Nucleated Cell Recovery Research Articles

A “No-wash” Albumin-Dextran Dilution Strategy for Cord Blood Unit Thaw: High Rate of Engraftment and a Low Incidence of Serious Infusion Reactions

Preparation of cord blood (CB) units for infusion by albumin-dextran dilution without centrifugation may be advantageous for adult patients to minimize cell loss and, unlike a bedside thaw, is still conducted in the controlled laboratory environment. Therefore, we studied CB transplantation (CBT) using this technique in 54 consecutive CBT recipients >20 kg. Patients (median age=42 years [range: 7-66 years]; median weight=71 kg [range: 24-109]) were transplanted for high-risk hematologic malignancies with myeloablative (n=35) or nonmyeloablative (n=19) conditioning and 4-6/6 human leukocyte antigen (HLA)-matched double-unit grafts. One hundred seven units were thawed with dilution, whereas 1 red blood cell (RBC)-replete unit was washed. A 5:1 dextran 40%/25% albumin solution was used. RBC-depleted units (n=104) were diluted >or=5.5-fold (median final volume 200 mL [range: 200-500]), whereas RBC-replete units (n=3) were diluted >or=4-fold (median final volume 400 mL [range: 400-535]). Total nucleated cell (TNC) recovery was 86%; the median infused TNC dose was 2.17x10(7)/kg/unit. Although 35 patients (65%) had a total of 45 infusion reactions (6 nausea, 31 hypertension, 3 pain, 1 rigors/fever, 2 transient hypoxia, 2 renal impairment) requiring additional therapy, there were no infusion-related serious adverse events, and reactions were not related to dimethyl sulfoxide (DMSO) dose/kg. Cumulative incidence of sustained donor engraftment was 94% (95% cumulative incidence [CI]: 87-100) with neutrophil recovery occurring at a median of 25 days (range: 13-43) in myeloablative and 10 days (range: 7-36) in nonmyeloablative recipients. CB thaw with albumin-dextran dilution reduces unit manipulation, and minimizes cell loss, speeds time to infusion, is associated with a tolerable infusion reaction profile, and a high rate of sustained engraftment in CBT recipients >or=20 kg.

Consistency of the initial cell acquisition procedure is critical to the standardization of CD34+ cell enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units and cryopreserved aliquots

The CD34+ cell content is a predictive factor for engraftment and survival after umbilical cord blood (UCB) transplantation. The high variability in the CD34 assay results in different recommended cell doses for infusion across transplant centers and also limits the clinical utility of the CD34+ cell counts provided by cord blood banks (CBBs). This bi-institutional study was intended to understand the sources of this variability. The level of CD34 agreement between the University of Minnesota (UM) and the Madrid CBB (MCBB) was evaluated on 50 UCB units before and after cryopreservation. Two cryopreserved vials per unit were thawed and processed at both laboratories. Dual-platform ISHAGE-based flow cytometry was used for CD34 enumeration. Postthaw nucleated cell recoveries were similar. However, whereas CD34+ cell enumeration before freezing was 0.35 +/- 0.22 percent, the results after thawing were 0.98 +/- 0.65 and 0.57 +/- 0.39 percent at UM and MCBB, respectively. Bland-Altman plots analysis ruled out the interchangeability of MCBB and UM CD34 values. Differences in the initial cell acquisition settings accounted for most of the CD34 discrepancy, which was no longer present after normalization of the forward scatter threshold for cell acquisition. The standardization of CD34+ cell enumeration by flow cytometry is strongly reliant on a consistent initial cell acquisition procedure. The interlaboratory variation can be minimized by using frozen cell aliquots as reference samples. Both requisites should be considered for CD34 testing and UCB unit selection by regulatory institutions involved with cord blood banking and transplantation.

A new automatic device for routine cord blood banking: critical analysis of different volume reduction methodologies

Volume reduction is the usual process in cord blood banking that has some advantages regarding reducing the storage space and dimethyl sulfoxide (DMSO) quantity in the final product. The volume reduction methodology must guarantee high cell recovery and red blood cell (RBC) depletion by reducing all the umbilical cord blood (UCB) units to a standard volume. We analyzed and compared critically three different volume reduction methods [hydroxyethylstarch (HES), top and bottom with Optipress II and Compomat G4, and AXP] used at the Valencia Cord Blood Bank over 10 years. The highest significant RBC depletion was achieved with the AXP system (P<0.001), while the top and bottom system with Compomat G4 and an adjusted buffy coat (BC) volume to 41 mL enabled the best total nucleated cell (TNC) recovery (P<0.001). TNC recovery and RBC depletion were similar for AXP and HES with an adjusted volume to 21 mL. In the multivariate analysis, when analyzing all cases, the BC volume set significantly influenced TNC, CD34+ and lymphocyte recoveries and RBC depletion (P<0.001). RBC depletion was significantly influenced by the initial volume and initial RBC content of UCB units (P<0.001). AXP is a highly efficient method for RBC depletion, providing the same TNC recovery as HES method with a final volume of 41 mL. AXP has the advantages of being an automatic and functionally closed system that shortens and better standardizes the proceedings. Top and bottom is a closed system that allows better TNC recoveries when the BC volume set is 41 mL.

