Bone Marrow Processing Research Articles

Red blood cell depletion in small-volume bone marrow processing using manipulation with third-party red blood cells: A comparison of the performance of the COBE spectra and the spectra Optia systems.

For pediatric recipients, red blood cells (RBCs) are added to bone marrow (BM) collections before low RBC volume BM processing using COBE Spectra (COBE) or Spectra Optia (Optia). However, the processing efficiency of this approach has not been evaluated. This study aimed to evaluate RBC depletion and nucleated cell subpopulation recovery rates in third-party RBC-manipulated BM products processed with the COBE or Optia. We retrospectively collected data on RBC depletion from low RBC volume BM with third-party RBCs (manipulated group) and on conventional large-volume, BM (unmanipulated group) processing performed between September 2010 and December 2021. All procedures were performed using COBE or Optia. The median residual RBC volume in the manipulated group was 9.5 ml in COBE and 2.5 ml in Optia (p=.01). The median total nucleated cell (TNC) and mononuclear cell (MNC) were comparable between the manipulated groups using each cell separator (TNC, 40.8 vs. 47.1%; MNC, 78.3 vs. 79.4%). The manipulation did not adversely affect TNC and MNC recoveries in either device. In addition, Optia achieved similar CD34+ cell recovery to that in large-BM-volume processing using the same device (147.5 vs. 184.5%, p=.112). During a follow-up period, neutrophil engraftment was achieved in all patients who received third-party RBC-manipulated grafts, and platelet engraftment was achieved in all cases, except one. The addition of third-party RBC to low RBC volume BM collections from or for pediatric patients does not have any negative impact on either RBC depletion or hematopoietic cell recovery during processing with the widely used cell separator.

Collection and Processing of Bone Marrow at 3% Oxygen Significantly Alters the Manifestation of Aged Mouse Hematopoietic Stem Cell Phenotype

Aging is an inevitable process associated with eventual deterioration of normal physiological functions. Aged hematopoiesis is associated with increased numbers of hematopoietic stem cells (HSC), but with decreased HSC functional activity (e.g. decreased engrafting capability in lethally irradiated mice and a shift in the myeloid:lymphoid bias of the engrafting HSC of the old mice, such that there are more myeloid but fewer lymphoid cells generated from HSC of the old mice). Production of HSC and progenitor (HPC) cells ex vivo is more efficient when cells are cultured in a hypoxic environment of ~ 5% oxygen tension than when cells are grown at ambient air (~21% oxygen). The bone marrow (BM) microenvironment niche that nurtures the survival and production of HSC and HPC and hematopoiesis during adult life is a hypoxic environment (~1-5% oxygen tension) compared to that of ambient air. However, almost all results of studies of young and aged mouse hematopoiesis have been based on numbers and activity of HSC and HPC that have been collected and processed in ambient air. Our recent work evaluating hematopoiesis in BM cells of young adult mice and with human cord blood cells found, through a phenomenon we designated Extra Physiological Oxygen Shock/Stress (EPHOSS), that there is a large loss of HSC with an increase of HPC within minutes of the collection of these cells in ambient air (Mantel et al., Cell, 2015). This led us to reason that perhaps what we know about aging hematopoiesis might not be entirely accurate and that a re-evaluation of aged HSC, HPC, and hematopoiesis was in order. We hypothesized that hematopoiesis in aged (~20-27 months of age) mice may not be as dysregulated as reported but that collection and processing of BM from the aged mice is more sensitive than similar cells from young (~6-16 weeks) mice to EPHOSS-induced events generated by the collection of the cells in ambient air. We evaluated BM from three different mouse strains (CB6, BALB/c, and C57Bl/6) at 20-25 months vs. 6-16 weeks of age, collected/processed in ambient air or hypoxia (3% oxygen). BM from old mice collected/processed under hypoxic conditions exhibited phenotypically increased long-term HSC and common lymphoid progenitor (CLP) numbers and decreased common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) numbers when compared to old BM collected/processed under ambient air conditions. BM collected from old C57Bl/6 mice under hypoxia had increased engrafting capability more closely matching that of young BM. This was associated with a 3.14-fold increase in the number of competitive repopulating units (representative of functional HSC) in old BM collected under hypoxic conditions compared to old BM collected in ambient air as determined through limiting dilution analysis. The myeloid:lymphoid ratio of old BM collected under hypoxia matched that of young BM collected under air. This was associated with decreased cycling of CFU-GM, BFU-E, and CFU-GEMM in old BM collected/processed in hypoxia. Enhanced numbers/function of old BM HSCs collected in hypoxia is associated with changes in expression of CXCR4 (and HSC homing capability), CCR5, stress protein levels (e.g. HSP40 etc) and ROS (both total and mitochondrial). All of these noted changes demonstrated that the old BM collected/processed under hypoxic conditions more closely resemble functionally young BM. Thus, age-related differences between the HSC/HPC populations are not as drastic as previously reported and reflect the increased sensitivity of hematopoiesis from aged mice to an artificial ambient air collection procedure. Disclosures No relevant conflicts of interest to declare.

