Peripheral Blood Progenitor Cell Samples Research Articles

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.

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.

­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.

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.

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.

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.

Detection and clinical significance of bone marrow involvement in patients with rhabdomyosarcoma

Childhood rhabdomyosarcoma (RMS) has two major histological subtypes: alveolar (aRMS) and embryonal. The aim of the study was to monitor minimal disseminated disease (MDD) using real-time quantitative reverse-transcription PCR (RQ-RT-PCR) of the PAX3-FKHR, PAX7-FKHR fusion genes and myoD1 gene. We prepared an assay using RQ-RT-PCR for a quantitative assessment of MDD in aRMS by using hydrolysis probe for quantification of PAX3-FKHR, PAX7-FKHR and myoD1 genes and beta-2-microglobulin housekeeping gene. Primary tumor samples (44), samples of local recurrences (26) from 48 patients with aRMS were examined by nested RT-PCR and RQ-RT-PCR techniques. Additionally, bone marrow samples (115), peripheral blood progenitor cell samples (27), and peripheral blood samples (25) from 33 aRMS patients were tested. PAX3/7-FKHR and myoD1 transcripts proved to be a sensitive tool for detection of MDD in RMS. We were able to identify 15/25 patients with bone marrow (BM) involvement at the time of presentation using RQ-RT-PCR. We analyzed PAX3-FKHR or PAX7-FKHR expression during the course of the disease. The RQ-RT-PCR results correlated well with nested RT-PCR results (p < 0.0001). The presence of metastases is the most adverse prognostic factor in RMS, and bone marrow is a frequent site of the tumor dissemination in RMS, especially in aRMS. Our results detecting the fusion transcripts or myoD1 transcript in the BM or peripheral blood suggest that patients with positive findings are at high risk of the tumor progression.

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.

Novel Efficient Primary Human Peripheral Blood Progenitor Cell-Targeted Recombinant Adeno-Associated Viral Vectors.

Introduction: G-CSF mobilized peripheral blood progenitor cells (PBPC) are an excellent target for the cure of acquired and inherited disorders of the hematopoietic system. AAV-based vectors have an advantageous safety profile, due to their low immunogenicity, the lack of any associated human disease and its episomal localization. Unfortunately, currently standard rAAV2-based vectors lack the required efficiency for gene transfer into human blood progenitor cells. To increase the gene transfer efficiency on primary hematopoietic cells we applied an AAV random peptide library on primary human CD34+ PBPC.Materials and Methods: The AAV random peptide library was applied on primary human CD34+ PBPC samples (3 selection rounds). Several mutant blood progenitor-targeted rAAV2 capsid clones were obtained and sequenced. Five new rAAV vectors with the most efficient clones were produced. Titers were verified using real time PCR-based titration. To determine efficiency and specificity, a panel of leukemia (CML and AML) and non-leukemia cell lines, as well as primary human CD34+ PBPC were transduced. GFP expression was determined against uninfected control cells.Results: On a panel of leukemia cell lines at least one capsid mutant was significantly more efficient (%GFP+; p<0.01) than standard rAAV2 vectors. On solid tumor cell lines rAAV2 was more efficient, suggesting the increased specificity of our capsid mutants for hematopoietic cells. Significantly higher gene transfer efficiency (up to 16% GFP+ cells) could be obtained with the newly generated vectors compared to standard rAAV2 (up to 9-fold higher; p<0.01). Using a different approach, up to 25% gene transfer into CD34+ PBPC was observed.Conclusion: These novel vectors may finally enable efficient (>20%) gene transfer using rAAV-based vectors into primary human PBPC and may offer a safe alternative to other vector systems which is applicable in vivo.

Flow Cytometric Analysis of Specimens by a Central Reference Laboratory in a Multi-Center Study: Factors Affecting Data Quality.