Avoidance of Post-Thaw Wash Prior to Transplantation of Plasma Depleted Cord Blood (PD CB) Is Associated with Improved Engraftment & Decreased Severity of Chronic GvHD (cGvHD) without Increased Relapse.

Post-thaw washing of frozen CB prior to transplantation is widely practiced to remove DMSO, red cell debris and free hemoglobin, however, washing a red cell containing product may result in cell loss. We analyzed outcomes of engraftment, survival, relapse and GvHD in 299 patients transplanted with plasma depleted but not red cell reduced (PD) CB where 154 were washed (W) and 145 were directly infused - (non-washed or NW). 207 (69%) of 299 patients had malignant diagnosis. The two groups were comparable for patient, disease and graft characteristics except for a higher % of Asian patients in the NW group in contrast to a higher % of Caucasian/Hispanic patients, higher % of US transplant centers (TC) and higher number of malignant diagnoses for the W group. Nucleated cell recovery after thawing, as reported by TC, was higher for NW (median 85% vs. 79%, p=0.003). The NW group had higher cumulative incidences of and faster neutrophil (ANC500) and platelet engraftment (Plt 20K and 50K) than the W group. No difference was seen for acute GvHD (aGvHD); however, the NW group had significantly lower rates of extensive (Ext) cGvHD and higher rates of limited (Ltd) cGvHD (p=0.0005 and <0.0001). Relapse rates were similar between W and NW. Multivariate analysis and matched pair analysis between W versus NW confirm these observations. In univariate analysis, overall survival (OS) and disease-free survival (DFS) were significantly higher for the NW group. This retrospective analysis represents the largest series comparing different thaw methods for PD CB and is the first to demonstrate a potential association of no post-thaw wash with significant decrease in extensive cGvHD rate without concomitant increase in relapse rate. These improvements may contribute to the apparently better survival for the NW group. Prospective trials which may include infusion of wash supernatants into patients are necessary to confirm and better understand these observations.ANC 500 & Plt 20K100 Day TRM1 & 3 Yr OS1 & 3 Yr DFS1 Yr RelapseII-IV/III-IV aGvHDLtd/Ext cGvHDOverall88±4/69±417±261±3 56±351±3 39±523±335/1420/11W87±5/64±517±354±4 48±443±5 30±627±536/159/17NW91±6/76±616±369±4 67±460±5 57±520±436/1436/1P value0.002/0.0010.050.020.050.290.93/0.80<0.0001/0.0005ANC 500 & Plt 20KTRMOSDFSRelapseII-IV/III-IV aGvHDLtd/Ext cGvHDMV adjusted Cox RR & P value1.51 0.002/1.70 0.0010.67 0.110.71 0.090.74 0.110.76 0.34N/A3.88/<0.001Matched Pairs P value0.008/0.00020.530.320.220.190.98/0.440.02/0.001All results in % except for p-values & where indicated. Engraftment presented as cumulative incidence, GvHD as proportions; all others estimated by Kaplan-Meier.

Evaluation of an Automated Ficoll Procedure To Enrich for Mononuclear Cells for Use in Clinical Trials.