Erythrocyte depletion from bone marrow: performance evaluation after 50 clinical-scale depletions with Spectra Optia BMC

BackgroundRed blood cell (RBC) depletion is a standard graft manipulation technique for ABO-incompatible bone marrow (BM) transplants. The BM processing module for Spectra Optia, “BMC”, was previously introduced. We here report the largest series to date of routine quality data after performing 50 clinical-scale RBC-depletions.MethodsFifty successive RBC-depletions from autologous (n = 5) and allogeneic (n = 45) BM transplants were performed with the Spectra Optia BMC apheresis suite. Product quality was assessed before and after processing for volume, RBC and leukocyte content; RBC-depletion and stem cell (CD34+ cells) recovery was calculated there from. Clinical engraftment data were collected from 26/45 allogeneic recipients.ResultsMedian RBC removal was 98.2% (range 90.8–99.1%), median CD34+ cell recovery was 93.6%, minimum recovery being 72%, total product volume was reduced to 7.5% (range 4.7–23.0%). Products engrafted with expected probability and kinetics. Performance indicators were stable over time.DiscussionSpectra Optia BMC is a robust and efficient technology for RBC-depletion and volume reduction of BM, providing near-complete RBC removal and excellent CD34+ cell recovery.

Mesenchymal Stem Cells Harvested from Vertebral Body and Iliac Crest Bone Marrow: Are They Equally Good? Comparison of Two Tissues and Two Harvesting Techniques

Introduction/Objectives Clinical applications of Mesenchymal Stem Cells (MSCs) harvested from bone marrow (BM) are well established. Iliac crest (IC) is considered to be the gold standard. Vertebral body (VB) BM represents an alternative source, promoting bone fusion in spinal operations. Existing literature, comparing ICBM and VBBM MSCs, presents limitations. Density gradient centrifugation (lymphoprep, Lmp) for BM processing is used, despite the known effectiveness on ICBM, of the red cell lysis technique (ammonium chloride, AC). This study compared the yield and differentiation capacities of MSCs in VBBM and ICBM, and evaluated the effectiveness of two harvesting procedures on both BM sources. Patient/Methods Eighteen patients undergoing idiopathic scoliosis surgery were recruited (median age 15.4 years, range 13–17). BM was aspirated intraoperatively from IC (10ml) and 12th thoracic vertebra (10ml). Aspirates were processed using AC and Lmp. MSCs were enumerated using standard CFU-F assay. Colony areas, MSCs doubling rates and outcomes of the differentiation assays were statistically analyzed. Results AC yielded more leukocytes from ICBM (23x106cells/ml), compared with VBBM (14.4x106cells/ml, p = 0.013). Lmp yielded similar numbers of mononuclear cells from ICBM (7.5x106cells/ml) and VBBM (5x106cells/ml). More MSCs were harvested from 1ml of BM aspirate using AC technique, from both anatomical sites (IC: Lmp 199MSCs/ml, AC 367MSCs/ml, p = 0.012; VB: Lmp 296MSCs/ml, AC 440MSCs/ml, p = 0.043). There were no significant differences in MSC numbers/ml and average colony areas between the sites (IC: AC 14mm2, Lmp 15mm2; VB: AC 11mm2, Lmp 13mm2) or MSCs doubling rates in days (IC: AC 2, Lmp 1.9, VB: AC 1.9, Lmp 1.8). Calcium production, after 14 days of osteogenic induction, was higher in VBBM MSCs with both harvesting methods (AC: 15-fold, p = 0.003, Lmp: 6.6-fold). Adipogenic differentiation, measured by Nile-Red absorbance on day 21 post-induction, was not significantly different between two BM sources. Glycosaminoglycan (GAG) production, after 21 days of chondrogenic induction, was higher in VBBM MSCs (AC 2.1μg/ml, Lmp 3.7μg/ml) compared with ICBM (AC 1.2μg/ml, Lmp 2.9μg/ml), but not statistically significant. Conclusion AC results in more effective MSC isolation and does not affect MSCs’ proliferation and differentiation. Compared with ICBM, VBBM is richer in MSCs with osteo- and chondrogenic differentiation capacities and can therefore represent the first choice for MSC harvesting in spinal surgery.

