Flow Cytometry Assessment of CD34+ Viability in Thawed Cord Blood Units: A Multi-Center Eurocord and Netcord Study

Abstract BACKGROUND. A set of Quality Controls (QC) of a Cord Blood Unit (CBU) at release is required by International Standards for Cord Blood banking, often including Flow Cytometry (FC) counting and viability assessment of thawed stem cells. Reliability and reproducibility of such tests across different CB banks is still an unmet need. Indeed, discrepancies between the thawed product characterized at the Transplant Center and the QC performed at the CB bank, might be addressed to different assessment technologies. Therefore, there is room for a multicenter exercise, aimed to check the reproducibility of the flow cytometry-driven QC and possibly to propose technical standardization and improvements. In this context, a protocol for manipulation and FC assessment of thawed CB samples was circulated and shared among 5 European CB banks(Dusseldorf, Barcelona, Florence, Cremona, Nottingham) , on behalf of Eurocord and Netcord. METHODS. Different techniques of sample preparation were tested on thawed samples, washed samples and the segment attached to the CB bag, respectively, as shown in Fig. 1. A solution of Dextran and Human Albumin as described by the New York CBB was used for dilution/washing the samples. In particular, one sample of each thawed Unit was processed for FC assay undiluted and diluted 1:3 and 1:10, respectively. Five CB Units (CBU) per CB bank unsuitable for banking (with TNC count≥1x109) were selected for the assay. The FC assay protocol was a standard TruCOUNT®, ISHAGE-based (SI), single-platform multigating strategy for CD34+ cells counting, shared by all the Banks in order to standardize the gating strategy. A mock sample was circulated before the exercise in order to check the assay reproducibility. A modification to the ISHAGE methodology (MI) was also proposed with the aim of improving the assessment of CD34+ cells viability in thawed samples. RESULTS A total of 25 CBU across the 5 labs were cryopreserved and thawed following local standards; pre-freezing and post-thawing samples were assessed for CD34+ recovery and viability. Data from all Banks were centralized and statistical analysis was performed. TNC recoveries resulted to be reproducible in all Centers for all the thawed products (undiluted, 1:3 and 1:10 dilution, segment 1:3 diluted and washed undiluted samples, respectively). Both SI and MI strategies showed no significant discrepancies when determining CD34+ absolute number and viability in pre-freezing CBUs (p>0.05 with pair T test). The recovery of viable CD34+ cells assessed by SI or MI in the thawed products did not show any significant difference in all the experimental conditions, whilst the recovery of total CD34+ cells was always lower in SI-analyzed samples than in MI-analyzed samples (* = pair T test p<0,05). One to ten dilution of the thawed product showed a higher variability, as well as segment data when compared to the bag, likely for the small size of the sample. Post-thawing CD34+ viability in MI methodology was significantly lower than in SI technique in all the experimental conditions (Fig.2). Finally, CFU recovery did not show significant differences in post thaw 1:3, washed 1:1 and segment 1:3 diluted samples when related to the pre-freeze clonogenic potential. Interestingly, MI showed a better correlation with CFU. CONCLUSIONS · Flow Cytometry-based assessment of CB graft before and after cryopreservation can provide reproducible results, following an adequate standardization of sample preparation and gating strategy. · The segment-related data were shown to be a reliable Quality Control of the stored CB Unit · Standard ISHAGE technology was less reliable in the CD34+ cell viability assessment in the thawed samples and might therefore lead to an overestimation of CD34+ viability. An adaptation of both the acquisition setting and the gating strategy is needed in order to detect viable and non viable CD34+ cells in thawed CB units. The dissemination of these findings might have a significant impact in CB transplantation, as a validated and standardized, single platform, CD34+ cell count methodology is an essential step to reduce the intra- and inter-laboratory variability. In particular, such standardization might reduce the discrepancies between QC at release in the CB banks and the thawed product at bedside. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Plerixafor Ex Vivo Mobilization of Placental Derived Haematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.4790.4790 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4790-4790

Author(s):