Mononuclear cell fraction (MNC) enrichment from hematopoietic progenitor cells (HPC) cells is required for a number of clinical applications. Previously, we have used a manual method for enrichment of these cells. We evaluated an automated procedure for better consistency and patient safety (reduce risk of contamination). We have been using the Sepax (BioSafe) for the past 18 months to process Cord Blood (RBC depletion) before cryopreservation. Since the instrument can also do density gradient enrichment of mononuclear cells, we evaluated the ability to process different sources of hematopoietic progenitor cells (HPC) on the Sepax.Methods: Bone marrow, non-mobilized apheresis and cord blood were evaluated as a source of HPC. Briefly, 100ml of Ficoll solution were added to the chamber of the Sepax disposable. After initiating centrifugation, 50–110 ml of HPC were then overlayed onto the density gradient. The cells were fractionated, mononuclear cell fraction isolated, and then washed three times before final resuspension in 50ml of Saline/5% albumin. In some experiments the HPC were divided into two fractions so that a direct comparison of the Manual vs Automated enrichment procedure could be compared. Finally, a recent publication from Seeger et al (European Heart Journal 28:766, 2007) suggested different density gradient solutions have an effect on MNC enrichment. Therefore, four different brands of density gradient solution (GE Ficoll-Paque Premium, Sigma Histopaque 1077, Accurate Lymphoprep and Lonza Lymphocyte Separation media) were evaluated to determine if there was a differential separation of subsets of cells.Results: The total nucleated cell (TNC), MNC and CD34 recoveries after MNC enrichment were similar for either bone marrow or cord blood HPC comparing the manual procedure and the automated procedure. The TNC and MNC recovery was higher for apheresis products (66% and 69% respectively) than the other two sources of HPC. This most likely reflects the enriched MNC starting population from an apheresis device. We evaluated the TNC and MNC recoveries over a range of initial TNC (7.5–20 × 108 cells) but could not see an effect of either cell number or cell concentration on recoveries. Nor could we see an effect of initial cell volume (50–100 mls) on recovery of cells post processing. Finally, we could not see a difference in either TNC or MNC recovery when the four different sources of density gradient solution were compared.Conclusion: We have shown that the Sepax instrument can be used for enrichment of mononuclear cells from HPC, Marrow, HPC, Apheresis and HPC, Cord. The benefits of an automated system are1.consistency,2.functionally closed system which reduces possibility of contamination3.documentation of run parameters and4.shorter processing time. [Display omitted]

Optimising methods of red cell sedimentation from cord blood to maximise nucleated cell recovery prior to cryopreservation

Human cord blood is now an established source of stem cells for haematopoietic reconstitution. Red blood cell (RBC) depletion is required to reduce the cord blood unit volume for commercial banking. Red cell sedimentation using hydroxy ethyl starch (HES) is a standard procedure in most cord blood banks. However, while standardising the procedure for cord blood banking, a significant loss of nucleated cells (NC) may be encountered during standard HES sedimentation protocols. This study compares four procedures for cord blood processing to obtain optimal yield of nucleated cells. Gelatin, dextran, 6% HES and 6% HES with an equal volume of phosphate-buffered saline (PBS) were compared for RBC depletion and NC recovery. Dilution of the cord blood unit with an equal volume of PBS prior to sedimentation with HES resulted in maximum NC recovery (99% [99.5±1.3%). Although standard procedures using 6% HES are well established in Western countries, they may not be applicable in India, as a variety of factors that can affect RBC sedimentation (e.g., iron deficiency, hypoalbuminaemia, thalassaemia trait, etc.) may reduce RBC sedimentation and thus reduce NC recovery. While diluting cord blood with an equal volume of PBS is a simple method to improve the NC recovery, it does involve an additional processing step.

A novel high-yield volume-reduction method for the cryopreservation of UC blood units

For the application of umbilical cord blood (UCB) units as hematopoietic grafts, a dose of 3.7 x 10(7) nucleated cells (NC)/kg body weight is required. NC can be lost during volume-reduction processing and during thawing. A novel modification of the double-processing protocol with the aim of minimizing NC loss is described and evaluated. One-hundred and fifty UCB were collected. The volume was reduced by a centrifugation step following double-processing in the presence of 2% HES 200/0.5. Pre- and post-processing cell counts and platelet parameters were measured with an automatic counter. The number of viable CD34+ hemopoietic stem cells was measured by flow cytometry. In 25 of the samples, colony-forming units (CFU) were also determined. The same samples were thawed 6 months after cryopreservation and re-evaluated. The volume was reduced to 6 +/- 1.5 mL. The recovery of NC, MNC, CD34+ hemopoietic stem cells, RBC depletion and CFU following double-processing was 93.6 +/- 3.2%, 95.8 +/- 2.2%, 98.4 +/- 1.5%, 96.8 +/- 1.1% and 107.1 +/- 6.1% (for 25 samples), respectively. The post-thaw recoveries of NC, MNC, CD34+ hemopoietic stem cells and CFU (for 25 samples) were 78.6 +/- 5.4%, 90.8 +/- 4.4%, 96.4 +/- 2.5%, 89.1 +/- 4.1%, respectively. No post-thaw cell aggregation was observed. A significant (P<0.05) post-thaw loss of platelets and signs of platelet activation was observed. The protocol uses non-expensive equipment and clinically approved materials and results in samples that can be used in patients with a mean weight of 32.7 kg.

Post-That Washing Prior to Transplantation of Umbilical Cord Blood (UCB) That Were Depleted of Plasma but Not of Red Blood Cells.