Central reference flow cytometry laboratories are used in multicenter trials to eliminate inter laboratory variation in testing. Yet, factors that materially affect the quality of data such as graft processing, sample composition, packaging, and shipment have been little studied. The Baylor Center for Cell and Gene Therapy (BCGT) was the central reference laboratory for the NMDP IRB-2002-0068 multicenter study on the correlation of graft composition on clinical outcome. Samples of processed or non-processed grafts of 489 bone marrow (BM) and 696 peripheral blood progenitor cell (PBPC) grafts from July 2003-March 2004 were shipped to BCGT using 2 types of shipping containers with frozen gel packs to keep samples cool during transit. On arrival samples were assessed for temperature of gel packs, sample integrity (clumps, clots requiring filtration), viability by flow cytometry using 7AAD, and immunophenotype (CD34+, CD3+, and CD3+ T-cell subsets, CD25, CD69, HLA-DR). The FDA “standard” of 70% viability was used to define acceptable sample viability. Integrity and viability of BM and PBPC samples were not influenced by the method of cell processing: non-processed, plasma depletion, mononuclear cell concentration or T-cell depletion. BM samples were not affected by either temperature or time to evaluation. Viability of PBPC samples was adversely affected by both gel pack temperature and time from collection to evaluation. A 55% mean viability was observed with gel packs arriving thawed-warm vs 89% with frozen gel packs. Additionally, the samples that arrived >48hr after collection also had mean viability less than the 70% standard value. Viability of samples was inversely correlated with gel pack temperature on arrival and time from collection to evaluation. Therefore, the quality of FACS data and by implication, the graft utility was largely influenced by cell type, temperature in transit, and the time from collection to evaluation. Thus, while the use of a central reference laboratory can produce reliable data on cells following a variety of processing methods, certain factors must be optimized in order to collect data that accurately reflects the samples under study. Shipping standards to maintain graft viability and reliability of laboratory assessments of graft composition must be established and validated against the success of transplantation outcomes.

Low-Level Laser Therapy at Different Energy Densities (0.1–2.0 J/cm2) and Its Effects on the Capacity of Human Long-Term Cryopreserved Peripheral Blood Progenitor Cells for the Growth of Colony-Forming Units

The aim of this research was to investigate the effects of low-level laser therapy (LLLT) at different energy densities (0.1-2.0 J/cm(2)) on the capacity of long-term cryopreserved peripheral blood progenitor cell (PBPC) for growth of colony-forming units (CFU) in vitro. There are no data concerning the effects of LLLT on human cryopreserved PBPC. Cryopreserved PBPC samples were thawed after 3 years in order to demonstrate the positive effect of LLLT and after 5 years in order to confirm the LLLT's proliferative effect. Cultures were plated in quadruplicate 35-mm-diameter Petri dishes in methylcellulose medium (2 x 10(5)/mL final concentration) and incubated for 14 days at 37 degrees C with 5% CO(2). A 685-nm diode laser with 25-mW optical power was used as the source of irradiation. Cultures were exposed to energy densities of 0.1, 0.5, 1.0, 1.5, and 2.0 J/cm(2) before incubation (10 irradiated and 10 controls at each energy density group). A higher number of CFU was observed at the dose of 1.0 J/cm(2) (control 21.3 +/- 8.5 x 10(5) cells, irradiated 40.1 +/- 10.5 x 10(5) cells, p < 0.001). No differences were observed in cultures exposed to doses of 0.1, 0.5, and 1.5 J/cm(2). A decreased number of CFU was demonstrated in samples exposed to the dose of 2.0 J/cm(2) (control 21.4 +/- 11.9 x 10(5) cells, p = 0.013). PBPC samples cryopreserved for 5 years were thawed for CFU assays and exposed to a single dose of 1.0 J/cm(2); once again the exposed group showed a higher number of CFU (control 8.8 +/- 7.8 x 10(5) cells, irradiated 18.1 +/- 13.1 x 10(5) cells, p = 0.026). Dependent upon the energy density, LLLT elevates (1.0 J/cm(2)) or decreases (2.0 J/cm(2)) the potential of long-term cryopreserved PBPC for growth of CFU in vitro.

Real-time polymerase chain reaction in multiple myeloma: quantitative analysis of tumor contamination of stem cell harvests.