M. Mansour Ceesay ◽

Sameer Tulpule ◽

Sergio Querol ◽

Linda Barber ◽

Terie Duffy ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cord Blood ◽

Ex Vivo ◽

T Test ◽

Haematopoietic Stem Cell ◽

Blood Collection ◽

Cell Dose ◽

Cd34 Cells ◽

Significant Difference

Abstract Abstract 4790 Introduction The utility of cord blood as an alternative source of stem cells for haematopoietic stem cell transplantation is well established in the paediatric population. Although it's use in adults has surged over the past few years there are still problems associated with a low stem cell dose which may lead to delayed engraftment, incomplete and inefficient immune reconstitution, and poor survival. Various techniques have been used to increase the stem cell dose including ex vivo expansion, the use of double cords, intrabone injections and the use of third party donor cells. Using standard collection techniques only about 25% of cord blood samples collected reach the target cell dose in adults. The placenta is a rich source of CD34+ cells which may not be fully mobilised using current cord blood collection techniques. A modified two-step collection technique in which the initial collection is followed by placental perfusion with 50 ml heparinised 0.9% saline has shown a 15% increase in the number of nucleated cells. We have modified this technique further by perfusing the placenta with Plerixafor, a CXCR4 antagonist that is currently licensed in the UK for mobilisation of autologous stem cells from adults. The study aim was to assess whether plerixafor could enhance recovery of CD34+ cells and change the immune profile of the perfusate. Method Ten cord blood units were collected as per our standard operating procedures. After cord blood collection, 5 placentas were perfused with 50 ml 0.9% saline plus 5,000 units of preservative free heparin by slow infusion over 50 minutes and perfusate collected after about 5 minutes of starting the infusion over 50 minutes (controls). The other 5 placentas were treated the same way except for the addition of 24 mg Plerixafor to the 50 ml 0.9% saline plus 5,000 units of preservative free heparin and collected as per control arm (plerixafor arm). The samples were analysed by multicolour flow cytometry using a combination of antibody cocktails to identify and enumerate CD34+ stem cells, T cells, T cell precursors, T cell subsets, T regulatory cells, B cells, NK and NK cell precursors, and Dendtritic cells. The CD34+ progenitor cells were further quantified by CFU – a methylcellulose media based assay. Results Successful recovery of perfusate was achieved from 9 placentas (mean volume 44 ml +/− SEM 4.7) as one of the collected samples clotted. There was no significant difference in the perfusate volumes between the 2 arms (48ml v 38ml, P=0.152). Percentage increase in numbers of cells recovered by perfusing the placenta was calculated from total numbers of CD34+ and CD45+ cells collected from each cord and placenta pair. Plerixafor did not significantly increase the recovery of cells from the placenta. Perfusion of the 5 control placentas increased recovery of CD34+ cell by 5.06% (mean +/− 0.89 SEM) and CD45+ cells by 4.69% (mean +/−1.09 SEM). Perfusion of 4 placentas with saline plus Plerixafor increased recovery of CD34+ cells by 5.79% (mean +/−2.17 SEM) and CD45+ cells by 2.61% (mean +/− 0.79 SEM). Viability of haematopoietic stem cell progenitors was assessed by colony formation assay with no significant difference detected between the control arm (mean 18.4 CFU +/− 8.26 SEM; n=5) and the plerixafor arm (mean 4.8 CFU +/− 2.7 SEM; n=4). There was no significant difference in the number of CD34+ cells recovered from the controls as compared to their corresponding cords (n=5, paired t test, p= 0.054). Similarly, there was no significant difference in the plerixafor arm (n=4, paired t test, p=0.091). There was no significant increase in the number of CD34+ stem cells recovered from the plerixafor group as compared to the control group (unpaired t test, p=0.74). There was no significant increase in the number of CD45+ cells in the plerixafor arm compared to the controls (unpaired t test, p= 0.4). Similarly there was no significant difference in the clonal efficiency of the between the 2 groups (unpaired t test, p=0.2). There were no significant differences in the CD3, CD4, CD8 T cells, Tregs, NK, DCs, progenitor T and NK cells between the 2 groups. Conclusion In this pilot study plerixafor does not seem to significantly increase the CD34+ stem cells and other cell subsets. It is possible that the duration of infusion was too short to mobilise the stem cells. Further studies are warranted to investigate this further. Disclosures: Off Label Use: Plerixafor for stem cell mobilzation from placenta. Shaw:Therakos, a Johnson and Johnson company: Honoraria, Speakers Bureau.

