CD34 Cell Recovery Research Articles

Abstract 23 A First Insight into the Validity of Post-Thaw Cell Recovery, Viability, and Potency Over Time

IntroductionTesting of a thawed segment or representative sample of a cord blood unit (CBU) for cell recovery, viability, and potency forms part of pre-release testing. Not all CBUs that have had pre-release testing completed are shipped for transplant, due to postponed or cancelled shipment request, and are therefore kept in the inventory until they are searched again. In an effort to identify these CBUs for future expedited release, the World Marrow Donor Association (WMDA) initiated a project to flag them as “Ready to Ship” in the WMDA Search & Match Service. However, it is not known how long post-thaw results are valid before they must be repeated on another thaw. Such information can be beneficial not only for transplant centers to better assess CBU suitability but also for Cord Blood Banks (CBBs) to avoid unnecessarily exhausting their precious samples.ObjectiveThis study aimed to provide a first insight into the validity of post-thaw results over various lengths of time.MethodsNineteen CBUs, collected between 2003 and 2015 at the BMDI CBB and that had post-thaw testing performed twice, were analyzed for change in the total nucleated cell (TNC) recovery, total CD34 cell recovery, CD34 cell viability, and CFU-GM counts between two thaws.ResultsThe 19 CBUs were grouped into 4 groups based on their storage time between thaws. An average decrease in TNC recovery of 5.3% from first thaw to second thaw was observed for the entire dataset (P = .015). Four CBUs with a greater than 10% decrease had a bag TNC greater than 25 × 108. Individual CBU data is shown in Figure 1. No significant change between thaws was seen with total CD34 recovery (P = .738) or CD34 viability (n = 9 pairs, P = .354). A large variation in CFU-GM counts was observed between thaws. It is worth noting that all CBUs met CBB release criteria of >60% TNC recovery and that CD34 viabilities were above 70% (FACT requirement) in both thaws.Figure 1.Comparison of first and second thaw TNC recovery (A), total CD34 recovery (B), CD34 viability (C), and CFU-GM counts (D) of individual cord blood unit, grouped into 4 groups based on their storage time between thaws: 0-6 months (i), 2-3.5 years (ii), 6-8.5 years (iii), and 12 years (iv).DiscussionWhile results need to be confirmed on a larger dataset, our preliminary data shows very little change in post-thaw cell recovery and viability over various lengths of time (2 months-12 years), suggesting post-thaw results may be valid for longer than 10 years.

Low CD45 mean fluorescence intensity predicts poor Post-Thaw CD34 cell viability

Background & Aim Background Reduced CD34 cell viability following cryopreservation is associated with suboptimal engraftment in stem cell transplantation. Collection of extra hematopoietic progenitor cells (HPC) can compensate for the potential loss, but subjects patients to unnecessary procedures, and drives up healthcare costs. Aim The goal of this study was to identify variables available on the day of HPC collection that help predict the likelihood of a significant post-thaw drop in viable CD34 cells (vCD34). A prediction model was generated to recommend appropriate extra-day collections. Methods, Results & Conclusion Methods We collected data on 235 consecutive autologous HPC apheresis collections in a single pediatric hospital and analyzed variables that are patient-related, product-related, and technical in nature (Table 1). Both continuous and discrete variables were analyzed and used to construct a prediction model with a false negative rate (missing a call to predict a significant drop in vCD34%) of zero. Results In 5.5% of apheresis products, post-thaw vCD34% decreased by >50%, 15% had 25-50% drop, 29% showed 50%) post-thaw vCD34% decrease included a diagnosis of Hodgkin lymphoma and a low mononuclear cell (MNC) percentage. A simple algorithm combining these three factors correctly identified 9 out of 11 (81%) unnecessary repeat collections, and did not miss any patients for whom a recollection was necessary. Retrospective analysis suggests that using this algorithm, 33% of repeat collections could be avoided. Conclusion Low CD45 nMFI of CD34+ cells is a novel marker of poor post-thaw CD34 cell recovery. A predictive model that combines CD45 nMFI, Hodgkin lymphoma disease status, and MNC percentage has shown good power for predicting unnecessary repeat collections in our patient population. Our predictive model of post-thaw CD34 cell recovery, implemented as a clinical support tool, could help spare unnecessary procedures in a significant percentage of patients. Prospective validation of the algorithm is ongoing, and data will be presented at the meeting.