Bone marrow processing for transplantation using Cobe Spectra cell separator

Concentration of bone marrow aspirates is an important prerequisite prior to infusion of ABO incompatible allogeneic marrow and prior to cryopreservation and storage of autologous marrow. In this paper we present our experience in processing 15 harvested bone marrow for ABO incompatible allogeneic and autologous bone marrow (BM) transplantation using Cobe Spectra® cell separator. BM processing resulted in the median recovery of 91.5% CD34+ cells, erythrocyte depletion of 91% and volume reduction of 81%. BM processing using cell separator is safe and effective technique providing high rate of erythrocyte depletion and volume reduction, and acceptable recovery of the CD34+ cells.

Single Cell Trajectory Detection Orders Hallmarks of Early Human B Cell Development

AbstractAbstract 1044 Background:Normal B cell development in the bone marrow (BM) is a seemingly well-understood, progressive process and thus represents a suitable test system in which to apply an algorithmic approach to modeling cellular differentiation. In humans, hematopoietic stem cells form lymphoid progenitor cells that develop into pro- then pre- B cells and finally those cells that escape negative selection become immature B cells that leave the BM for the peripheral immune organs. Flow cytometry can track these stages using the expression of immunophenotypic cell surface markers, including those for progenitors (CD34, CD38), early B cell populations (CD10), as well as those of more mature B cells (CD20, IgM). Expression of the B cell transcription factor PAX5, and immune diversity conferring enzymes terminal deoxynucleotidyl transferase (TdT) and recombination-activating gene (RAG) can also be tracked at the single cell level. Regulatory signaling by factors in the BM orchestrates critical checkpoints in the B cell developmental program, such as Interleukin (IL)-7-mediated STAT5 phosphorylation and signaling downstream the preB cell receptor/B cell receptor (BCR) (p-BLNK, p-Syk, p-PLCγ2, p-Erk). Successful coordination of these signals with immunoglobulin gene rearrangement results in a burst of proliferative expansion prior to maturation/exit to the periphery. Failure of any one of these processes results in B cell deletion while certain dysregulations driven by oncogenic processes can result in malignancy.While much of this core understanding has been founded in murine models, the rarity of early B cell progenitors and lack of genetic tools has complicated definition of B cell development in humans. Using 42 parameter mass cytometry in combination with a novel single-cell trajectory finding algorithm, we have now laid a human B cell developmental process in primary human BM to an unprecedented level of detail, mapping out the expression pattern of virtually all relevant B cell immunophenotypic markers as well as intracellular enzyme, transcription factor and regulatory modification simultaneously, at the single cell level. Methods:The mononuclear cell fraction of multiple healthy human marrows was characterized by simultaneously analyzing 42 antibody parameters with mass cytometry targeting a multitude of phenotypic markers, intracellular signaling molecules, hallmarks of cell cycle and apoptosis all in the context of in vitro perturbations relevant to B cell development (including IL-7 and BCR crosslinking). The resulting multidimensional data was modeled using a novel, scalable, robust graph-based trajectory algorithm that iteratively refines a solution trajectory using random landmarks to reduce variability. Populations of interest were prospectively isolated and a novel qPCR assay was created to quantitate immunoglobulin heavy chain (IgH) rearrangement in genomic DNA. Results/Conclusion:Modeling of the resulting data was undertaken using this algorithm (termed Wanderlust) that devised and ordered cellular relationships based on the average phenotypic progression from our defined starting point, in this case, CD34+CD38- hematopoietic stem cells, in order to calculate a developmental trajectory. The predicted trajectory was then used to inform a traditional 'gating' analysis of the data and provide a higher resolution view of human B cell development than previously published. It both confirmed established steps in human B cell progression, and importantly, revealed new populations of early B cell progenitors based on expression of CD34, CD38, CD24 and TdT. These populations were corroborated to be of B-lineage and ordered as predicted based on the progressive rearrangement of the IgH locus by qPCR of extracted genomic DNA. We aligned previously unregistered key developmental checkpoints such as STAT5 activation in response to IL-7 and proliferation in response preBCR expression with traditional immunophenotypic cell populations. While predicted in silico, and then molecularly verified and staged in vitro, these regulatory events all lay within discrete cell subsets that can now be demarcated using conventional cytometric methods. Together, this provides a backbone on which to further examine both healthy regulatory events as well as the corruption of this developmental process such as in malignant or immunodeficient states. Disclosures:No relevant conflicts of interest to declare.

P5-18-07: Presence of Disseminated Tumor Cells after Adjuvant Chemotherapy in Breast Cancer and Disseminated Tumor Cells Monitoring during Secondary Adjuvant Treatment.

