Peripheral Blood Progenitor Cell Products Research Articles

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis

BackgroundGraft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products.MethodsNeuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture.ResultsPDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft.ConclusionThese results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

Label-free neuroblastoma cell separation from hematopoietic progenitor cell products using acoustophoresis - towards cell processing of complex biological samples

Processing of complex cell preparations such as blood and peripheral blood progenitor cell (PBPC) transplants using label-free technologies is challenging. Transplant-contaminating neuroblastoma cells (NBCs) can contribute to relapse, and we therefore aimed to provide proof-of-principle evidence that label-free acoustophoretic separation can be applied for diagnostic NBC enrichment and removal (“purging”) from human blood and PBPC products. Neuroblastoma cells spiked into blood and PBPC preparations served as model systems. Acoustophoresis enabled to enrich NBCs from mononuclear peripheral blood cells and PBPC samples with recovery rates of up to 60–97%. When aiming at high purity, NBC purities of up to 90% were realized, however, compromising recovery. Acoustophoretic purging of PBPC products allowed substantial tumour cell depletion of 1.5–2.3 log. PBPC loss under these conditions was considerable (>43%) but could be decreased to less than 10% while still achieving NBC depletion rates of 60–80%. Proliferation of cells was not affected by acoustic separation. These results provide first evidence that NBCs can be acoustically separated from blood and stem cell preparations with high recovery and purity, thus indicating that acoustophoresis is a promising technology for the development of future label-free, non-contact cell processing of complex cell products.

Feasibility and Cost Analysis of Day 4 Granulocyte Colony-Stimulating Factor Mobilized Peripheral Blood Progenitor Cell Collection from HLA-Matched Sibling Donors

Feasibility and Cost Analysis of Day 4 Granulocyte Colony-Stimulating Factor Mobilized Peripheral Blood Progenitor Cell Collection from HLA-Matched Sibling Donors

Feasibility and Cost Analysis of Day 4 Granulocyte Colony-Stimulating Factor Mobilized Peripheral Blood Progenitor Cell Collection from HLA-Matched Sibling Donors

Introduction: Published guidelines recommend mobilization of allogeneic peripheral blood progenitor cells (PBPC) with 4-5 days of granulocyte colony-stimulating factor (G-CSF) followed by PBPC collection on day 5. Given that some autologous transplant patients are adequately mobilized by day 4, and concern that a subset of allogeneic donors may be maximally mobilized before day 5 of G-CSF administration, we performed a feasibility study evaluating day 4 allogeneic PBPC collection.Methods: This single-center prospective study included adult patients with malignant diseases who received a first allogeneic hematopoietic stem cell transplant (HCT) at our center between 7/1/2013 - 6/30/2017. HLA-matched sibling donors received 10 μg/kg of G-CSF subcutaneously prior to PBPC collection. Donors were collected on day 4 of G-CSF if peripheral blood (PB) CD34+ counts were ≥0.04x106/ml. Donors with day 4 PB CD34+ counts <0.04x106/ml were collected on day 5, and those with inadequate collected CD34+ cells/kg recipient weight underwent repeat collection over two days. Patient and donor characteristics, PBPC product composition, transplant outcomes, and cost analysis of the PBPC collection procedure were compared to a historical cohort of donors collected on day 5 according to our prior algorithm.Results: One hundred and one patients/donors were eligible for inclusion on study, and 75 were included in the historical cohort. Of the 101 donors on study, 50 (49.5%) had adequate PBPC collection on day 4 of G-CSF, 26 (25.7%) were collected on day 5, and 25 (24.8%) required two-day collections on days 4 and 5. The median day 4 donor PB CD34+ cell count was 0.06 (range 0.03-0.13) x 106/ml among donors collecting on day 4, 0.03 (range 0.007-0.09) x 106/ml for donors collecting on day 5, and 0.019 (range 0.004-0.04) x 106/ml for donors collecting over two-days (p<0.001).The most common reported donor symptoms were bone pain, myalgia/malaise, headache, and circumoral tingling/electrolyte abnormalities. Compared to the historical cohort, donors with PBPC collection on day 4 were significantly more likely to develop bone pain, require narcotics, and take over-the-counter (OTC) medications, day 5 donors were significantly more likely to develop bone pain and take OTC medications, and two-day donors were significantly more likely to develop bone pain and circumoral tingling/electrolyte abnormalities.The median collected PBPC CD34+ cell count (7.25 x 106/kg recipient weight) and infused PBPC product CD34+ cell count (5.67 x 106/kg recipient weight) were significantly greater in products collected on day 4, compared to products collected on other days. In comparison, the infused PBPC product total nucleated cell (TNC) count (7.13 x 108/kg recipient weight), mononuclear cell (MNC) count (5.84 x 108/kg recipient weight), and CD3+ cell count (2.14 x 108/kg recipient weight) were significantly lower in products collected on day 4 compared to other collection cohorts (p<0.001, p<0.001, and p=0.002, respectively for day 5, two-day, and historical cohort).There were no significant differences in time to neutrophil or platelet engraftment, or in the incidence of acute or chronic graft-versus-host-disease, based on day of collection. There was a non-significant trend towards improved 1-year survival among patients transplanted with PBPC collected on day 4 (p=0.077). Cost analysis revealed a 6.7% savings in direct costs per collection procedure for the day 4 collection method versus historical cohort.Conclusions: Using a day 4 PB CD34+ threshold of ≥0.04x106/ml identifies donors with a high likelihood of adequate PBPC collection. While differences in PBPC product composition and donor symptoms were seen based on day of collection, we found no evidence of adverse post-HCT outcomes associated with day 4 PBPC collection. Day 4 may be the optimal day of stem cell collection for a population of healthy donors without adverse effect on transplant outcomes, and with an anticipated cost savings. Ongoing analyses include resource utilization review, matched cohort analysis of day 4 versus 5 collection and correlation with transplant outcomes, more detailed PBPC production composition analysis among the collection cohorts, and longer post-HCT follow-up and association with outcomes based on day of PBPC collection. These findings merit further analysis in larger HCT cohorts. [Display omitted] DisclosuresCook:Syros Pharmaceuticals: Consultancy. Maziarz:Incyte: Consultancy, Honoraria; Kite Therapeutics: Honoraria; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Athersys, Inc.: Patents & Royalties; Juno Therapeutics: Consultancy, Honoraria.