Fully automated, clinical-grade bone marrow processing: a single-centre experience.

Clinical grade processing of harvested bone marrow is required in various clinical situations, particularly in the management of ABO mismatching in allogeneic haematopoietic stem cell transplantation (HSCT) and in regenerative medicine. We report a single-centre experience using a fully automated, clinical grade, closed system (Sepax, Biosafe, Switzerland). From 2003 to 2015, 125 procedures were performed in our laboratory, including buffy-coat production for HSCT (n=58), regenerative medicine in an orthopaedic setting (n=54) and density-gradient separation in a trial for treatment of critical limb ischaemia (n=13). Buffy coat separation resulted in a median volume reduction of 85% (range, 75-87%), providing satisfactory red blood cell depletion (69%, range 30-88%) and a median recovery of CD34 cells of 96% (range, 81-134%) in the setting of allogeneic HSCT. Significantly greater volume reduction (90%; range, 90-92%) and red blood cell depletion (88%; range, 80-93%) were achieved by the new SmartRedux software released for Sepax2, validated in the last eight allogeneic HSCT. The density gradient separation programme resulted in complete red blood cell depletion associated with high CD34 recovery (69%; range, 36-124%). No reactions related to the quality of the product were reported. Time to engraftment following allogeneic HSCT was in the normal range. No cases of microbiological contamination related to the manipulation were reported. Clinical grade, automated bone marrow manipulation with Sepax was shown to be effective, giving operator-independent results and could be used for a broad range of clinical applications.

Automated CD34+ cell isolation of peripheral blood stem cell apheresis product

Background aimsImmunomagnetic enrichment of CD34+ hematopoietic “stem” cells (HSCs) using paramagnetic nanobead coupled CD34 antibody and immunomagnetic extraction with the CliniMACS plus system is the standard approach to generating T-cell-depleted stem cell grafts. Their clinical beneficence in selected indications is established. Even though CD34+ selected grafts are typically given in the context of a severely immunosuppressive conditioning with anti-thymocyte globulin or similar, the degree of T-cell depletion appears to affect clinical outcomes and thus in addition to CD34 cell recovery, the degree of T-cell depletion critically describes process quality. An automatic immunomagnetic cell processing system, CliniMACS Prodigy, including a protocol for fully automatic CD34+ cell selection from apheresis products, was recently developed. We performed a formal process validation to support submission of the protocol for CE release, a prerequisite for clinical use of Prodigy CD34+ products. MethodsGranulocyte-colony stimulating factor–mobilized healthy-donor apheresis products were subjected to CD34+ cell selection using Prodigy with clinical reagents and consumables and advanced beta versions of the CD34 selection software. Target and non-target cells were enumerated using sensitive flow cytometry platforms. ResultsNine successful clinical-scale CD34+ cell selections were performed. Beyond setup, no operator intervention was required. Prodigy recovered 74 ± 13% of target cells with a viability of 99.9 ± 0.05%. Per 5 × 10E6 CD34+ cells, which we consider a per-kilogram dose of HSCs, products contained 17 ± 3 × 10E3 T cells and 78 ± 22 × 10E3 B cells. ConclusionsThe process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.