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Mesenchymal Stem Cell (MSC) Based Cord Blood (CB) Expansion (Exp) Leads to Rapid Engraftment of Platelets and Neutrophils

Blood ◽

10.1182/blood.v116.21.362.362 ◽

2010 ◽

Vol 116 (21) ◽

pp. 362-362 ◽

Cited By ~ 8

Author(s):

Marcos De Lima ◽

Simon Robinson ◽

John McMannis ◽

Amin M Alousi ◽

Rima M Saliba ◽

...

Keyword(s):

Cord Blood ◽

Platelet Transfusion ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Chronic Gvhd ◽

Hla Antigens ◽

Median Number ◽

Transfusion Independence ◽

Cd34 Cells ◽

Grade Ii

Abstract Abstract 362 Delayed engraftment and low rates of platelet transfusion independence are frequently observed after CB transplantation (CBT). We conducted a study of ex-vivo co-culture of CB mononuclear cells with either third party haploidentical family member marrow derived MSCs (N=8) or off-the-shelf mesenchymal progenitor cells (MPCs) from Angioblast (N=24). Patients received a double cord blood transplant, with one of the 2 units undergoing ex vivo expansion using this system. MSCs create a microenvironment that promotes expansion and fosters the differentiation of hematopoietic cells. Patients must have had two CB units matched in at least 4/6 HLA antigens, with a minimum of 1×107 TNC/Kg per unit. Method: Diagnoses were AML/MDS (n=21), ALL (n=6), NHL (n=2), CLL (n=2), and HD (n=1). Fourteen patients (44%) were in CR (CR1, n=3, CR2 or more, n=11) and 18 (56%) had active disease at CBT. Preparative regimen: myeloablative fludarabine, melphalan, thiotepa and ATG (n=32), with rituximab in the 4 NHL/CLL cases. GVHD prophylaxis: tacrolimus and MMF. Median weight was 75.2 Kg (range, 15–118) and median age was 35.3 years (2.8-62 years). Donor-recipient HLA matching was 6 of 6 in 5%, 5 of 6 in 28% and 4 of 6 in 67% of the cases, respectively. Ex-vivo EXP: 100 ml of marrow was aspirated from the family donor and MSCs generated in ten T175 flasks, which took ∼21 days (n=8) or one vial of Angioblast MPCs was thawed and expanded to confluence in 10 flasks within 4 days (n=24). The CB unit with the lowest TNC dose was then thawed, divided into 10 fractions, and each placed into 1 flask containing the confluent layers of MSCs in expansion media with SCF, FLT3-ligand, G-CSF and TPO. After 7 days at 37°C, the non-adherent cells were removed from each flask, placed into each of ten one-liter Teflon-coated culture bags (American Fluoroseal) and cultured for an additional 7 days (14 days total), while 50 ml of media/growth factors was added to the flasks to culture the remaining adherent layer during that time period. On day 14 the cells from the bags and the flasks were combined, washed and infused along with a second unmanipulated CB unit. Result: The median number of total nucleated cell (TNC) and CD34+ cells infused/Kg in unmanipulated CB was 2.35 × 107 (range 0.2–8.2) and 0.95 × 105 (range 0–4). The median number of TNC and CD34+ cells infused/Kg after EXP was 5.8 × 107 (range, 0.3–14.4) and 8.7 × 105 (range, 0–93.4). This represented a median expansion of 14-fold (range 1–30) for TNC and 40-fold (range 4–140) for the CD34+ cells. Median time to neutrophil and platelet engraftment was 15 days (range 9–42) and 40 days (range 13–62). There were no toxicities attributable to the EXP cells. Thirty-one (97%) and 26 (81%) of all patients engrafted neutrophils and platelets, respectively. One patient died before engraftment. Thirty and one-hundred day non-relapse mortality is respectively 6% and 19%. Median donor(s) chimerism was 100% in the mononuclear, T lymphocyte and myeloid cell populations. On transplant day+21, EXP unit contributed with a mean of 19% of mononuclear cell, 16% of T cell, and 14% of myeloid chimerism. Subsequently, hematopoiesis was increasingly derived from the unexpanded unit with long-term engraftment provided by the unexpanded unit by six months posttransplant. Acute grade II-IV and III-IV GVHD rate was 50% and 16%; 25% of the grade II-IV GVHDs occurred beyond 100 days, and two patients developed chronic GVHD. With a median follow-up of 9 months, 11 patients are alive; actuarial one-year survival is 40%. Mortality was due to relapse in 26% and non-relapse causes in 74% of patients. Conclusion: MSC-CB Exp is feasible and leads to fast engraftment of neutrophils and platelets, and high-rates of platelet transfusion independence. Disclosures: No relevant conflicts of interest to declare.