Affinity-Bead-Mediated Enrichment of CD8+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Acoustophoresis.

Acoustophoresis is a technique that applies ultrasonic standing wave forces in a microchannel to sort cells depending on their physical properties in relation to the surrounding media. Cell handling and separation for research and clinical applications aims to efficiently separate specific cell populations. Here, we investigated the sorting of CD8 lymphocytes from peripheral blood progenitor cell (PBPC) products by affinity-bead-mediated acoustophoresis. PBPC samples were obtained from healthy donors (n = 4) and patients (n = 18). Mononuclear cells were labeled with anti-CD8-coated magnetic beads and sorted on an acoustophoretic microfluidic device and by standard magnetic cell sorting as a reference method. CD8 lymphocytes were acoustically sorted with a mean purity of 91% ± 8% and a median separation efficiency of 63% (range 15.1%–90.5%) as compared to magnetic sorting (purity 91% ± 14%, recovery 29% (range 5.1%–47.3%)). The viability as well as the proliferation capacity of sorted lymphocytes in the target fraction were unimpaired and, furthermore, hematopoietic progenitor cell assay revealed a preserved clonogenic capacity post-sorting. Bead-mediated acoustophoresis can, therefore, be utilized to efficiently sort less frequent CD8+ lymphocytes from PBPC products in a continuous flow mode while maintaining cell viability and functional capacity of both target and non-target fractions.

Multigene MRD Assessment Improves AML Relapse Risk Stratification in Autologous Hematopoietic Cell Transplantation