Abstract Introduction: Detection of disseminated tumor cells (DTC) after completion of systemic adjuvant treatment is a strong predictor of early systemic relapse and death. This analysis can discover early failure of a chosen adjuvant systemic treatment. In this study, we wanted to evaluate the value of DTC detection in bone marrow (BM) as a surrogate marker for response to docetaxel rescue treatment, to predict the effect of this treatment. Further, we wanted to compare disease free survival between patients treated with docetaxel resulting in eradication of DTC after treatment and patients treated with docetaxel where DTC persists after treatment. The follow up of the study is still ongoing. Here, we present the preliminary descriptive data from the study. Materials and Methods: A total of 1128 pts with node positive or high risk node negative disease (T1c/T2GII-IIIN0) was enrolled in the period from October 2003 to May 2008. All patients had completed primary surgery and received 6 cycles of adjuvant antracycline containing chemotherapy. The first BM aspiration was performed 8–12 weeks after termination of adjuvant chemotherapy (BM1). A second BM aspiration was performed 6 months later (BM2). The processing of BM and DTC analysis (by ICC) were performed as previsously described (Wiedswang G et al, J Clin Oncol 2003). If BM2 was positive (+) for DTC, the patient was treated with docetaxel (3qw, 6 courses) Docetaxel-treated patients were reexamined at the inclusion hospital with new BM analysis at approximately 1 month (BM3) and 13 months (BM4) after the last docetaxel infusion. Results: Of 997 patients with conclusive DTC results for both BM1 and BM2, 83 patients (8.3%) were BM1 positive and 78 (7.8%) BM2 positive. Among the BM1+ patients, 15 (18.1%) were BM2+. The concordance between BM1 and BM2 were 87%. Of the patients positive at one or both time points, the concordance was 10% (15/146). The BM1 was not significantly associated with primary tumor characteristics (although borderline significance for Grade and ER status), whereas for BM2, DTC+ patients had increased frequency of node positive disease and pN2-3 stage (p=0.001, chi-square), and were positively associated with lobular carcinoma (p=0.01, chi-square). At BM1 24.7% of the DTC+ patients had >1 DTC, and 9.9% had ≥3 DTC. For BM2, 48.2% had >1 DTC, and 25.7% had ≥3 DTC. For patients with positive BM2 receiving docetaxel, the BM3 turned DTC negative in 55 of 66 evaluable cases (83.3%), and 48 of 59 were negative in BM4 (81.4%). Of 67 patients with a conclusive BM result at either BM3 or BM4, 12 were BM positive. Of 16 patients with ≥3 DTC before docetaxel treatment, only 4 patients were positive after treatment (25%). Conclusions: DTC status after adjuvant antracycline containing chemotherapy changes during the first 9 months of FU, with increased DTC positivity among patients with pN+ disease and lobular carcinomas. After docetaxel rescue treatment, the majority of patients experience disappearance of the DTCs. The clinical significance of these results awaits mature FU data, but the present results may indicate possibility for eradication of residual disease by alternative chemotherapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-18-07.

Very Small Embryonic-Like Stem Cells with Maximum Regenerative Potential Get Discarded During Cord Blood Banking and Bone Marrow Processing for Autologous Stem Cell Therapy

Very small embryonic-like stem cells (VSELs) are possibly lost during cord blood banking and bone marrow (BM) processing for autologus stem cell therapy mainly because of their small size. The present study was conducted on human umbilical cord blood (UCB, n=6) and discarded red blood cells (RBC) fraction obtained after separation of mononuclear cells from human BM (n=6), to test this hypothesis. The results show that VSELs, which are pluripotent stem cells with maximum regenerative potential, settle along with the RBCs during Ficoll-Hypaque density separation. These cells are very small in size (3-5 μm), have high nucleo-cytoplasmic ratio, and express nuclear Oct-4, cell surface protein SSEA-4, and other pluripotent markers such as Nanog, Sox-2, Rex-1, and Tert as indicated by immunolocalization and quantitative polymerase chain reaction (Q-PCR) studies. Interestingly, a distinct population of slightly larger, round hematopoietic stem cells (HSCs) with cytoplasmic Oct-4 were detected in the "buffy" coat, which usually gets banked or used during autologus stem cell therapy. Immunohistochemical studies on the umbilical cord tissue (UCT) sections (n=3) showed the presence of nuclear Oct-4-positive VSELs and many fibroblast-like mesenchymal stem cells (MSCs) with cytoplasmic Oct-4. These VSELs with nuclear Oct-4, detected in UCB, UCT, and discarded RBC fraction obtained after BM processing, may persist throughout life, maintain tissue homeostasis, and undergo asymmetric cell division to self-renew as well as produce larger progenitor stem cells, viz. HSCs or MSCs, which follow differentiation trajectories depending on the somatic niche. Hence, it can be concluded that the true stem cells in adult body tissues are the VSELs, whereas the HSCs and MSCs are actually progenitor stem cells that arise by asymmetric cell division of VSELs. The results of the present study may help explain low efficacy reported during adult autologous stem cell trials, wherein unknowingly progenitor stem cells are injected rather than the pluripotent stem cells with maximum regenerative potential.