Washing after thawing of frozen UCB used for hematopoietic stem cell transplantation (HSCT) is widely practiced for the purpose of removal of the cryoprotectant DMSO and free hemoglobin from lysed red cells. An updated retrospective audited analysis was performed on the outcomes of 208 PD UCB used in 186 patients with known thaw conditions that have engraftment and/or survival data −106 washed (W) and 80 non-washed (NW). When the DMSO dose was kept under the recommended 1 g per kg of recipient weight, the only severe adverse reaction was a seizure and encephalopathy following infusion of a NW PD UCB that resolved without any sequelae. Total nucleated cell recovery after thawing as reported by transplant centers was higher for NW (median 89% vs. 75%). The Kaplan-Meier estimate of 3-month neutrophil (ANC500) engraftment were 91±4% for NW versus 88±4% for W with median time to neutrophil of 21 versus 24 days (p = 0.02). The K-M estimate for 6-month platelet 20K engraftment were 86±6% for NW and 78±5% for W with median time to engraftment at 46 days for NW and 55 days for W (p = 0.002). Acute grade III–IV GvHD were similar at 12% (NW) and 13% (W), but extensive chronic GvHD were 4% (NW) and 19% (W). 1-year TRM were 25±5% for NW and 34±5% for W (p=0.75), with relapse rate at 16±5% for NW and 28±5% for W (p=0.09). K-M estimates of 1-year OS were 63±6% versus 53±5% (p = 0.54), and for 1-year DFS were 62±6% versus 48±5% (p = 0.23) for NW and W respectively. To our knowledge, this was the largest transplant outcome study comparing no post-thaw wash versus wash for UCB, though the conclusions are confined to PD CBU. No clear benefits of post-thaw washing were seen for PD UCB. HSCT with NW PD UCB was at least as efficacious as that using W PD UCB with respect to GvHD, TRM, relapse rate, 1-year OS and DFS. Moreover, washing may have a negative impact on neutrophil and platelet engraftment of PD UCB.

Automated separation of cord blood units in top and bottom bags using the Compomat G4

Cord blood (CB) has become a real alternative source of haematopoietic stem cells for bone marrow reconstitution in a variety of malignant disorders. As a response to this increasing activity, CB banks have been developed to guarantee the quality of processed CB units. Volume reduction of CB units maximizes storage space and also has other advantages. The aim of this study was to develop a program for the volume reduction of CB in the Compomat G4 device. We also compared two different top and bottom systems for CB fractionation (Compomat G4 and Optipress II). We empirically designed three different programs for volume reduction of CB with Compomat G4: two for final BC volume of 41 ml (CB1 and CB2) and the other one for buffy coat (BC) volume of 25 ml (CB3). Significantly worse recoveries were achieved for CB processed with program CB3. A RBC depletion of >or=50%, >or=60% and >or=70% were achieved for 67%, 39% and 9% of all units respectively. When comparing Compomat G4 and Optipress II, total nucleated cell recovery was similar for both methods, while lymphocytes recovery was significantly better for Optipress II.

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.

682. Collection and Genetic Correction of Fanconi Complement Type A Hematopoietic Stem Cells: Results of Two Pilot Studies

Fanconi anemia (FA) is a genetic syndrome characterized by the uniform development of aplastic anemia. Current therapy for patients lacking HLA-identical sibling hematopoietic stem cell (HSC) donors are not optimal. We have opened two phase I clinical trials to determine if potentially useful numbers of CD34+ cells can be collected early in the course of the disease (collection study) and if these cells, once corrected, can engraft without cytoreduction and demonstrate proliferative advantage in vivo over un-corrected cells (gene transfer study). To date, 7 FA patients have undergone a 20 ml/kg bone marrow harvest (BMH) with an average of 1.9|[times]|106 CD34+ cells/kg (range 0.3|[ndash]|2.9|[times]|106 CD34+ cells/kg) collected, suggesting that collection of adequate numbers of cells is possible but challenging, even early in the disease. In the gene transfer study, 3 FA patients with genotype A (FAA) have enrolled, meeting eligibility criteria of FAA, no evidence of malignancy and a minimum of 1|[times]|105 viable CD34+ cells/kg for ex vivo culture and gene transfer. BMHs from the 3 patients (2 fresh and one previously cryopreserved) were CD34+ cell selected. Despite collection before significant pancytopenia, an average of only 5|[times]|105 CD34+ cells/kg (range 1.5|[ndash]|10|[times]|105 CD34+ cells/kg) was purified from these 3 cases representing |[sim]|10% of the expected yield from normal individuals. These cells underwent ex vivo gene transfer using cytokine prestimulation in serum-free medium followed 2 exposures to a GALV-MSCV-FANCA vector. Transduction efficiency of the final product determined by real-time PCR analysis of progenitors averaged 48% (range 40|[ndash]|62%). Equivalent correction of mitomycin C hypersensitivity in progenitor cells confirmed this analysis at the functional level. Nucleated cell recovery after ex vivo manipulation was 82|[ndash]|110% of input nucleated cells using freshly harvested bone marrow derived CD34+ cells (N=2). However, despite good CD34+ cell recovery and viability after CD34+ selection, only 6% of input nucleated cells were recovered utilizing CD34+ cells purified from the previously cryopreserved bone marrow and these cells were not re-infused. In the two patients who did receive gene corrected cells, the total cell dose re-infused was 2.5|[ndash]|3.5|[times]|105 nucleated cells/kg, reflecting the low number of initial CD34+ cells placed in culture. FAA vector sequences were detected in the PB of one patient early post-infusion (+4 weeks) but none was detected after 8 weeks. Despite no long term detection of the corrective gene in the PB and BM, both patients exhibited a transient but marked improvement (10|[ndash]|20% increase) in their peripheral blood hemoglobin (> 6 months) and platelets counts (2|[ndash]|3 months). The data demonstrates that gene transfer efficiency in the clinical setting has been significantly improved, however the collection of adequate numbers of HSC may be the critical barrier to the success of genetic correction attempts in FA.

Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries

Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The leukocyte-containing fraction with the stem/progenitor cells was recovered from the filters by reverse flushing. Utilizing the routine traditional processing and testing procedures of each laboratory, in vitro parameters were determined, with samples obtained after collection, after processing and after freezing/thawing. The results were expressed as the percentage recovery of viable cells in processed vs. collected samples (performance 1; PF1) and in thawed vs. processed samples (performance 2; PF2). The composite results obtained by the seven laboratories were summarized. The median PF1 percentage recovery of total nucleated cells (TNC) was comparable with both traditional methods (HES 79%, T&B 86%) and statistically reduced with both filtration procedures (filter A 58%, filter B 61%). Mononuclear cell (MNC) PF1 recovery was highest statistically with the T&B method (91%) and reduced on using filter A (77%) and filter B (70%) and the HES method (72%). CD34+ cell recovery was judged to be essentially comparable with the four methods, although the range of unit recoveries differed. The percentage recovery of TNC and MNC in PF1 was influenced by the volume of the collected cord blood, especially with use of the filtration procedures. This correlated with TNC content. A greater percentage of red cells and platelets was removed during processing with both filter methods. The time to process cord blood preparations with filter A was significantly shorter than the other methods. Processing with the HES method took the longest time. The recoveries for TNC, MNC and CD34+ cells in PF2 did not appear to be influenced by the specific processing procedure. These data indicate that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.

Early Results of the Clinical Collection and Genetic Correction of Fanconi Complement Type A Hematopoietic Stem Cells.

Fanconi anemia (FA) is a genetic syndrome characterized by almost uniform development of aplastic anemia. Current therapies for patients lacking HLA-identical sibling hematopoietic stem cell (HSC) donors have shown high morbidity and mortality in clinical trials. Genetic correction of FA HSC using viral vectors has been demonstrated in animal models. However, harvesting of sufficient CD34+ cells at the time that HSC therapy is clinically indicated is difficult due to the severe bone marrow hypoplasia that accompanies pancytopenia. We have opened two phase I clinical trials that seek to determine if potentially useful numbers of CD34+ cells can be collected early in the course of the disease (collection study) and if these cells, once corrected, can engraft without cytoreduction and demonstrate proliferative advantage in vivo over un-corrected cells (gene transfer study). These studies are being conducted with approval by the FDA/NIH-RAC/Institutional IRB and monitored by an independent DSMB. To date, 4 FA patients have undergone a 20 ml/kg bone marrow harvest (BMH) with an average of 1.3x106 CD34+ cells/kg (range 0.3–2.9x106 CD34+ cells/kg) collected, suggesting that collection of adequate numbers of cells will be challenging, even early in the disease. In the gene transfer study, 3 FA patients with genotype A (FAA) have enrolled, meeting eligibility criteria of FAA, no evidence of malignancy and a minimum of 1x105 viable CD34+ cells/kg for ex vivo culture and gene transfer. BMHs from the 3 patients (2 fresh and one previously cryopreserved) were CD34+ cell selected using the CliniMACS device. Despite collection before significant pancytopenia, an average of only 5x105 CD34+ cells/kg (range 1.5–10x105 CD34+ cells/kg) was purified from these 3 cases representing ~10% of the expected yield from normal individuals. These cells underwent ex vivo gene transfer using cytokine prestimulation in serum-free medium followed 2 exposures to a GALV-MSCV-FANCA vector. Transduction efficiency of the final products determined by real-time PCR analysis of CD34+-derived progenitors averaged 48% (range 40–62%). Equivalent efficiency of correction of mitomycin C hypersensitivity in progenitor cells confirmed this analysis at the functional level. Nucleated cell recovery after ex vivo manipulation was 82–110% of input nucleated cells using freshly harvested bone marrow derived CD34+ cells (N=2). However, despite good CD34+ cell recovery and viability after CD34+ selection, only 6% of input nucleated cells were recovered utilizing CD34+ cells purified from the previously cryopreserved bone marrow and these cells were not re-infused. In the two patients who did receive gene corrected cells, the total cell dose re-infused was 2.5–3.5x105 nucleated cells/kg, reflecting the low number of initial CD34+ cells placed in culture. One patient is now 6 months post re-infusion with no evidence of gene marking observed in her PB or BM. The second patient had detectable FAA vector sequences in her PB early post-infusion (+4 weeks) but had none detected +8 weeks. The data suggest that while gene transfer efficiency in the clinical setting has been significantly improved, collection and expansion (either in vitro or in vivo) of adequate number of HSC may be critical to the success of genetic correction attempts in FA.