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Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice.

Blood ◽

10.1182/blood.v116.21.3722.3722 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3722-3722

Author(s):

Li Ming Ong ◽

Xiubo Fan ◽

Pak Yan Chu ◽

Florence Gay ◽

Justina Ang ◽

...

Keyword(s):

Cord Blood ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Insulin Growth Factor ◽

Nsg Mice ◽

Nonobese Diabetic ◽

Cd34 Cells

Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.

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Quantitative Expression Analysis of WT1 Main Isoforms in AML

Blood ◽

10.1182/blood.v118.21.1469.1469 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1469-1469

Author(s):

Irene Luna ◽

Esperanza Such ◽

Jose Cervera ◽

Eva Barragan ◽

Marta Llop ◽

...

Keyword(s):

Cord Blood ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Wilms Tumor ◽

Mrna Level ◽

Prognostic Impact ◽

Cell Selection ◽

Healthy Donors ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract Abstract 1469 The Wilms Tumor 1 (WT1) gene was first described as a tumour suppressor gene, but its accurate role in leukemia development has not been completely elucidated. Some authors support the role of WT1 as a prognostic marker in acute myeloid leukemia (AML) based on the assessment of its expression at the mRNA level. However, the prognostic value of the main isoforms of WT1 has been less studied. The aim of this study was to develop a specific quantitative assay to estimate the ratio of expression of the four major WT1 isoforms (A, 5-/KTS-; B, 5+/KTS-; C, 5-/KTS+; D, 5+/KTS+) and to evaluate their prognostic impact. WT1 expression was analyzed in bone marrow samples from 108 patients with AML at diagnosis (65 male/46 female, median age: 61 yr, range: 17 – 91). Likewise, peripheral blood samples of 20 healthy donors and 6 samples of cord blood CD34+ cell selection were analyzed as normal controls. We performed a new method to quantify the ratios of the four major isoforms of WT1. Briefly, to amplify all isoforms within a PCR reaction, specific WT1 primers flanking exon 4 to exon 10 were used in cDNA samples, followed by capillary electrophoresis with laser-induced fluorescence analysis on an ABIPRISM 310 DNA Analyzer (Applied Biosystems, Foster City, CA) and lastly analyzed with the Gene Mapper 4.2 software (Applied Biosystems). The amount of each isoform was calculated by the area under the curve. Subsequent comparisons of isoform ratios were made by standardized calculation of percentage. All values are given as the mean of duplicate PCRs. In parallel, RQ-PCR for total WT1 detection was performed as previously described by Barragan et al. (Haematologica 2004; 89: 926–933). GUS gene was used as housekeeping gene. Eighteen patients (17%) did not express WT1, while 90 patients (83%) overexpressed WT1 above background levels. The median value of each WT1 isoform was: 18% (range: 2 – 73) for A isoform; 16% (range: 7 – 63) for B isoform; 24% (range: 2 – 52) for C isoform; and 33% (range: 3 – 55) for D isoform. None of healthy donors had detectable WT1 levels in peripheral blood. All samples of CD34+ cells expressed the four isoforms of WT1: 21% (range: 2 – 26) for A isoform; 16% (range: 1 – 64) for B isoform; 24% (range: 1 – 47) for C isoform; and 36% (range: 25 – 44) for D isoform. These data reveal that, in our series, the most predominant isoform was +5/+KTS, both in AML and in cord blood CD34+ cell selection samples. There were no significant differences when comparing the proportion of each isoform between the cord blood CD34+ cell selection samples and the cohort of AML patients. There was not significant correlation between the overexpression of total WT1 with the ratio of each isoform, and we were unable to demonstrate that the overexpression of WT1 is due to a particular isoform overexpression. A significant lower event-free survival (EFS) was observed in those patients overexpressing total WT1, taking a cut-off value of 3000 WT1 copies/ GUS copies × 104 (75th percentile, P =.001). However, when the same cut-off as well as the median value for each one of the isoforms was used, we found no significant differences in EFS and in overall survival. To sum up, none of the isoforms were correlated with overexpression of total WT1 or survival. We were unable to find differences between the expression of each isoform of WT1 in CD34+ cells from normal cord blood and in AML patients. Further studies including larger controls need to be carried out. Disclosures: No relevant conflicts of interest to declare.