How do I collect and process stem cells?

How do I collect and process stem cells?

Red blood units collected from bone marrow harvests after mononuclear cell selection qualify for autologous use

After large volume bone marrow (BM) harvest, donors and patients can develop severe anaemia, because collected BM can contain up to 20% of their red cell mass. In a prospective analysis, we investigated the feasibility to recover red blood cells (RBCs) from the harvested BM and investigated whether these RBC units meet the quality requirements of the European Council. From 19 patients (median age 51 yrs, range 31-77) with acute myocardial infarction, who participated in the MYSTAR study, a median volume of 1299 ml (range, 700-1870 ml) BM was collected. During BM processing, mononuclear cells (MNC) were separated using the Cobe Spectra apheresis system and the residual RBCs were collected in a separate bag. The quality of the collected RBCs was assessed by measuring LDH, free haemoglobin, potassium and lactate. Haemolysis was calculated and the intracellular concentration of ATP, ADP, AMP was determined by HPLC. RBC units recovered from BM after MNC separation had a mean volume of 312 +/- 95 ml with a haematocrit of 47 +/- 8.9%, a haemoglobin content of 51 +/- 15 g per unit, a haemolysis of 0.15 +/- 0.005%, a pH of 6.8 +/- 0.007 and an intracellular ATP concentration of 135 pmol/10(6) RBC +/- 41, which is comparable with freshly collected packed red blood cells (PRBCs). RBCs, collected from bone marrow harvests, can be used for autologous blood support to minimize allogeneic blood transfusions in donors and patients after large volume BM donation.

The utility of flow cytometric immunophenotyping in cytopenic patients with a non-diagnostic bone marrow: A prospective study

Cytopenia is a common problem in hematology outpatient clinics and among hospitalized patients. A bone marrow (BM) aspirate and biopsy are often performed to rule out an infiltrative versus intrinsic BM process, such as myelodysplastic syndrome (MDS). We have previously described a flow cytometric (FCM) assay useful in diagnosing MDS and demonstrated its good correlation with “gold standard” morphologic and cytogenetic criteria. In this study, we prospectively tested the utility of the FCM assay in 102 cytopenic patients with BMs showing neither diagnostic morphological dysplasia nor abnormal cytogenetics. FCM, following our published criteria, was positive in 22 cases (21.6%), intermediate in 11 cases (10.8%) and negative in 69 cases (67.6%). With a median follow-up period of 11 months (range, 4–24 months), 12 (11.8%) patients were proven to have or/develop MDS or related BM diseases (group-1); 61 (59.8%) patients had their cytopenia(s) attributed to various medical causes (group-2). In the remaining 29 patients, the causes of cytopenia(s) were not found, and some had the features consistent with the recently defined clinical entity – idiopathic cytopenia of uncertain significance. A positive FCM result was significantly more prevalent (9/12, 75%) in group-1 patients; while a negative FCM result was significantly more frequent in group-2 patients (4/61, 7%) ( p < 0.0001) with a positive predictive value of 69% and a negative predictive value of 95%. We conclude that FCM analysis of myelomonocytic maturation has diagnostic utility in cytopenic patients who have an inconclusive BM examination by morphologic and cytogenetic evaluation, and may therefore be a useful adjunct in clinical management of these patients.

Separation of adult bone marrow mononuclear cells using the automated closed separation system Sepax

The Düsseldorf-based cardiologist Professor Strauer was the first to present a therapeutic concept for the repair of acute infarcted myocardium in 2001: the autologous intracoronary transplantation of unfractionated human bone marrow (BM) mononuclear cells (MNC). The Division of Cardiology, Pneumology and Angiology, University of Duesseldorf Medical School, Duesseldorf, Germany, was also able to show the regenerative potential of BM stem cell transplantation in patients with chronic heart disease (CHD) and peripheral arterial disease (PAD). In the mean time, several clinical trials have been set up worldwide, predominantly by using MNC isolated manually from BM aspirates via density-gradient centrifugation; 374 patients have been treated here with unselected BM MNC since 2001. Altogether 217 BM aspirates have been processed manually. In order to maintain the high standards required for cellular therapeutics, the Sepax cell-separation system was implemented into routine BM processing in 2006. The closed Sepax system provides a reproducible MNC isolation method, and 157 BM samples have been processed with the Sepax device. The results of manual MNC isolation were compared with the Sepax-mediated MNC isolation. The manual Ficoll separation method was compared with the Sepax density gradient-based separation (DGBS) protocol using Ficoll with the kit CS-900 and the Sepax S-100 main processing unit from Biosafe. Nucleated cell and MNC recovery were significantly higher after Sepax processing (P<0.0001) whereas no significance was found for red blood cell depletion. The Sepax cell-separation system is a time-saving method providing clinical-grade MNC isolated automatically from human BM by Ficoll density centrifugation.