Autologous transplantation of bone marrow (BM) and peripheral blood progenitor cells (PBPC) is commonly used for treatment of multiple myeloma (MM). Although both stem cell sources harbor residual clonal cells, a quantitative evaluation of their level of tumor contamination (LTC) still needs to be performed through highly accurate and reproducible approaches. In this study, we used a validated real-time polymerase chain reaction (PCR) strategy to evaluate LTC of BM and PBPC samples obtained from MM patients. The patients underwent two different mobilization courses (defined as early or late course) following two cycles of cyclophosphamide 5 g/m(2). LTC was evaluated by measuring the number of clonal immunoglobulin heavy-chain rearrangements followed by normalization of samples using the GAPDH gene. Overall, 26 PBPC and 12 BM samples were analyzed. Main results are as follows. 1) PBPC harvests are less contaminated than BM samples taken immediately after each mobilization course (median difference 2.68 logs; range 1.7 to 4.6) (p < 0.0001). 2) LTC of PBPC harvests has only minimal variation among different leukaphereses performed during the same mobilization course (median difference 0.45 logs; range 0.22 to 1.2). 3) No difference was observed among PBPC and BM samples obtained after the late mobilization course as compared to the early mobilization course (median reduction 0.21 logs; range -0.39 to 1.3) (p = 0.84). 4) In PBPC but not in BM samples, there is a clear overestimation of the percentage of plasma cells when flow cytometric evaluation of CD38(bright) cells is compared to real-time PCR results. This suggests that in PBPC, most CD38(bright) cells do not belong to the neoplastic clone. Real-time PCR using the IgH rearrangement proved an effective tool for monitoring LTC in stem cell harvests from MM patients. The smaller LTC of PBPC harvests supports the role of PBPC as stem cell rescue for MM patients compared to BM cells.

Human mammaglobin RT-PCR assay for detection of occult breast cancer cells in hematopoietic products

BackgroundThe aim of this study was: (i) to evaluate the sensitivity and specificity of mammaglobin as a reverse transcriptase polymerase chain reaction (RT-PCR) marker of breast cancer cells; (ii) to determine the incidence of tumor cell contamination of hematopoietic samples from patients with breast cancer. Materials and methodsA nested RT-PCR assay for mammaglobin was developed. Sensitivity was determined by serial dilution assays with breast cancer cell lines, human breast cancers and normal breast tissue. Specificity was evaluated in hematopoietic samples from healthy volunteers and patients with hematological malignancies or solid tumors other than breast cancer. ResultsThe mammaglobin transcript was detected in all 15 breast cancers, one benign breast tumor and five normal breast tissues studied, as well as in three breast cancer cell lines, in dilutions as low as 10–8. The transcript was not detected in any of 47 peripheral blood samples, 15 bone marrow aspirates and 28 peripheral blood progenitor cell samples from the three control populations. Mammaglobin mRNA was detected in 19 of 78 peripheral blood samples from patients with breast cancer starting systemic chemotherapy, as well as in five of 30 repeat samples collected before the fourth cycle of treatment. The transcript was also present in six of seven bone marrow aspirates from patients with metastatic disease, two of five with loco-regional disease, but not in the aspirate of two patients with thrombocytopenia and a previous history of breast cancer. ConclusionsHuman mammaglobin mRNA is a sensitive and specific marker of breast cancer cells and should be further studied as a molecular marker of tumor cell contamination of hematopoietic tissues.

Enumeration of CD34 cells in mobilized peripheral blood and apheresis collections: Comparative analyses of imagn 2000 steller® assay and flow cytometry results