Effect of Post-Thaw Washing Prior to Transplantation of Umbilical Cord Blood Units (UCB) That Were Depleted of Plasma but Not of Red Blood Cells.

Background: Washing after thawing of frozen UCB used for hematopoietic stem cell transplantation (HSCT) is widely practiced for the purpose of removal of the cryoprotectant DMSO and free hemoglobin from lysed red cells; however, frozen peripheral blood stem cells with similar loads of free hemoglobin and DMSO are often thawed and infused directly without washing. Recently, similar clinical outcome and post-thaw viability were seen with the standard red cell depleted (RCD) UCB whether washing was employed after thawing or not. However, no data exist on the utility of post-thaw washing for UCB that were depleted of plasma (PD) but not depleted of red blood cells.Hypothesis: Myeloid and platelet engraftment, speed of engraftment, overall survival (OS), disease-free-survival (DFS), and transplant related mortality (TRM) will be similar in two comparable groups of patients who received frozen PD CBU that were washed (W) or not washed (NW) after thawing.Methods: A retrospective analysis was performed on the outcomes of 84 patients in remission without history of prior transplants, who received either washed (n=43) or non-washed (n=40) PD UCB units for HSCT.Results: Adverse events of any grade occurring more than once during infusion include hemoglobinuria (9NW, 1W), hypertension (6NW, 4W), hives (1NW, 1W), nausea/vomit (2NW) and dyspnea (1NW, 1W). One patient developed seizure and encephalopathy, although relationship to infusion was uncertain. Total nucleated cell recovery after thawing is higher for NW (median 95% vs. 75%). Unadjusted cumulative incidence (C.I.) of neutrophil engraftment was similar for both groups, 91±5% for NW (n=36) versus 93±4% for W (n=41), but median time to neutrophil (20 vs 26 days) and platelet engraftment (platelet 20K 47 vs. 55 days & platelet 50K 55 vs 63 days) occurred earlier for NW. Additionally, C.I. for platelet 20K engraftment was higher for NW (n=28; 92±6%) than W (n=39; 75±7%). Acute grade III–IV GVHD were 10% (NW) and 19% (W), and extensive chronic GVHD were 0% (NW) and 22% (W). TRM was 18±6% for NW and 20±7% for W, with the relapse rate for malignant cases at 11±7% for NW and 25±8% for W. One-year OS was 75±7% (n=40) versus 72±8% (n=43), and 1-year DFS was 69±10% (n=23) versus 54±9% (n=34) for NW and W respectively.Conclusion: No clear benefits of post-thaw washing for PD UCB prior to HSCT was found, except for the anticipated lower incidence of transient hemoglobinuria. HSCT with NW PD CBU was at least as efficacious as that using W PD units with respect to neutrophil engraftment, TRM, relapse rate, 1-year OS and DFS. Moreover, washing may have a negative impact on C.I. of platelet engraftment and the speed of neutrophil and platelet engraftment. The questions of PD versus RCD UCB and post-thaw W versus NW have important clinical consequences and prospective randomized studies are needed to clarify these issues.

