β-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.

The Composition of Adipose-Derived Regenerative Cells Isolated from Lipoaspirate Using a Point of Care System Does Not Depend on the Subject’s Individual Age, Sex, Body Mass Index and Ethnicity

Uncultured, unmodified, autologous, adipose-derived regenerative cells (UA-ADRCs) are a safe and effective treatment option for various musculoskeletal pathologies. However, it is unknown whether the composition of the final cell suspension systematically varies with the subject's individual age, sex, body mass index and ethnicity. UA-ADRCs were isolated from lipoaspirate from n = 232 subjects undergoing elective lipoplasty using the Transpose RT system (InGeneron, Inc.; Houston, TX, USA). The UA-ADRCs were assessed for the number of nucleated cells, cell viability and the number of viable nucleated cells per gram of adipose tissue harvested. Cells from n = 37 subjects were further characterized using four-channel flow cytometry. The present study shows, for the first time, that key characteristics of UA-ADRCs can be independent of the subject's age, sex, BMI and ethnicity. This result has important implications for the general applicability of UA-ADRCs in regeneration of musculoskeletal tissue. Future studies must determine whether the independence of key characteristics of UA-ADRCs of the subject's individual age, sex, BMI and ethnicity only applies to the system used in the present study, or also to others of the more than 25 different experimental methods and commercially available systems used to isolate UA-ADRCs from lipoaspirate that have been described in the literature.

Skewed fate and hematopoiesis of CD34+ HSPCs in umbilical cord blood amid the COVID-19 pandemic.

Umbilical cord blood (UCB) is an irreplaceable source for hematopoietic stem progenitor cells (HSPCs). However, the effects of SARS-CoV-2 infection and COVID-19 vaccination on UCB phenotype, specifically the HSPCs therein, are currently unknown. We thus evaluated any effects of SARS-CoV-2 infection and/or COVID-19 vaccination from the mother on the fate and functionalities of HSPCs in the UCB. The numbers and frequencies of HSPCs in the UCB decreased significantly in donors with previous SARS-CoV-2 infection and more so with COVID-19 vaccination via the induction of apoptosis, likely mediated by IFN-γ-dependent pathways. Two independent hematopoiesis assays, a colony forming unit assay and a mouse humanization assay, revealed skewed hematopoiesis of HSPCs obtained from donors delivered from mothers with SARS-CoV-2 infection history. These results indicate that SARS-CoV-2 infection and COVID-19 vaccination impair the functionalities and survivability of HSPCs in the UCB, which would make unprecedented concerns on the future of HSPC-based therapies.

EVALUATION OF PREDICTABILITY OF BONE MARROW CELL CELLULARITY FROM RELATED AND UNRELATED DONORS

Introduction: For successful hematopoietic stem cell transplantation, a minimum number of total nucleated cells should be obtained (TNC) during the bone marrow (BM) harvest procedure. Objectives: Evaluate the predictability of the BM TNC collected from an interim sample collected during the procedure and the factors related to high-cellular harvest. Methodology: This is a retrospective observational study including BM donors and recipient from 2017 to 2019. The final TNC was based on an interim quantification of TNC and was compared with the actual final TNC obtained. Results: 81 donors were included and interim TNC of 53 donors were available. Based on linear regression, a significant correlation was found between the volume of BM collected and the interim and final TNC (n=53; R2=0.83; P&lt;0.001). The relationship between donor and recipient weight significantly influenced the collection yield. There was also a positive correlation between the volume of BM collected and the interim and final count of TNC (n=53; r=0.88; p&lt;0.001). The difference in donor and recipient weight also had a positive correlation (p&lt;0.02). Conclusion: Our results showed that interim TNC quantification can help to achieve a better performance during the procedure, allowing real-time re-estimation of the volume needed to be harvested.

The 48th Annual Meeting of the European Society for Blood and Marrow Transplantation: Physicians - Poster Session (P001 - P578).