▪While commonly used in other hematological malignancies, high dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-HCT) has not been widely adopted in acute myeloid leukemia (AML) due to concerns regarding high post-transplant relapse rates. These relapses may be due, in part, to autograft contamination with AML. High sensitivity methods to detect residual AML have demonstrated the ability to correctly identify patients in morphological complete remission (CR) at risk of relapse. We sought to determine if testing of the graft prior to transplantation could predict post auto-HCT relapse. Prior studies investigating the utility of measurable residual disease (MRD) in auto-HCT have been limited by small patient cohorts and/or by the use of a single biomarker to detect AML, a heterogeneous cancer. We report here the largest retrospective study to date of adult auto-HCT AML patients tested for measurable residual disease (MRD) by both molecular methods (RQ-PCR) and multi-parameter flow cytometry (MPFC).Seventy-two patients transplanted between 2004-2013 at a single academic medical center (UCSF) were eligible for this study based on availability of cryopreserved GCSF mobilized autologous peripheral blood progenitor cell (PBPCs) specimens. All samples were collected on IRB approved research protocols.The median age at time of transplant was 48 (24-69), 54% were female, cytogenetics were known in 94% (of which 24% were favorable, 69% intermediate, 7% poor), 99% were in CR at the time of HCT (64/72 in CR1, 6 in CR2, 1 in CR3). Following auto-HCT 1 year RFS was 50% (1 year relapse rate 43%) and 2 year RFS was 40% (2 year relapse rate 47%).Wilms tumor 1 (WT1) is expressed in up to 90% of AML, but sufficiently over-expressed in peripheral blood to have utility as a sensitive marker of MRD in less than 50% of cases. RQ-PCR detectable WT1 above the previously established European LeukemiaNet threshold was found in 9 patients, of whom 6 relapsed in the first year after auto-HCT (19% sensitivity, 93% specificity, PPV: 67%, NPV: 60%).We have previously reported that multi-gene testing can augment WT1 based MRD detection in AML. We were unable however to use one component of our previously reported AML MRD panel, PR3, due to increased baseline expression in these GCSF stimulated PBPC products. Increased PR3 expression following GCSF administration, correlating with neutrophilia, was also observed in an independent cohort of healthy donors.Testing for PRAME, MSLN, CCNA1, t(8,21), Inv16, t(15:17) and NPM1 mutations A, B and D, as a supplement for WT1, in pre-HCT PBPCs resulted in substantially improved ability to predict post auto-HCT relapse (52% sensitivity, 80% specificity, PPV: 67%, NPV: 69%). Addition of these extra genes allowed for correct identification of 10 additional MRD+ patients who relapsed within 1yr after transplant, and added 5 false positive patients (1 of whom suffered early non-relapse mortality, and another who did relapse but more than 1 year after HCT).Finally, in expert hands, flow cytometry can identify residual AML with high sensitivity. Forty PBPC samples from the above cohort were also assessed for MRD using MPFC. CD34 positive cells comprised 0.05-12.5% of autograft specimen mononuclear cells. Due to immunophenotypic changes likely attributable to GCSF mobilization, and without leukemia associated immunophenotypes from diagnosis available, MPFC was unable to identify MRD in any of 40 patients tested.In summary, no single MRD test on autografts could completely predict post-HCT AML relapse. Auto-HCT presents unique challenges for AML MRD testing due to masking effects of GCSF on MPFC and RQ-PCR gene expression signatures. Additionally, detection of any AML autograft contamination must be extremely sensitive to be useful for predicting relapse given the absence of any potentially protective graft versus leukemia effect following an auto-HCT. Here we show combinations of molecular MRD assays can overcome some, but not all, of these limitations (Figure). [Display omitted] DisclosuresRadich:Incyte: Consultancy; Ariad: Consultancy; Novartis: Consultancy; Novartis: Other: Lab Contract. Andreadis:Cellerant: Consultancy; Novartis: Consultancy; McGraw Hill: Other: Publishers; Pharmacyclics: Honoraria. Damon:Sunesis: Research Funding; McGraw Hill: Other: Chapter Royalties; Atara: Consultancy; Sigms Tau: Research Funding. Logan:Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy; Pharmacyclics: Consultancy. Martin:Sanofi: Consultancy.

Hydroxyethyl starch as a substitute for dextran 40 for thawing peripheral blood progenitor cell products

Background aimsRemoving DMSO post-thaw results in: reduced infusion reactions, improved recovery and stability of viable CD34+ cells. Validated methods use 5%–8.3% Dextran 40 with 2.5%–4.2% HSA for this purpose. Recent shortages of clinical grade Dextran require identification of suitable alternatives. MethodsPBPC were used to compare a standard 2X wash medium of 5 parts 10% Dextran 40 in saline (DEX) with 1 part 25% HSA (8.3% DEX/ 4.2% HSA) with Hydroxyethyl Starch (HES)-based solutions. Cells in replicate bags were diluted with an equal volume of wash solution, equilibrated 5 minutes, the bag filled with wash medium, pelleted and the supernatant expressed. Bags were restored to the frozen volume in wash medium and tested by single platform flow cytometry and CFU. Total viability, viable TNC, MNC, and CD34+ cell recovery, and CD34+ cell viability were compared immediately post-thaw and after 90 minutes. Results5.2% HES/4.2% HSA did not differ from our standard in CD34 recovery or viability. Due to concerns that high concentrations of HES could affect renal function we tested 0.6% HES/2.5% HSA resulting in significantly poorer CD34 recovery and viability. Results improved using 2.4% HES/4.2% HSA and when 0.6% HES/4.2%HSA was used no significant differences were seen. CFU assays confirmed no differences between the standard dextran arm and HES at 2.4% or 0.6% so long as HSA was at 4.2%. ConclusionsWe conclude that HES from 0.6% to 5.2% with 4.2% HSA is a suitable substitute for Dextran 40 as a reconstitution/washing medium for PBPC products.