Quantification of nucleated red blood cells in allogeneic marrow graft and impact of processing on recovery

In allogeneic bone marrow transplantation (BMT), a higher nucleated and CD34+ cell dose has been reported to improve various outcomes. Other cell types, such as lymphocyte subsets, also influenced BMT results. While nucleated red blood cells (NRBCs) represent a subset of bone marrow (BM) cell subpopulation, the question of their quantification in BM grafts and the impact of BM processing on their recovery has not been addressed. In a prospective study on 77 BM products, NRBCs were enumerated by flow cytometry and the recovery analyzed after manipulation. Because NRBCs could compromise white blood cell count, the impact of NRBC count on CD34+ cell percentage and total nucleated cell (TNC) dose were also determined. The mean percentage of NRBCs in BM grafts was 21.6 percent (range, 7.8%-40.9%). Mean NRBC recoveries after BM concentration or RBC depletion were 98.4 and 28.7 percent, respectively, close to those obtained for TNC cells (88.6 and 31.3%, respectively). When corrected with NRBC count, the mean percentages of corrected CD34+ cell and TNC dose were significantly modified when compared with uncorrected values, whatever the type of BM manipulation. Our data show that NRBC quantification might be of importance to improve quality control of BM products and to evaluate the influence of NRBCs cell dose on outcomes after BMT.

Predictive value of the original content of CD34+ cells for enrichment of hematopoietic progenitor cells from bone marrow harvests by the apheresis procedure

We retrospectively investigated the feasibility of the apheresis procedure for red blood cell (RBC) reduction with a closed-bag system. We also sought to determine the optimal processing volume for the maximal recovery of hematopoietic progenitor cells (HPC). Twelve bone marrow (BM) harvests were processed for major ABO-incompatible allogeneic transplantation and one BM harvest was processed for autologous transplantation. The processing was performed through seven apheresis cycles with a two-bag system using COBE Spectra Version 6.1. The mean recovery rates were compared in the products after four cycles and seven cycles of BM processing. Mean cell recovery rates were 79.2% (67.6-97.5%) and 87.3% (68.9-111.9%) for the mononuclear cells (MNC) and 84.5% (69.4-109.5%) and 92.0% (79.0-107.7%) for the CD34(+) cells after four and seven cycles, respectively. A mean of 96.3% (93.0-98.1%) of the RBCs were finally removed. The yield of CD34(+) cells after seven cycles of processing (median: 10.35 x 10(7) cells) was 7.9% greater than that after four cycles of processing (median: 9.65 x 10(7) cells), exhibiting a less-than-significant enhancement in yield. The CD34(+) cell contents recovered in the concentrates up to four cycles (r = 0.989) and up to seven cycles (r = 0.993) were strongly correlated with the original content of the CD34(+) cells. Engraftment was obtained in all patients except one patient infused with purified CD34(+) cells. This latter result confirmed the hematopoietic potential of the cell populations recovered. Granulocyte recovery (defined as an absolute neutrophil cell count > or = 500/microL for a period of three consecutive days) ranged from 8 to 25 days (median: 16 days) post-transplantation. No hemolytic reaction was observed in any of the patients. Our results confirmed the efficacy of BM processing cycles with the COBE Spectra device. However, we could not conclude that the large-volume apheresis for BM processing significantly enhanced the yields of HPC. The final recovery of CD34(+) cells after processing could be predicted from the CD34(+) cell content of the original collected marrow.

ABO‐mismatched marrow processing for transplantation: results of 114 procedures and analysis of immediate adverse events and hematopoietic recovery

Red cell (RBC) depletion is needed to bypass ABO mismatch in allogeneic bone marrow transplantation (BMT). Technical and clinical data obtained after bone marrow (BM) processing with a continuous-flow cell separator (Cobe Spectra, Gambro BCT) are reported. RBC depletion and recovery of nucleated cells, CD3+ cells, CD34+ cells, and colony-forming unit-granulocyte-macrophage were calculated. Bacteriologic contaminations, side effects of graft infusion, and hematopoietic recovery were analyzed. A total of 114 BM samples were processed. The mean volume collected was 1099 mL (range, 390-2450 mL). Initial and residual mean RBCs volumes were 309.9 and 4.0 mL corresponding to a depletion of 98.6 +/- 0.78 percent. Before processing, the mean numbers of nucleated cells, granulocytes, CD3+ cells, CD34+ cells, and CFU-GM were 20.28 x 10(9), 12.79 x 10(9), 1.96 x 10(9), 356.7 x 10(6), and 195.6 x 10(5), respectively. The mean corresponding recoveries after processing were 33.66, 48.98, 82.02, 82.2, and 93.9 percent. Limited side effects were observed in 14 patients without correlation with residual RBCs volume. All but two patients engrafted. BM processing with the Cobe Spectra cell separator provides high rates of RBC depletion without significant side effects after BMT.