The Cell Loss after Thawing in Cord Blood Units

Umbilical cord blood (CB) are increasingly used for allogeneic transplantation as an alternative source to bone marrow or peripheral stem cell for patients who lack a human leucocyte antigen (HLA)-matched donor. Cell dose is one of the main determinant factor for selection of the unit and it has been shown to correlate with time and probability of engraftment. Cell doses available for selection of the unit are the ones evaluated before cryopreservation. Processing of the graft either with cryopreservation and/or thawing inevitably lead to cell loss but the data showing to what extent is very limited although this has great importance especially for the units with critical cell counts. To evaluate the extent of the cell loss after thawing and the effect of preservation duration on cell loss and the viability, we retrospectively analyzed our single center experience in a cohort of patients receivingunrelated CBT.Sixty cord blood units used for unrelated CBT in fifty-eight patients in our Pediatric Stem Cell Transplantation Unit were included in the study. One of the patient was transplanted with double cord blood unit and one had recurrent CBT because of previous graft failure.Median number of declared TNC per kilogram of recipient body weight was 16x107/kg (range: 3,16-59,1). After thawing the median TNC dose decreased to 11,1x107/kg (range:1,5-34), with median cell viability at 80%. The median TNC recovery was found as 70%. Median CD 34+ cell count declared by the banks per kilogram body weight of recipient was 6,0x105/kg (range: 0,5-37). The CD 34 cell count of one of the units used in a patient having double cord blood infusion was not available. The CD34 cell count of the other complementary unit was 0,48x105/kg. Since the total cell counts received bu the recipient was given, the CD34 cell count of the patient having double CB infusion was not included in statistical analysis. The median postthawed CD 34 cell count was 3,4 x105/kg (range: 0-21)and median CD34 cell recovery was %58.The storage duration of the CB units in liquid nitrogen was available for 59 units and that was 52±30 months (range:3-130 months). Correlation analysis did not show any significant correlation between CB unit storage duration and viability (r=-0,2 p=0.07). Also any correlation between storage time and also TNC and CD34 cell recovery were not identified.Our study indicated that approximately 30% of cell loss is expectable in cord blood transplantation because of either cryopreservation or thawing. It should be kept in mind while assessing acceptibility of cord blood units with limited cell counts DisclosuresNo relevant conflicts of interest to declare.

FT1050 (16,16-dimethyl Prostaglandin E2)-Enhanced Umbilical Cord Blood Accelerates Hematopoietic Engraftment After Reduced Intensity Conditioning and Double Umbilical Cord Blood Transplantation