Phenotypic and functional comparison of mobilized peripheral blood versus umbilical cord blood megakaryocyte populations

Background aimsHematopoietic stem cell transplantation of mobilized peripheral blood progenitor cell (PBPC) products results in rapid platelet engraftment, whereas the use of cord blood (CB) shows significant delays. The difference in the quality and number of megakaryocyte (MK) progenitors that may be responsible for the delay in platelet engraftment has not been fully defined. The objective of this study was to quantify the cells of the MK lineage in PBPC and CB products to determine whether potential differences exist. MethodsWe examined PBPC or CB for differences in surface markers and subpopulations as well as polyploidization status within the MK lineage. Colony-forming assays were used to determine whether differences exist in the clonogenic MK progenitor cell. Finally, we transplanted PBPC and CB mononuclear cells into NOD/SCID/IL2Rγ−/− (NSG) mice to study platelet engraftment rates. ResultsEquivalent MK populations and polyploidization was observed in PBPCs and CB. MK progenitors were present only in CD34+ cells and had little difference in colony growth between PBPC and CB. Additionally, MK subpopulations were similar in either product with a slightly more progenitor-enriched phenotype in CB. Finally, when PBPC or CB was transplanted at similar doses, equivalent platelet engraftment rates were observed. ConclusionsPBPC and CB contain similar frequencies of MK populations, and, when transplanted in comparable doses, CB is as effective as PBPCs in producing platelet engraftment in vivo. Understanding the differences in MK populations between PBPC and CB could help generate protocols to improve platelet engraftment after CB transplantation.

­Separation of Lymphocyte Populations from Peripheral Blood Progenitor Cell Products Using Affinity Bead Acoustophoresis

Introduction: Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations. We have previously reported the use of a novel ultrasound-based sorting technology, called acoustophoresis, for sorting of platelets (Dykes et al., PloS one 2011) and CD4+ cells from PBPC products (Lenshof et al., Cytometry Part A 2014). Here, we investigated the performance of microfluidic acoustophoresis for the separation of CD8+ lymphocytes from PBPC, and present a method for affinity-bead-mediated acoustic separation of cells which otherwise cannot be discriminated acoustically. In an acoustic standing wave field radiation forces induce movement of particles depending on particle and medium properties, such as for example particle size, density and compressibility. Targeting of cells by affinity specific beads generates cell-bead complexes that exhibit distinct acoustic properties relative to non-targeted cells and, thus, become possible to isolate.Method: PBPC samples (n=16) were obtained from patients and healthy donors. Following density gradient centrifugation, mononuclear cells were labelled with anti-CD8 microbeads (Dynal) and sorted either on an acoustophoresis-microchip (Figure 1) or standard magnetic cell sorting technique for comparison. PBPC samples, target and waste fractions were analysed for purity, separation efficiency, recovery, T-cell function and progenitor cell content.Results: PBPC products contained a mean of 11.6 ± 7.1% CD8+ cells before sorting. Purities obtained with acoustic sorting of CD8+ lymphocytes were 93.3 ± 6.8% compared to 94.4 ± 8.6% for magnetic sorting (n=16). Viabilities of sorted cells were 97.0 ± 3.9% (acoustic) and 97.5 ± 3.5% (magnetic). Mean separation efficiency recovery of acoustic sorted CD8+ cells was 57 ± 19% of the total CD8+ cells compared to a median recovery of magnetic sorted CD8+ cells of 43 ± 17%. Leukocyte subpopulation analysis performed after CD8 selection showed a relative increase of CD4 cells in the non-target fractions due to the removal of CD8 cells. Functional testing of sorted CD8+ lymphocytes showed unimpaired mitogen mediated proliferation capacity after 2-day, 4-day and 6-day stimulation with CD3/CD28. Furthermore, hematopoietic progenitor cell assays revealed a preserved colony forming ability of the post-sorted non-target cellsConclusion: Acoustophoresis is a promising technology to efficiently sort bead-labelled lymphocyte populations from PBPC samples with high purity and recovery without impairing lymphocyte function. Affinity-bead acoustophoresis is, thus, an interesting technology for stem cell processing in PBPC. [Display omitted] DisclosuresLaurell:Acousort AB: shareholder Other. Scheding:Acousort AB: Co-founder and board member Other.