Efficacy and kinetics of bone marrow processing and enrichment of haematopoietic progenitor cells (HPC) by a large-volume apheresis procedure.

We investigated the efficacy of bone marrow (BM) processing by an automated large-volume apheresis procedure (6 x original BM volume) in 10 paediatric and adult patients undergoing BM harvesting before myeloablative therapy. Volume-dependent kinetics during apheresis were analyzed by sequential collection of processed cells into a six-fold collection bag system with consecutive analysis of the single bags. BM processing resulted in an 83.3% (+/- 21) recovery of mononuclear cells (MNC), a 97.9% (+/- 1.1) reduction of erythrocytes (RBC) and a 87.7% (+/- 2.9) volume reduction. To determine volume-dependent kinetics of haematopoietic progenitor cell (HPC) enrichment during apheresis, leukocytes (WBC), mononuclear cells (MNC), CD34 cells and colony-forming cells (CFU-GM) were serially quantitated in subsequent collection bags. Large-volume BM processing significantly enhanced absolute yields of CD34+ cells (mean: 4.01 (+/- 2.81) x 10(6)/kg bw) and CFU-GM (mean: 1.92 (+/- 1.47) x 10(4)/kg bw) compared with the standard procedure (3 x BM volume) by 26.9% (+/- 10.9) and 27.2% (+/- 11.6), respectively. We concluded that large-volume apheresis for BM processing is an efficient technique significantly improving the yields of haematopoietic progenitor cells (HPC) without any relevant changes in the purity of the final product. Moreover, sequential collection and analysis of HPC represents a good model to investigate the volume-dependent kinetics and efficacy of BM processing.

Preparation of autologous bone marrow grafts for cryopreservation using the AS104 cell separator.

We evaluated the AS104 cell separator (Fresenius AG, Bad Homburg, Germany) for ex vivo processing of bone marrow (BM) grafts of 43 patients suffering from germ cell cancer (GCC, n = 22), acute lymphocytic leukemia (ALL, n = 13) and malignant lymphoma (ML, n = 8). Recoveries of total nucleated cells (TNC), mononuclear cells (MNC) and colony-forming units granulocyte-macrophage (CFU-GM) were determined in the BM concentrates prepared for cryopreservation. Hematopoietic reconstitution was analyzed in patients who underwent autologous transplantation following high-dose radio-/chemotherapy (HDRCT). Processing of the BM suspension with a median volume of 1,013 ml (range: 422-1,574) resulted in 156 ml (80-186) within 50-120 min (median: 90). In the BM concentrates, medians of 28.6% TNC (10.6-69.6), 37.9% MNC (22.3-86.4), and 52.4% CFU-GM (20.8-96.4) were recovered. Twenty-six patients underwent HDRCT with reinfusion of autologous BM and were evaluable for engraftment. They received a median of 0.8 x 10(8) MNC/kg (0.3-1.6 x 10(8)) and 2.2 x 10(4) CFU-GM/kg (0.6-12.8 x 10(4) for hematopoietic rescue. Engraftment with neutrophils > 500/microliter occurred in a median time of 12 days (8-33) in all patients. We conclude that ex vivo processing of autologous BM with median recovery rates of 37.9% for MNC, and 52.4% for CFU-GM, results in a cell population that can rescue patients from HDRCT. The described technique is convenient, time-efficient, and provides reliable results in preparing BM autografts for cryopreservation.

Sources and sequelae of bacterial contamination of hematopoietic stem cell components: implications for the safety of hematotherapy and graft engineering.