Prevention of Apoptosis as a Possible Mechanism behind Improved Cryoprotection of Hematopoietic Cells by Catalase and Trehalose

Our previous in vitro work has shown the usefulness of membrane stabilizers and antioxidants as additives in conventional freezing medium to freeze mouse and human hematopoietic cells. The present work was carried out using murine model to test the in vivo engraftment ability of mouse bone marrow frozen with (test cells) or without (control cells) addition of a combination of trehalose and catalase in the medium containing 10% dimethyl sulfoxide (DMSO). Viability, nucleated cell recovery, and progenitor content of revived cells were measured. Freezing efficacy was tested by in vivo assays like colony forming unit-spleen (CFU-S), pre-CFU-S, and short-term engraftment of frozen marrow in irradiated mice. Long-term engraftment ability of frozen marrow was assessed using a Ly5.1-Ly 5.2 chimera model. Levels of apoptosis and reactive oxygen species (ROS) generated in revived cells were estimated. The former by Annnexin V, TUNEL, and DNA laddering and the latter by DCFH-DA probe. Our results show that the combination of catalase and trehalose with 10% DMSO improves freezing efficacy not only in terms of viability, cell recovery, and progenitor content but also by in vivo assays like CFU-S, pre-CFU-S, and short- and long-term engraftment. Both the level of apoptosis and ROS generation were considerably reduced in test set as compared to control set. We conclude that inclusion of a combination of trehalose and catalase in conventional freezing medium leads to enhanced engraftment potential of cryopreserved mouse bone marrow cells probably by preventing apoptotic cell death. Our observation using animal model may have significant clinical implications.

Evaluation of an automated cell processing device to reduce the dimethyl sulfoxide from hematopoietic grafts after thawing

The direct transfusion of thawed hematopoietic progenitor cells (HPCs) is associated to transfusion-related side effects that are thought to be dose-dependent on the infused dimethyl sulfoxide (DMSO). Both the effectiveness of a fully automated cell processing device to washing out DMSO and the effects of DMSO elimination over the recovered cells were evaluated. Twenty cryopre-served peripheral blood HPC bags (HPC apheresis [HPC-A]) were thawed and processed for washing with an automated cell-processing device. Viability, colony-forming units (CFUs), and absolute count of recovered cells were evaluated by flow cytometry immediately after washing as well as at different times after washing and compared with a sample taken just after thawing (control) but maintained at 4 degrees C. DMSO content was measured by high-performance liquid chromatography and the osmolarity with an osmometer. The median recovery of viable total nucleated cells, viable CD34+ cells, and CFU colonies was 89 (range, 74-115), 103 (range, 62-126), and 91 percent (range, 46%-196%), respectively, in the washing group. Recovery of viable CD3+ cells was 97 percent (range, 42%-131%) and CD14+ cells was 82 percent (54%-119%). The percentages of DMSO elimination and osmolarity reduction were 98 (range, 96-99) and 90 percent (range 86%-95%), respectively. Moreover, elimination of the cryoprotectant improved CFU count, viability, and cell recoveries along the time when compared with the control group. Washing out DMSO in thawed HPC-A by use of this approach is safe and efficient in terms of recovery and viability of nucleated and progenitor cells. Additionally, the removal degree of DMSO is very high and therefore might ameliorate the transfusion-related side effects.

Basic investigation of the lectin method for separation and recovery of nucleated red blood cells in maternal blood, and a study into the frequency of nucleated red blood cells in fetomaternal disorders

We previously reported the separation and recovery of nucleated red blood cells (NRBCs) in maternal blood using the lectin method. In the present study, we verified the lectin method and investigated the appearance of NRBCs during pregnancy. For the concentration of lectin soy bean agglutinin, 7 mL of maternal peripheral blood was collected from 20 subjects, and the relative fluorescence intensity was measured using flowcytometry; 50 mg/mL, used in previous studies, was the optimal concentration. The number of cells recovered at each step of the lectin method was also investigated by FACS using fluorescence-labeled CD11a and CD33, and the results showed the usefulness of the method. Next, 7 mL of maternal peripheral blood was collected from 292 women with a normal single pregnancy (389 specimens), and NRBCs were separated and recovered using the lectin method. NRBCs slightly increased over the course of pregnancy (y = 4.29x + 5.03, r2 = 0.11). When blood was collected multiple times in the same subjects, NRBCs increased in 63 of 77 subjects (83.1%, percent change: 2.4 +/- 19.0). No NRBCs were recovered in 17 subjects (4.7%). Regarding the relationship between fetomaternal disorders and the frequency of NRBCs, 89.4 +/- 92.6 cells appeared per 10 mL of maternal blood in the normal group, but NRBCs increased in patients with 18 trisomy, placenta previa, pre-eclampsia, intrauterine fetal death, and 21 trisomy. NRBC examination may play an assisting role not only in fetal diagnosis but also in fetomaternal diagnosis.