Volume 85A, Number 11, November 2014 Cover Image

On the cover: This hypnotic image almost enables you to hear the sound waves it is intended to depict. The sound waves used in a novel method for affinity‐bead‐mediated acoustic separation of cells. The new method was tested successfully to purify CD4+ lymphocytes from peripheral blood progenitor cell products, demonstrating its unique ability for simultaneous separation of multiple cell populations not presently available in standard magnetic‐based approaches. Read the accompanying article by Lenshof et al. in this issue.Cover design by Bärbel Beran [www.beran‐design.de].

Affinity-bead-mediated acoustophoresis: a novel tool in cytometry.

ACOUSTOPHORESIS combined with biofunctionalized capture beads—a method called affinity-bead-mediated acoustophoresis—can be used for highly selective isolation of CD41 lymphocytes from peripheral blood progenitor cell products (PBPCS). This opens possibilities for improved hematopoietic stem cell transplantation. Acoustophoresis is a method based on the migration of suspended particles or cells in an acoustic field caused by the acoustic radiation force. When implemented in a flowthrough setting, acoustophoresis can be used for separation of particles from a fluid medium, or separation of particles having different size and acoustic properties (1). Over the last 10–15 years, acoustophoresis applied to microfluidic devices has been rapidly developing (2), primarily due to its simplicity and low cost relative alternative methods. For cytometry purposes, acoustophoresis-based technology was recently (in year 2010) implemented commercially for improving cell alignment, as an alternative to the standard sheath-flow alignment [Attune

Efficient purification of CD4+ lymphocytes from peripheral blood progenitor cell products using affinity bead acoustophoresis.

Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations, utilizing primarily magnetic or fluorescence activated sorting methods. Here, we investigated the performance of microfluidic acoustophoresis for the separation of lymphocyte subsets from PBPC, and present a novel method for affinity-bead-mediated acoustic separation of cells which can otherwise not be acoustically discriminated. As the acoustic force on a particle depends on particle size, density and compressibility, targeting of cells by affinity specific beads will generate cell-bead complexes that exhibit distinct acoustic properties relative to nontargeted cells and are, thus, possible to isolate. To demonstrate this, PBPC samples (n = 22) were obtained from patients and healthy donors. Following density gradient centrifugation, cells were labeled with anti-CD4-coated magnetic beads (Dynal) and isolated by acoustophoresis and, for comparison, standard magnetic cell sorting technique in parallel. Targeted CD4+ lymphocytes were acoustically isolated with a mean (±SD) purity of 87 ± 12%, compared with 96 ± 3% for control magnetic sorting. Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic), respectively. The mean acoustic separation efficiency of CD4+ lymphocytes to the target fraction was 65 ± 22%, compared with a mean CD4+ lymphocyte recovery of 56 ± 15% for magnetic sorting. Functional testing of targeted CD4+ lymphocytes demonstrated unimpaired mitogen-mediated proliferation capacity and cytokine production. Hematopoietic progenitor cell assays revealed a preserved colony forming ability of nontarget cells post sorting. We conclude that the acoustophoresis platform can be utilized to efficiently isolate bead-labeled CD4+ lymphocytes from PBPC samples in a continuous flow format, with preserved functional capacity of both target and nontarget cells. These results open up for simultaneous affinity-bead-mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technology. © 2014 International Society for Advancement of Cytometry.

Optimized Processing of Growth Factor Mobilized Peripheral Blood CD34+ Products by Counterflow Centrifugal Elutriation

Cell separation by counterflow centrifugal elutriation has been described for the preparation of monocytes for vaccine applications, but its use in other current good manufacturing practice (cGMP) operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cell products were collected from healthy donors and processed by elutriation using a commercial cell washing device. Fractions were collected for each product as per the manufacturer's instructions or using a modified protocol developed in our laboratory. Each fraction was analyzed for cell count, viability, and blood cell differential. Our data demonstrate that, using standard elutriation procedures, >99% of red blood cells and platelets were removed from apheresis products with high recoveries of total white blood cells and enrichment of CD34+ cells in two of five fractions. With modification of the basic protocol, we were able to collect all of the CD34+ cells in a single fraction. The CD34-enriched fractions were formulated, labeled with a ferromagnetic antibody to CD34, washed using the Elutra device, and transferred directly to a magnetic bead selection device for further purification. CD34+ cell purities from the column were extremely high (98.7 ± 0.9%), and yields were typical for the device (55.7 ± 12.3%). The processes were highly automated and closed from receipt of the apheresis product through formulation of target-enriched cell fractions. Thus, elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP and current good tissue practice-compliant cell processing.