It is important to compare the incidence of bacterial contamination of components collected from the peripheral blood or bone marrow (BM), as well as of components processed with or without cell selection or depletion, and to evaluate the sequelae of such contamination. Bacterial contamination rates were compared in 1380 untreated autologous peripheral blood progenitor cells (PBPCs), 291 untreated autologous BM samples, 916 monoclonal antibody (MoAb)-treated autologous and allogeneic BM samples, and in 45 autologous PBPC components from which the CD34+ cells were selected. Bacterial cultures were performed at sequential time points during the processing of MoAb-treated BM. Bacterial contamination was documented in 44 of 2632 components from 1593 patients (1.67% of components, 2.76% of patients) before cryopreservation. Although only 0.65% of untreated PBPCs were contaminated before cryopreservation, each patient was more likely to have given a contaminated PBPC component than a contaminated BM component (2.41% vs. 0%, p < 0.01). Bacterial contamination of MoAb-treated BM was greater during or after manipulation than it was before (2.33% vs. 0.77%, p < 0.05). At thawing, contamination was documented in 42 (1.97%) of 2136 components cultured. Ten (13.7%) of 73 patients who received hematopoietic progenitor cells that were contaminated before cryopreservation or at thawing developed fever or positive blood cultures within 48 hours of transfusion. Fever was associated with bacteremia in two cases, but no irreversible clinical sequelae were noted. These studies suggest that, despite careful attention to sterile procedures, low-level contamination of hematopoietic stem cell components can be introduced before or during manipulation as well as at thawing, and that standards for monitoring of the procedures for collection, processing, cryopreservation, thawing, and transfusion of hematopoietic progenitor cells are necessary.

Analysis for recovery and loss of mononuclear cells and colony-forming units granulocyte-macrophage during ex vivo processing of autologous bone marrow.

During ex vivo processing of autologous bone marrow (BM) substantial loss of stem and progenitor cells should be avoided to achieve rapid and sustained hematopoietic reconstitution after high-dose radio-/chemotherapy. We processed 25 autologous BM grafts with the Fresenius AS104 cell separator for cryopreservation and we determined recoveries for mononuclear cells (MNC) and colonyforming units granulocyte-macrophage (CFU-GM) in the BM concentrates. To identify cell loss in BM fractions not cryopreserved, we investigated the MNC and CFU-GM content of BM fat and BM blood. MNC and CFU-GM recovery yielded a mean ( +/- SEM) of 42 +/- 12 and 54 +/- 20% in the BM concentrate. BM fat showed a mean loss of 7 +/- 5% for MNC and 4 +/- 3% for CFU-GM, BM blood 30 +/- 12% for MNC and 13 +/- 13% for CFU-GM, respectively. CFU-GM recovery was significantly higher in the BM concentrate of patients with hematologic malignancy (HM) compared with patients suffering from germ cell cancer (GCC): 66 +/- 21 vs. 43 +/- 12% (p < 0.02). Seventeen patients (7 GCC, 10 HM) underwent high-dose chemotherapy or radio-/chemotherapy and were autografted with 0.8 +/- 0.2 x 10(8) MNC/kg and 3.7 +/- 2.0 x 10(4) CFU-GM/kg. All patients achieved engraftment with neutrophils > 0.5 x 10(9)/l at a mean of 14 +/- 6 days. We conclude that: (1) ex vivo processing of autologous BM with a mean of recovery of 42% for MNC and 54% for CFU-GM in the BM concentrate can result in a cell population capable of sustained hematopoietic reconstitution, (2) CFU-GM recovery is significantly higher in patients with HM than in patients with GCC and (3) 37% MNC and 17% CFU-GM represent in fact cell losses recovered from BM fractions not cryopreserved (BM fat, BM blood). Furthermore, it is likely that MNC and CFU-GM not recovered from BM concentrate, BM fat and BM blood are cell losses related to the cell separator.

Adhesion receptors on bone marrow stromal cells: in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice

Cell adhesion molecules (CAMs) play a key role in interactions between stromal and hematopoietic cells in bone marrow (BM) and in cell traffic through vascular endothelium. To examine the identity of CAMs involved in these processes in mouse BM, we have investigated the in vivo expression of vascular cell adhesion molecule-1 (VCAM-1) and its counter-receptor, very late antigen-4 (VLA-4). Radioiodinated monoclonal antibodies (MoAbs) detecting VLA-4 and VCAM-1 were injected intravenously. Antibody binding was detected in BM by light and electron microscope radioautography. VCAM-1 labeling was restricted to stromal reticular cells and endothelial cells lining BM sinusoids. VCAM- 1+ reticular cells formed patchy concentrations, especially in subosteal regions, associated with lymphoid, granulocytic, and erythroid cells. After gamma-irradiation to deplete hematopoietic cells, reticular cells and endothelial cells all showed VCAM-1 labeling in apparently increased intensity. VLA-4 labeling was shown by undifferentiated blast cells and lymphohematopoietic cells both in BM cell suspensions and in vivo, especially at reticular cell contact points. The results demonstrate that VCAM-1 is expressed in vivo by certain BM reticular cells, suggesting that the molecule mediates adhesion to multiple lineages of lymphohematopoietic cells. The finding that VCAM-1 is also expressed constitutively by BM sinusoidal endothelium, unlike its inductive expression by endothelia elsewhere, suggests that VCAM-1 and VLA-4 may be involved in regulating the normal cell traffic between BM and the blood stream.