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Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽

10.1182/blood.v118.21.4808.4808 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4808-4808

Author(s):

Young-Ho Lee ◽

Young-hee Kwon ◽

Kyoujung Hwang ◽

Hyunju Jun ◽

Byungbae Park ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Conflicts Of Interest ◽

T Cell Subsets ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference ◽

Stromal Cell Derived Factor ◽

And Control ◽

Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

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CD34+ SSCloALDHbr – a New Marker for Improved Characterization of Poor Myeloid Regeneration in the Autologous Hematopoietic Transplantation Setting?,

Blood ◽

10.1182/blood.v118.21.4007.4007 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4007-4007

Author(s):

Katrine Nielsen ◽

Lea Bjerre ◽

Hanne Oestergaard Larsen ◽

Peter Hokland ◽

Anne Stidsholt Roug

Keyword(s):

Conflicts Of Interest ◽

Autologous Bone Marrow Transplantation ◽

Autologous Bone Marrow ◽

Post Transplantation ◽

Peripheral Blood Stem Cells ◽

Platelet Recovery ◽

Organ Systems ◽

Cd34 Cells ◽

Significant Difference

Abstract Abstract 4007 Background: An increasing body of evidence in different organ systems suggests that the presence of the cytosolic enzyme Aldehyde Dehydrogenase (ALDH) correlates to stemcellness. Thus, in mobilized peripheral blood stem cells (PBSCs), the side scatter low ALDH bright (SSCloALDHbr) population seems to be highly enriched for HSC as determined e.g. by Fallon et al1. However, these interesting preliminary findings need confirmation. Problem formulation: The generally accepted indirect relationship between CD34 content on the one hand, and successful hematopoietic regeneration after transplant on the other, is at least to a certain extent marred by the observation that, despite receiving sufficient weight-correlated amounts of CD34 positive HSC, some patients experience prolonged time to regeneration. Hypothesis: We hypothesized that the number of CD34+SSCloALDHbr is predictive of time to short and long-term regeneration following autologous bone marrow transplantation compared to the number of CD34+ cells alone. Results: PBSCs from 30 patients with refractory or relapsed diffuse large cell B-lymphoma referred for autologous stem cell transplantation after having failed at least three cytoreductive regimens were analyzed for ALDH expression. Laboratory results were registered on day 14 and day 100 post transplantation. Time to absolute neutrophil count (ANC) above 0,5 × 106/mL on two consecutive days was registered for each of the 30 patients. While a trend was noted for number of reinfused viable CD34+ cells (106/kg) to be correlated to ANC > 0,5 × 106/mL r=0.33, P = 0.07), only the CD34+SSCloALDHbr population correlated significantly to time to ANC > 0,5 × 106/mL (r=0.41, P =0.024). Moreover, while a positive correlation was observed between both of the analyzed subpopulations and CFU-GM proliferation, this correlation was strongest for the number of reinfused CD34+SSCloALDHbr/kg (r= 0.84, P <0.001). While neither of the analyzed subpopulations could be correlated to time to short term platelet recovery (defined as platelets > 20 × 106/mL on two consecutive days) an interesting finding emerged, when we considered day 100 platelet recoveries. Here, 11 patients were grouped as poor mobilizers (PM) having not attained a platelet count of 100 × 106/mL at day 100, and 16 patients were grouped as good mobilizers (GM) while in three cases no data were available. While mere CD34 enumeration was once more not informative, we observed a trend towards low numbers of CD34+SSCloALDHbrcells in the grafts in PM (p=0.06). With regard to long-term erythroid engraftment (defined as Hgb<100 g/L at day 100) 6 were PM (21 GM and data missing in 3) a significantly lower fraction of reinfused CD34+SSCloALDHbrwere administered to PM compared to GM (p=0.01). Once more, no significant difference in number of infused viable CD34+ cells between the groups was observed. Conclusion: In the autologous transplant setting the addition of SSCloALDHbrto standard CD34 enumeration seems to constitute a valuable marker for the identification of both time to ANC > 0,5 × 106/mL and inferior day +100 hematopoietic regeneration. While the assay does entail extra lab efforts, this layout is probably amply repaid by the possibility to institute pre-emptive therapy in PM patients. Moreover, application of the assay at the onset of leukapheresis sessions could provide a window of opportunity to administer alternative mobilization regimens, i.e. containing Plerixafor. Disclosures: No relevant conflicts of interest to declare.

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A Better Mobilization Regimen for Newly Diagnosed Multiple Myeloma Patients,

Blood ◽

10.1182/blood.v118.21.4051.4051 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4051-4051

Author(s):

Ahmed Y Abuabdou ◽

Eric R Rosenbaum ◽

Saad Usmani ◽

Bart Barlogie ◽

Michele Cottler-Fox

Keyword(s):