Efficient Removal of Platelets from Peripheral Blood Progenitor Cell Products Using a Novel Micro-Chip Based Acoustophoretic Platform

BackgroundExcessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC) apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties.Study Design and MethodsPBPC samples were obtained from patients (n = 15) and healthy donors (n = 6) and sorted on an acoustophoresis-chip. The acoustic force was set to separate leukocytes from platelets into a target fraction and a waste fraction, respectively. The PBPC samples, the target and the waste fractions were analysed for cell recovery, purity and functionality.ResultsThe median separation efficiency of leukocytes to the target fraction was 98% whereas platelets were effectively depleted by 89%. PBPC samples and corresponding target fractions were similar in the percentage of CD34+ hematopoetic progenitor/stem cells as well as leukocyte/lymphocyte subset distributions. Median viability was 98%, 98% and 97% in the PBPC samples, the target and the waste fractions, respectively. Results from hematopoietic progenitor cell assays indicated a preserved colony-forming ability post-sorting. Evaluation of platelet activation by P-selectin (CD62P) expression revealed a significant increase of CD62P+ platelets in the target (19%) and waste fractions (20%), respectively, compared to the PBPC input samples (9%). However, activation was lower when compared to stored blood bank platelet concentrates (48%).ConclusionAcoustophoresis can be utilized to efficiently deplete PBPC samples of platelets, whilst preserving the target stem/progenitor cell and leukocyte cell populations, cell viability and progenitor cell colony-forming ability. Acoustophoresis is, thus, an interesting technology to improve current cell processing methods.

Ex vivo graft purging and expansion of autologous blood progenitor cell products from patients with multiple myeloma.

Autologous peripheral blood progenitor cell (PBPC) transplantation is the treatment of choice for selected myeloma patients. However, tumor cells contaminating the apheresis product are a potential source of relapse. Here we report a sequential purging strategy targeting mature and immature clonogenic myeloma cell populations in the autograft. Thawed PBPC products of myeloma patients were treated with rituximab to kill CD138(-)20(+) B cells (highly clonogenic immature cells), and bortezomib to target CD138(+) cells (normal and differentiated myeloma plasma cells), followed by coculture with allogeneic mesenchymal stem cells (MSC) from normal donors. After 7 days of coculture, nonadherent cells were removed and cultured in the absence of MSC for an additional 7 days. Then, efficacy of purging (removal of CD138(-)20(+) and CD138(+) cells) was assessed by flow cytometry and PCR. We used our ex vivo purging strategy to treat frozen aphereses from 16 patients. CD138(+) and CD138(-)20(+)(19(+)) cells present in the initial products were depleted more than 3 and 4 logs, respectively based on 10(6) flow-acquisition events, and to levels below the limit of detection by PCR. In contrast, total nucleated cell (TNC), CD34(+) cell, and colony-forming cell numbers were increased by approximately 12 to 20, 8-, and 23-fold, respectively. Overall, ex vivo treatment of apheresis products with rituximab, bortezomib, and coculture with normal donor MSC depleted mature and immature myeloma cells from clinical aphereses while expanding the normal hematopoietic progenitor cell compartment.

Analysis of Cell Aggregation In Thawed Hematopoietic Stem Cell Products Using Micro-Flow Imaging.