Quantitative measurement of CD34 cells has become the gold standard for determining the sufficiency of peripheral blood progenitor cell (PBPC) collections to reconstitute patients after high-dose therapy. Flow cytometry is the accepted method for quantifying CD34 cells. However, there are concerns about the accuracy and reproducibility of this method. The IMAGN 2000 STELLer assay offers an alternative approach, with minimal sample manipulation and operator involvement. For this study we compared our flow results with STELLer on 50 mobilized PB samples obtained immediately prior to apheresis and the 50 matched PBPC harvests. Calibration and STELLer assays were run according to manufacturer's instructions. Flow staining and analyses consisted of two-color staining with CD14-FITC and CD34-PE reagents, acquisition of 100,000 events on a FACSCAN, and analysis using Winlist. Linear regression analyses indicated a strong correspondence between STELLer and flow results. On PBPC r2 = 0.88 for products ranging from 13-6000 CD34 cells/μl, for PB r2 = 0.75 (range 2–180 CD34/μl), and for all samples r2 = 0.92. As a general finding, STELLer counts were 10–20% lower than flow values. However, at lower cell numbers (counts < 10 CD34/μl) there was a tendency to report slightly higher results. We conclude that the STELLer assay can provide a highly accurate enumeration of CD34 cells in PB and PBPC samples, highly comparable with that obtained by flow cytometry.

The influence of patient-related factors on ex vivo retroviral-mediated gene transfer into mobilized peripheral blood myeloid progenitors.

Mobilized human peripheral blood progenitor cells are potential alternatives to bone marrow cells as targets for ex vivo gene transfer. We report the transduction efficiency of retroviral-mediated gene transfer into myeloid progenitors of peripheral blood progenitor cell (PBPC) harvests, mobilized by high-dose cyclophosphamide and GM-CSF, compared with nonmobilized bone marrow (BM). Eleven PBPC samples were enriched for CD34+ cells, preincubated with IL-3 (10 ng/ml), IL-6 (50 ng/ml), and 10% autologous plasma for 42 hr, and transduced over a 6-hr incubation with IL-3 + IL-6 and a retroviral vector carrying the NeoR gene. NeoR-specific sequences were detected by polymerase chain reaction in 10 cell pellets (91%). Gene expression in CFU-GM colonies was found in nine transduced samples (82%), with a mean transduction efficiency of 5.2% (95% CI, 1.3-11.8%) CFU-GM per PBPC sample. In univariate analysis, a higher transduction efficiency into CFU-GM correlated significantly with a higher CFU-GM concentration in the CD34+-enriched sample (p = 0.009), a shorter interval from diagnosis (p < 0.001), and fewer months of prior cytotoxic treatment (p = 0.001); correlation with younger age was of borderline statistical significance (p = 0.077). In multivariate analysis a shorter interval from diagnosis and, to a lesser degree, a higher CFU-GM concentration in the CD34+-enriched sample were independent predictors of higher transduction efficiency. Twelve BM samples were similarly transduced; 11 pellets were PCR positive. CFU-GM NeoR gene expression was 4.2% (95% CI, 2.0-7.2%) CFU-GM per BM sample, which was not significantly different from the transduction efficiency into PBPC cells. No correlation was found between the transduction efficiency of CFU-GM in BM samples and CFU-GM concentration in the CD34+-enriched sample, time from diagnosis, months of prior cytotoxic treatment, and/or patient age. Our data suggest that the transduction efficiency ex vivo may be influenced by time from diagnosis, CFU-GM concentration in the sample, and possibly by the extent of prior cytotoxic administration.

An intralaboratory quality control program for quantitation of CD34+ cells by flow cytometry.

This study was undertaken to develop a quality control protocol to monitor instrument, operator, and CD34 assay performance. A dual level control system was established by cryopreserving aliquots of cells from peripheral blood progenitor cell (PBPC) collections exhibiting different percentages of CD34+ cells. Twenty-five samples from each control specimen were assayed to establish a control range (mean +/- 2 SD). Levey-Jennings graphs were prepared for each control specimen to plot multiple measurements of CD34%. No significant differences were observed between fresh or cryopreserved PBPC aliquots in terms of light scatter properties or CD34 antigen density within the gated cell population. Cryopreserved PBPC samples are ideal for serving as a positive methodology control for daily CD34 analysis. Furthermore, such a system can help identify problems with assay reagents, sample preparation technique, or incorrect data analysis.