Multiple Myeloma ◽

Conflicts Of Interest ◽

Optimal Number ◽

P Value ◽

Newly Diagnosed ◽

The Poor ◽

Cd34 Cells ◽

Significant Difference ◽

Minimum Number ◽

Poor Mobilizer

Abstract Abstract 4051 Introduction: What constitutes an acceptable mobilization regimen for collecting CD34+ cells depends on whether the goal of collection is to obtain a minimum number versus optimal number of cells. When treating patients with high-risk myeloma it may be important to obtain an optimal number. Here we compare retrospectively our earlier mobilization regimen, VTD-PACE, with MVTD-PACE in newly diagnosed, previously untreated multiple myeloma patients. Materials and Methods : We reviewed data for all patients who collected hematopoietic progenitor cells on Total Therapy protocols TT3a/TT3b with VTD-PACE (n=394) from February 2004 to September 2008 (138 females and 256 males, median age 59y; range 31–75), and on TT4/TT5 with MVTD-PACE (n=188) from August 2008 to May 2011 (78 females and 110 males, median age 61y, range 30–76). Based on their predicted first day collection with a large volume leukapheresis (30L processed), using our center's predictive formula (Blood 2010; 116(21):1182a), patients were stratified into 4 mobilizer types: poor (<2×106 CD34+ cells/kg), intermediate (≥2 to 10×106), good (>10 to 20×106) and excellent (>20×106). Variables examined included number of CD34+ cells/μl blood on day 1 and day 2 of collection (we have a minimum 2 day collection requirement), number of collection days to reach our minimum goal of 20×106 CD34+ cells/kg, and total CD34+ cells/kg collected for both chemotherapy groups. Variables for both groups stratified by mobilizer type were compared using two-tailed student's t-tests, except for the poor mobilizer group, where population size was too small for formal statistical analyses (VTD-PACE n=7, MVTD-PACE n=4), although averages were calculated. Results : There was no significant difference between VTD-PACE and MVTD-PACE for CD34+ cells/μl blood on day 1 of collection among the excellent [mean 368.9 (n=184) vs. 434.6 x106 (n=92); p-value 0.07], good [mean 138.6 (n=102) vs. 128.6 x106 (n=40); p-value 0.19], and intermediate [mean 60.1 (n=100) vs. 55.9 x106 (n=52); p-value 0.39] groups. A statistically significant difference between VTD-PACE and MVTD-PACE was found for CD34+ cells/μl blood on day 2 of collection for excellent mobilizers [mean 333.8 (n=184) vs. 460 ×106 (n=92); p-value <0.001], but not for the good [mean 165.7 (n=102) vs. 189.5×106 (n=40); p-value 0.21] and intermediate [mean 80.1 (n=101) vs. 102.3 ×106 (n=52); p-value 0.07] groups. When CD34+ cell/kg