AbstractAbstract 1187 Background:Cell aggregation can be observed after thawing cryopreserved hematopoietic stem cell products used in transplantation. Micro-flow imaging (MFI) is a novel method of imaging biologic fluids and provides a means of quantifying cell aggregation. Method:Umbilical cord blood (UCB) was collected following informed consent and cryopreserved in 5% DMSO and 37% (w/v) pentastarch in liquid nitrogen. Aliquots of cryopreserved peripheral blood progenitor cell (PBPC) products were cryopreserved under similar conditions. Cells were thawed rapidly and filtered before analysis using MFI. Result:Large cellular aggregates were identified in all thawed PBPC samples analyzed (n=4) with a mean reduction in total cells of 19.7 ± 9.8% at 1 hour and 49.8 ± 15.9% at 24 hours. Cellular aggregates ≤ 170 um were visualized, sized and digitally imaged with MFI. Analysis of larger clumps was limited due to size of the analysis chamber. Cell transparency and circularity were unchanged over 24h after thawing and similar to freshly isolated PBPCs. Mononuclear cell yields did not diminish over 24 hours in freshly isolated PBPC (n=3, p>0.05) or in fresh UCB (n=3, p>0.05). Thawed UCB samples did not demonstrate significant clumping and maintained higher yields of total cells compared with PBPCs (p=0.02). Filtering of thawed PBPC products using standard blood transfusion filters (Baxter, 170 um) did not change the qualitative nature of observed cellular aggregates. The loss of cells in PBPC products correlated with early apoptotic mononuclear cells (7AAD-AnnV+). Treatment with DNase I eliminated the presence of observable cell clumping in thawed PBPCs at 24h with a marked improvement in cell yield at 24h (p<0.0001). Conclusion:Micro-flow imaging provides novel insight regarding cellular aggregation of blood stem cell products. Significant cell clumping occurs in cryopreserved PBPC after thawing and aggregates are not removed by standard transfusion filters. Aggregation increases with time after thawing and may influence the final dose of cells available for engraftment after transplantation. Fresh PBPC collections contain few aggregates and both fresh and thawed cryopreserved UCB demonstrated minimal cell clumping. Apoptosis induced by cryopreservation and thawing may contribute to increased aggregation in PBPCs and DNase I appears to preserve higher yields of total cells. New cryoprotectants that limit apoptosis are needed and methods to reduce cell clumping appear warranted. Disclosures:Martin:Brightwell Technologies: Employment.

Transportation of peripheral blood progenitor cell products: effect of ambient temperature

Peripheral blood progenitor cell (PBPC) products are often transported at high cell concentrations (>200 × 10⁹/L) over long distances, requiring >36 h transport time. Fresh PBPC samples from eight healthy donors were studied with two viability assays for effects of temperature outside the transport container (ambient temperature). The Coleman 5272 container, routinely used by the National Marrow Donor Program (NMDP) with two -20°C gel packs, was compared with the Coleman 6216 container, which can hold four -20°C gel packs. The temperature inside the smaller transport container (5272) proved to be sensitive to ambient temperature, whereas the larger container (6216) was less sensitive. The viability of CD34(+) cells, and the survival of granulocyte-macrophage colony-forming units (GM-CFU), was more dependent on the ambient temperature for the smaller than for the larger container. PBPC products are most often transported at approximately 2-8°C. The inside temperature of the container currently used by the NMDP appears to be more sensitive to increases in temperature when exposed to higher ambient temperature for prolonged periods of time. Increasing the number of gel packs from two to four improves the stability of the temperature inside the container but would require a different container.

A Strategy for Reducing CD3+ Content of Peripheral Blood Progenitor Cell Products for Matched Related Donor Allografts

A Strategy for Reducing CD3+ Content of Peripheral Blood Progenitor Cell Products for Matched Related Donor Allografts

Transportation of peripheral blood progenitor cell products: effects of time, temperature and cell concentration

Peripheral blood progenitor cell (PBPC) products are often transported at high cell concentrations (>200x10(9)/L) over long distances, requiring >36 h transport time. Fresh PBPC samples from 12 healthy donors were studied with various viability assays regarding the effects of temperature, cell concentration and duration of storage. Trypan blue exclusion was far less sensitive to cell damage than two-color fluorescence for CD34 and 7-AAD, and colony-forming unit-granulocyte-macrophage (CFU-GM) assays; the latter assay proved the most sensitive. All products stored at 4 degrees C maintained their viability for up to 4 days. Thus, at 96 h, recovery of viable CD34(+) cells was still 82%, and of CFU-GM 57%, even at concentrations of 200x10(9)/L. Higher storage temperatures rapidly decreased the viability, with extensive variation between donors. At room temperature 80% of viable CD34(+) cells and >90% of CFU-GM were lost after 48 h of storage at 200x10(9)/L. Lower cell concentrations allowed storage at higher temperatures: at 17 degrees C a concentration of 50x10(9)/L resulted in only 5% loss of viable CD34(+) cells after 48 h, while the loss was >30% at 200x10(9)/L. PBPC products should be transported at 4 degrees C. Dilution of the product may partly compensate for slightly higher temperatures. Trypan blue exclusion should be abandoned as a method for assessing viability after prolonged transportation. Proliferative assays should be used to validate transportation conditions.