Fate of Contaminating t(14;18)+ Lymphoma Cells During Ex Vivo Expansion of CD34-Selected Hematopoietic Progenitor Cells

The use of ex vivo expanded CD34-selected hematopoietic progenitor cells (HPCs) for autologous stem cell support or gene therapy is a major area of research and is likely to increase in the future. At present, little is known about the fate of contaminating malignant cells during ex vivo expansion of CD34-selected HPCs. We established a competitive polymerase chain reaction (PCR) titration assay to determine the number of residual lymphoma cells before and after selection and ex vivo expansion of CD34-selected HPCs in patients with t(14; 18) translocation carrying non-Hodgkin's lymphoma. Seven bone marrow (BM) and 2 mobilized peripheral blood progenitor cell samples from 8 patients without histologic BM involvement at the time of the harvest were analyzed by competitive PCR titration assay and determined to contain between < or = 10 and 4,000 lymphoma cells/ 10(6) mononuclear cells (MNCs). Immunoadsorption enriched CD34+ cells from a mean of 5% (range, 1% to 9%) to a mean of 88% (range, 76% to 94%) of MNCs and resulted in a 1 to 4 log depletion of contaminating tumor cells. Two HPC samples became PCR negative after CD34 selection, whereas 7 samples still contained < or = 10 to 200 residual lymphoma cells/10(5) MNCs. CD34-selected cells were consecutively expanded in suspension culture in the presence of stem cell factor, interleukin-1 beta (IL-1 beta), IL-3, and IL-6. The mean increase of cells was 13-fold (range, 4- to 22-fold) at day 7 and 65-fold (range, 43- to 110-fold) at day 14 of culture. Expansion resulted predominantly in myelomonocytic differentiation, whereas B-cell antigen-expressing cells became undetectable. Six of the seven PCR-positive CD34-selected samples became PCR-negative for the t(14; 18) translocation at day 7 and/or 14 of expansion. One PCR-positive and one PCR-negative CD34-selected sample were PCR-positive after ex vivo expansion, but the number of residual lymphoma cells remained at the limit of detection. We conclude that CD34-selection does not eliminate contaminating lymphoma cells in the majority of t(14; 18)+ HPC harvests. However, during ex vivo expansion of CD34-selected HPCs, residual t(14; 18)+ lymphoma cells do not proliferate and become undetectable by PCR in the majority of cases.

Similar breast cancer cell contamination of single-day peripheral-blood progenitor-cell collections obtained after priming with hematopoietic growth factor alone or after cyclophosphamide followed by growth factor.

To evaluate tumor-cell contamination of peripheral-blood progenitor-cell (PBPC) collections obtained after priming with granulocyte colony-stimulating factor (G-CSF). Immunocytochemical (ICC) and tumor clonogenic (TCA) assays were used to analyze tumor-cell contamination of pretreatment peripheral-blood (PB) and bone marrow (BM) samples, and of PBPC collection samples obtained after priming with G-CSF 5 micrograms/kg/d for 5 or 7 days in 38 women with advanced breast cancer undergoing high-dose chemotherapy (HDC). Results were compared with 37 historical control patients who underwent PBPC mobilization with cyclophosphamide (4 g/m2) followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) 5 micrograms/kg/d for 14 days. Before PBPC priming with G-CSF, only one of 37 (3%) PB and four of 36 (11%) BM samples had tumor cells detected by ICC. Tumor-cell contamination of PBPC collections obtained after 5 or 7 days of G-CSF priming was observed in only three of 38 patients (8%). All patients with tumor cells detected in the PBPC collection had stage IV disease. Cells with in vitro clonogenic potential were detected only in the pretreatment BM sample in one patient, and another two patients had ICC- and TCA-positive PBPC samples despite tumor-negative PB and BM before priming. These results are similar to those previously reported for PBPC primed with cyclophosphamide and GM-CSF. In patients with advanced breast cancer responsive to cytotoxic chemotherapy, tumor-cell contamination is not increased in PBPC collected after 5 or 7 days priming with G-CSF and appears similar to that seen when PBPC are primed with cyclophosphamide followed by GM-CSF.