collection totals with VTD-PACE and MVTD-PACE were compared, a significant difference was seen for the intermediate mobilizer group only [mean 23.6 (n=101) vs. 26.3 ×106 (n=52); p-value 0.03]. For the poor mobilizer group, VTD-PACE had an average CD34+ cells/μl blood of 13.5×106 for day 1 of collection and 17.0 ×106 for day 2, with a total of 14.5×106 CD34+cells/kg collected; while MVTD-PACE had an average of 13.2×106 CD34+ cells/μl blood for day 1 of collection, 24.9×106 for day 2, with a total of 24.2×106CD34+ cells/kg collected. The number of collection days was similar between VTD-PACE and MVTD-PACE in the excellent mobilization group (2 days), but was slightly more for VTD-PACE compared to MVTD-PACE for the good (2.1 vs. 2 days), intermediate (3.2 vs. 2.9 days), and poor (6.1 vs. 5.8 days) groups. Conclusion : Both regimens allow more than minimum collections, but MVTD-PACE provides a higher peak number of CD34+ cells/μl blood, resulting in a slightly lower mean number of days of collection than VTD-PACE to reach an optimal collection. Disclosures: No relevant conflicts of interest to declare.

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Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Blood ◽

10.1182/blood.v122.21.2030.2030 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2030-2030

Author(s):

Nishant Tageja ◽

Neha Korde ◽

Constance Yuan ◽

Kristen Cole ◽

Jennifer Hsu ◽

...

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

High Risk ◽

Tumor Cell ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cell ◽

Myeloma Cells ◽

Cd34 Cells ◽

The Mean

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare.

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Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽

10.1182/blood.v126.23.3464.3464 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3464-3464

Author(s):

Yang Song ◽

Yu-tong Wang ◽

Xiao-jun Huang ◽

Yuan Kong

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Endothelial Cells ◽

Th17 Cells ◽

Immune Thrombocytopenia ◽

Perivascular Cells ◽

Platelet Production ◽

Cd34 Cells ◽

Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

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Stem Cell Graft Cellular Composition, Hematological and Immune Recovery in NHL Patients Mobilized with or without Plerixafor: A Prospective Comparison

Blood ◽

10.1182/blood.v124.21.1193.1193 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1193-1193

Author(s):

Jaakko Valtola ◽

Ville Varmavuo ◽

Antti Ropponen ◽

Marja Pyörälä ◽

Anne Nihtinen ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Prospective Study ◽