Independent and Interdependent Influence of Number of CD34+ Cells and Use of G-CSF on Outcomes in Autologous PBSCT Patients

Background: Recovery of neutrophilic granulocytes after autologous peripheral blood stem cell transplantation (PBSCT), and thus overall outcome, depends on 2 main factors: the quality and quantity of mobilized peripheral blood progenitor cell products (CD34+ cells) and the use of myeloid growth factors, such as granulocyte colony stimulating factor (G-CSF).Methods: We performed a 5-year (from February 2003 to January 2008) retrospective analysis of data to evaluate independent and interdependent influence of number of CD34+ cells and use of G-CSF on outcomes in autologous PBSCT patients. At the time of analysis, the practice at our institution was as follows: Autologous PBSCT patients receiving infusion of < 5×106 CD34+ cells/kg were treated with daily subcutaneous injection of G-CSF (filgrastim 300 mg for < 80 kg; 480 mg for ≥ 80 kg). In these patients, G-CSF was started on Day +5 and was continued until the ANC was > 500/μl. On the other hand, autologous transplant patients who received ≥ 5×106 CD34+ cells/kg did not typically receive G-CSF. If engraftment did not occur after an “expected” length of time, G-CSF treatment was initiated at the discretion of the treating physician. The definition of “expected” length of time, however, varied from practitioner to practitioner. For the analysis, patients were divided in 3 groups: patients who collected < 5×106 CD34+ cells/kg and received G-CSF (group 1, n=103), patients who were infused with ≥ 5×106 CD34+ cells/kg and did not receive G-CSF (group 2, n=155), and patients who received ≥ 5×106 CD34+ cells/kg and were given G-CSF (group 3, n=47). Time to neutrophil engraftment (ANC >500/ml), time to platelet engraftment (platelets > 20,000/ml), and post-transplant length of hospital stay were compared.Results: Median time to neutrophil engraftment was significantly shorter in patients who were treated with G-CSF (11 days) in groups 1 and 3, compared to those who were not (13 days) in group 2 (table 1). Similarly, median post-transplantation hospital stay was significantly longer in patients who did not receive G-CSF (14 days) in group 2 compared to patients who were treated with G-CSF (13 days) in groups 1 and 3. There was no significant difference in time to neutrophil engraftment and post-transplant hospital stay between groups 1 and 3, suggesting that these outcome parameters did not significantly depend on number of CD34+ cells infused in our patients if G-CSF was used. Median time to platelet engraftment was significantly longer in patients receiving < 5×106 CD34+ cells/kg (12 days) in group 1 compared to patients infused with ≥ 5×106 CD34+ cells/kg (10 days) in groups 2 and 3. There was no significant difference in time to platelet engraftment between groups 2 and 3, indicating that G-CSF use did not influence platelet engraftment.Summary: These results suggest that a higher number of CD34+ cells helps accelerate platelet engraftment, but does not influence neutrophil engraftment and post-transplant length of hospital stay, as long as G-CSF treatment is instituted. The use of G-CSF accelerates neutrophil recovery, regardless of the number of CD34+ cells infused, without affecting platelet engraftment in patients undergoing autologous PBSCT. Based on this analysis, the practice at our institution has been revised to use G-CSF in all autologous transplant patients, regardless of the number of CD34+ cells, since this practice reduces the length of hospital stay.Table 1. A retrospective data analysis for patients treated at the UCSD BMT unit with autologous PBPCT from February 2003 to January 2008. The data is represented as a median value with a range indicated in parenthesis. * indicates significant difference from group 1, † indicates significant difference from group 2, and ‡ indicates significant difference from group 3 (p < 0.001, Mann Whitney U test; Graph Pad Prism, version 3.02 (Graph Pad Software, San Diego, CA)). Abbreviations: ANC-absolute neutrophil count, LOS-length of hospital stay.Group 1 < 5×106/kg (G) (N = 103)Group 2 ≥5×106/kg (no G) (N = 155)Group 3 ≥5×106/kg (G) (N = 47)CD34+ cells (×106/kg)3.2 †,‡ (1.4–4.98)6.8 * (5.0–16.7)7.0 * (5.0–12.3)Initiation of G-CSFDay +5N/ADay +5 (day 0–day +16)Time to ANC > 500/ml (days)11 † (9–28)13 *,‡ (9–21)11 † (8–17)Time to Platelet > 20,000/ml (days)12 †,‡ (6–42)10 * (0–29)10 * (0–27)Post-Transplant LOS (days)13 † (10–38)14 *,‡ (1–43)13 † (10–18)