Genistein Stimulates Hematopoiesis and Increases Survival in Irradiated Mice

Radiation protection from death and stimulating hematopoietic recovery by oral administrations of genistein, 160 mg/kg b.w., once daily for seven consecutive days before whole-body gamma-rays irradiation, were confirmed by tests with adult male BALB/c mice. Moreover, the protective action of genistein was compared to that of diethylstilbestrol (DES). Based on the studies of survival, behavior of hematograms, endogenous hematopoietic spleen colony formation (endoCFUs), and numbers of nucleated cell, granulocyte-macrophage colony forming units (CFU-GM) in bone marrow following irradiation, it was demonstrated that genistein was an effective radioprotector. The survival of irradiated mice protected by genistein was significantly increased and statistically higher than that of mice pre-treated with DES. Stimulated recovery of leukocytes, erythrocytes, lymphocytes and thrombocytes were observed in mice pre-treated with genistein or DES, however, the effects of genistein on promoting recovery of bone marrow nucleated cells, leukocytes and lymphocytes were significantly higher than those of DES. Enhanced endoCFUs, numbers of bone marrow nucleated cells and CFU-GM were also found in mice pre-treated with genistein as well as DES. Meanwhile, endoCFU numbers in mice pre-treated with genistein was 3.47-fold higher than that in the irradiated control group, although no significant difference was found between genistein administration and DES administration. It could be deduced that the radioprotective action against death is induced by a possible process of enhanced regeneration of the hematopoietic stem cells due to not only strengthened radioresistance and increased numbers of remained hematopoietic cells, but also enhanced post-irradiation repair or promoted proliferation of the hematopoietic stem cells. These effects of genistein may have some therapeutic implications for radiation-induced injuries.

Studies on preliminary concentration methods for recovery of fetal nucleated red blood cells in maternal blood

Non-invasive prenatal diagnostic methods posing no danger to the embryo have been desired for many years in the field of prenatal medicine. We are in the process of improving the lectin method that we developed for recovering fetus-derived nucleated red blood cells (NRBC) in the maternal peripheral blood. We previously used Ficoll density-gradient centrifugation for preliminary concentration in the lectin method. In the present study, we developed a molecular filter method, compared it with the Ficoll method, and tested its applicability to prenatal diagnosis. We tested the usefulness of a high molecular filter method for preliminary concentration. First, in a basic study, we prepared three kinds of non-woven cloth (NWC1-3) and a multi-porous filter (MP) to determine the optimal filter. Next, we compared the recovery rates of the Ficoll and filter methods as preliminary concentration methods in 34 normal pregnant women. Then, to examine whether the recovered NRBC were derived from the fetus, we attempted prenatal diagnosis by the fluorescence in situ hybridization (FISH) technique in 12 women pregnant with a male fetus (determined later by ultrasound) at between 8 and 16 weeks of gestation. Among the four devices used in the basic study of the high molecular filter method, NWC-2 had the best recovery rate. Therefore, we compared the numbers of NRBC recovered by the lectin method after preliminary concentration with NWC-2 or by Ficoll centrifugation, and found that the mean recovery rate of NWC-2 was 4.2 +/- 5.0 times as high as that of the Ficoll method, indicating that the NWC-2 filter method is superior as a preliminary concentration method. Next, FISH analysis of the 12 pregnant women with a male fetus for the Y chromosome showed that 19.5 +/- 12.8 NRBC were recovered, in 12.7 +/- 8.1 (63.6%) of which a Y signal was confirmed, suggesting the NWC-2 filter method can be applied to prenatal diagnosis. We consider the filter-lectin method to be a superior method for isolation and recovery of NRBC in the maternal blood which can be applied to prenatal diagnosis.

Washing of cord blood grafts after thawing: high cell recovery using an automated and closed system*

Cord blood (CB) progenitor cells are an alternative source of haematopoietic stem cells for bone marrow reconstitution. The critical importance of cell dose in the clinical outcome has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility. This aim of this study was to evaluate an automated CB washing protocol of thawed cord blood units using the Sepax device. After an initial 1:1 dilution using a dextran/albumin-containing buffer, the cells were washed in order to obtain a final product ready for transplantation. The automatic method was compared with the conventional manual washing procedure. Blood samples were taken after thawing and after washing. The processing time, viability and mean recovery of nucleated cells (TNC) and progenitors were determined. The automatic procedure resulted in a median recovery of 93% CD34+ cells and 89% TNC; no significant differences were observed between methods. In addition, median viability, as assessed by annexin V and 7-aminoactinomycin D (7-AAD), was 98% and 94%, respectively, within CD34+ cells. The automatic washing method described is as effective as the manual method in terms of viability and progenitor cells recovery, but faster and easier for the operators to perform. Overall, our data suggest that the automatic method is safe and suitable for the routine washing of thawed CB grafts in the clinic.