Nk Cells ◽

Immune Reconstitution ◽

Control Group ◽

Immune Recovery ◽

Cellular Composition ◽

Cd34 Cells ◽

Significant Difference

Abstract Introduction: Plerixafor, a reversible CXCR4 antagonist, may be used to enhance mobilization of CD34+ cells after G-CSF or chemotherapy plus G-CSF. There is a paucity of prospective data regarding cellular composition of grafts collected after plerixafor in chemomobilized patients. Also the data concerning hematopoietic or immune recovery after high-dose chemotherapy in plerixafor treated lymphoma patients is limited. Patients and methods: Thirty-one patients with NHL were included into this prospective study. There were 14 males and 17 females with a median age of 62 years (range 19 - 73). Altogether fourteen patients received plerixafor (plerixafor group) for poor or late mobilization whereas 17 did not (control group). All patients were mobilized with chemotherapy plus G-CSF. Cryopreserved graft samples were analyzed with flow cytometry for T and B cells (CD3/CD8/CD45/CD19) as well as for NK cells (CD3/CD16+CD56). Also CD34+ cell subclasses were analyzed (CD34/CD38/CD133). Complete blood counts were evaluated at +15 days, 1, 3, 6 and 12 months post-transplant. To evaluate immune reconstitution, flow cytometry of lymphocyte subsets (T, B, NK) was performed at 1, 3 and 6 months after the graft infusion with the same antibody panel as for graft analysis. Results: The median number of infused viable CD34+ cells was higher in the control group (3.1 x 106/kg vs. 2.0 x 106/kg, p = 0.036) (Table 1). The median percentage of the most primitive stem cells (CD34+CD133+CD38-) in the grafts was higher in the plerixafor group (3.5 % vs 1.2 %, p = 0.001), but there was no significant difference in the absolute counts (0.07 x 106/kg vs 0.05 x 106/kg, p = 0.620). The median amounts of CD3+CD4+ and CD3+CD8+ T cell subsets, CD3+ and NK (CD3-CD16/56+) cells were all significantly higher in the plerixafor group (Table 1). The neutrophil counts at +15 days after the graft infusion were lower in the plerixafor group (2.1 x 109/l vs. 4.8 x 109/l, p = 0.013). Otherwise there was no significant difference in the hematological reconstitution between the groups. The immune reconstitution was comparable except for the higher number of NK cells in the plerixafor group at one month (0.4 x 109/l vs. 0.1 x 109/l, p = 0.001). Also a trend towards faster recovery of blood CD4+ T cells was observed after one month in the plerixafor group (0.2 x 109/l vs. 0.1 x 109/l, p = 0.097). Conclusions: This prospective study evaluating cellular composition of grafts confirms that the apheresis products collected from plerixafor-treated NHL patients contain a greater proportion of the more primitive stem cells and a greater number of T lymphocytes and NK cells compared to patients mobilized without plerixafor. Hematopoietic reconstitution was comparable between the groups except for slower neutrophil recovery in the plerixafor-group. Immune reconstitution was comparable but NK cell as well as CD4+ T cell recovery was faster in the plerixafor group. These results will be further evaluated in a larger set of patients. Also the possible effect of graft composition on the progression free and overall survival will be evaluated in the ongoing GOA (Graft and Outcome in Autologous transplantation) study. Table 1. Cellular composition of freezed grafts of NHL patients mobilized with or without plerixafor. Mobilization with plerixafor (n = 14) Mobilization without plerixafor (n = 17) P - value Blood graft analysis (x106/kg) CD34 w/a 7AADCD34 w 7AADCD34+CD38-Proportion of CD34+ CD133+CD38- cells from all CD34+ cells (%)CD3+CD4+CD8+CD19+NK 2.1 (0.8 – 5.3)2.0 (0.6 – 5.5)0.07 (0.01 – 0.17)3.5 (0.80 – 10.80)178.3 (49.2 – 454.4)82.1 (29.1 – 267.1)75.4 (16.5 – 279.1)0.0 (0.0 – 0.0)21.5 (0.4 – 39.5) 3.4 (1.9 – 7.2)3.1 (1.5 – 6.7)0.05 (0.11 – 0.18)1.2 (0.44 – 5.3)66.2 (16.6 – 415.4)35.6 (8.0 – 114.3)23.3 (8.4 – 301.8)0.0 (0.0 – 3.2)6.6 (0.6 – 20.7) 0,0070.0360.6200.0010.0010.0010.0030.1920.001 7AAD = 7-Aminoactinomycin D w/a = without w = with Disclosures Jantunen: Sanofi: Employment.

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