Background: Bone marrow donation is a procedure that takes place in a hospital operating room. In many hospitals, autologous red cell units are collected from donor before the harvest and reinfused immediately after the donation to reduce the risk of anemia. The blood donation stimulates hematopoiesis, so it's possible that autotransfusion has an impact on number of collected cells. The aim of this study is to assess whether blood donation before the harvest has an impact on the total number of nucleated cells in the product. Methods: Study design: Most of the donors undergo preharvest autologous blood collection. The procedure was not carried out if the weight difference is greater than 30kg in favor of the donor. Until March 2020, due to COVID-19, routine blood donations for autotransfusion were suspended. This situation gave an opportunity to assess, if the donation of blood has an impact on the efficiency of BM collection and hemoglobin level in donors' blood after harvest. Bone marrow was aspirated under general anesthesia, from both pelvic bones. According to the local protocol the volume of marrow and WBC was measured during donation;if the total number of nucleated cells was sufficient, the donation was finished. The collection was also completed, if the total volume of BM reached 15 ml per kg of donor's body weight. Laboratory analyses: The blood samples for morphology analyses were taken in the day of qualification, one day before and one day after harvest. The samples of BM were taken during donation to measure number of lymphocytes. Statistics: TheMann-Whitney U-test was used to determine the significance between the cohorts. Differences with p values <0.05 were considered as statistically significant. Results: Between March and October 2020, 15 BM donations from 15 healthy donors (10 men and 5 women, median age: 35 years, range 20-49 years) were performed;these donors were not referred for autotransfusion. The control group consisted of 34 donors, who underwent harvest between January 2019 and March 2020 (23 men and 11 women, median age 25 years, range 18-41 years). Total volume collected BM was comparable in both groups (median: 1182 ml in first vs 1277 ml in control group). The level of hemoglobin on the day of qualification was similar in both group: median 15.2 g/dL for donors who had blood donation;and 15.4 g/dL, for donors not referred to autotransfusion. One day before harvest, the first group had statistically lower level of hemoglobin (median 14.1 g/dL vs 14.8 g/dL, p < 0.001), however one day after BM collection both groups were comparable (median 11.1 g/dL vs 11.5 g/dL). Total number of nucleated cells in the product was comparable in both groups: median 204 × 108 in donors referred for autotransfusion vs 219 × 108 in control group. Conclusions: Autologous blood transfusion had no impact on Hb levels in blood after BM donation and had no impact on the efficiency of BM harvest. This does not support the routine use of autologous blood transfusion for unrelated BM donors.

Animal Model Establishment and Its Mechanism of Cytarabine-Iduced Myelosuppression

To establish an optimized model of bone marrow suppression induced by cytarabine (Ara-C) in C57BL/6 mice and preliminarily explore the mechanism of myelosuppression based on the cycle and apoptosis of BMNC. C57BL/6 mice were intraperitoneally injected with Ara-C 50, 100 and 200 mg/kg for 7 days, respectively. The survival rate and body weight of C57BL/6 mice were monitored. The number of peripheral blood cells and bone marrow nucleated cells (BMNC) was detected, and the morphology of bone marrow, thymus and spleen were measured on the 7th, 14th and 21st day of the experiment. The cycle and apoptosis of BMNC were also detected by flow cytometry. Ara-C 200 mg/kg caused 46.7% mortality in mice, and other doses had no significant effect on mortality. All doses of Ara-C induced bone marrow suppression in mice, as shown by a decrease in the number of peripheral blood cells (WBC, Neu, RBC, PLT) and BMNC (P<0.05), decrease in bone marrow hyperplasia, accompanied by immunosuppression and compensatory hematopoiesis of the spleen, and the above manifestations and duration were dose-dependent. Among them, the myelosuppression caused by Ara-C 50 mg/kg recovered quickly, and caused by Ara-C 200 mg/kg was too severe. The result of flow cytometry showed that Ara-C could cause S and G2/m arrest and increased apoptosis in BMNC. Ara-C can induce myelosuppression in mice with a dose-dependent severity and duration, and the model of myelosuppression with Ara-C 100 mg/kg is more optimized. The mechanism is related to the inhibition of BMNC proliferation and the promotion of apoptosis.