Abstract 653▪▪This icon denotes a clinically relevant abstractUmbilical cord blood (UCB) transplantation relies on a small number of hematopoietic stem cells (HSCs) to restore hematopoiesis. Even with double umbilical cord blood transplantation, engraftment times are prolonged and immune reconstitution delayed. Preclinical studies have suggested that ex vivo treatment of HSCs with FT1050 (16,16-dimethyl PGE2) can enhance engraftment of HSCs without stem cell expansion or differentiation of HSCs to committed progenitors. Using both murine and human-xenograft model systems, the mechanisms of action of FT1050 treatment of HSCs have been demonstrated to involve improved homing via CXCR4-SDF-1, increased proliferation and entry into the cell cycle, and decreased rates of apoptosis (North 2007; Goessling 2009; Hoggatt 2009; Goessling 2011). A Phase Ib trial was designed to evaluate the safety of FT1050-treated HSCs and whether this ex vivo treatment can enhance engraftment after double UCB transplantation in adults. The competitive engraftment dynamic of double UCB transplantation also permits determination of whether FT1050-HSCs are able to out-compete untreated HSCs. Methods: Patients without a matched sibling or matched-unrelated donor were conditioned with fludarabine (30 mg/m2/day IV Day −8 to −3), melphalan (100 mg/m2/day IV Day −2), and rabbit ATG (1 mg/kg/day Days −7, −5, −3 and −1). GvHD prophylaxis was with sirolimus (target 3–12 ng/mL) and tacrolimus (target 5–10 ng/mL). Patients received 2 UCB units: the first UCB unit (FT1050-UCB) was thawed, washed and treated with FT1050 for 2 hours at 37°C. After an additional wash to remove residual FT1050, the FT1050-UCB was infused without further manipulation. The second untreated UCB was thawed, washed and infused 2–6 hours later. The primary objective of the study was to determine the safety of FT1050-UCB based upon engraftment by Day 42 with > 5% chimerism of the FT1050-UCB unit. Secondary objectives included time to engraftment, the rates of non-hematologic toxicity, graft failure, acute and chronic GvHD, relapse, treatment related mortality (TRM), fractional chimerism, and relapse-free and overall survival. Results: A total of 12 subjects received FT1050-UCB in this fashion, of which 11 are currently evaluable. The median age was 57.5 years (range 19–66) and 67% were male. Diagnoses included: AML(5), MDS(4) and NHL/CLL(3). The median precryopreservation UCB sizes were FT1050-UCB: 2.7×107 TNC/kg (range 2.0–5.1) with 1.3×105 CD34/kg (range 0.3–6.3); untreated UCB: 2.0×107 TNC/kg (range 1.8–5.0) with 1.1 × 105 CD34/kg (range 0.5–3.4). The median combined cell dose was 4.7×107 TNC/kg (range 3.9–10.1) with 2.1×105 CD34/kg (range 1.4–9.7). The FT1050 incubation did not result in significant cell loss, with a mean viable CD34 cell recovery of 90%. The adverse events attributed to FT1050-UCB consisted of five Grade 1/2 infusion-related events in 4 subjects, consisting of chills, flushing, abdominal pain, and cough. One additional subject with known coronary artery disease experienced transient Grade 4 ST-elevation following infusion and evidence of myocardial ischemia by cardiac troponin assay. The median time to neutrophil recovery (> 500 cells/μL) was 17 days (range 15–27 days), which compares favorably to a median of 21 days for a historic control group of similarly treated patients at our institution (n=53; p= 0.025). The median time to an unsupported platelet count of 20,000/μL was 42 days (n=9 evaluable). There were no instances of primary or secondary graft failure. The FT1050-UCB was the dominant source of hematopoiesis in 9 of the 11 evaluable subjects, and the median total chimerism of FT1050-UCB at day 14 was 90%, with long-term dominance again by the FT1050-UCB unit. To date, 2 cases of Grade 2 acute GvHD have been observed and one patient developed NIH mild chronic GvHD. No cases of EBV-lymphoproliferative disease were noted. TRM was 9% (1 subject) and 1 patient has relapsed; 9 subjects remain alive without relapse with a median follow-up of 5.0 months (range 1.6–9.4). Conclusions: These data support the potential benefit of a novel ex vivo modulation approach to improving engraftment in patients undergoing UCB transplantation. Additional studies exploring the potential use of FT1050-HSCs in other low cell dose settings are planned, including whether FT1050-UCB can enable single UCB transplant in adults. Disclosures:Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment. Desponts:Fate Therapeutics: Employment. Goessling:Fate Therapeutics: Consultancy. North:Fate Therapeutics: Consultancy. Mendlein:Fate Therapeutics: Employment, Equity Ownership. Multani:Fate Therapeutics: Employment, Equity Ownership.

Positive selection of autologous peripheral blood stem cells

The development of monoclonal antibodies against differentiation antigens on human haematopoietic cells has led to a new concept in stem cell purification: the positive selection. In terms of autologous PBSC transplantation, the immature stem cells are identified by their expression of a specific antigen, the CD34. The CD34 antigen is expressed on early lymphohaematopoietic stem cells and progenitor cells, but not on mature blood cells or on tumour cells of several diseases. CD34+cells are found in low numbers in bone marrow (<2%) and in even lower numbers in steady state blood (<0.01%) but may increase from 1 to 5% after mobilization using chemotherapy and/or growth factors.Several techniques have been set up to enrich PBSC grafts in CD34[mop]stem cells. The quality of each system is here analysed in terms of CD34 purity of the selected cell fraction, the CD34 cell recovery, the tumour cell depletion efficiency and the functional capacity ex vivo and in vivo of the selected cells. The final CD34+cell purity of the selected fractions is correlated to the concentration of CD34+cells before selection. The optimal recoveries and the highest purities were generally obtained when the initial CD34 content was roughly over 1%. Below this figure, the final purity seems to be less predictable. Besides the better tolerance resulting from the reduction in the number of autologous cells, and consequently the total volume of DMSO reinfused to the patient, the selective enrichment of the CD34 cell population offers a new approach to tumour purging. The procedure by itself results in elimination of about 99% in the total number of initial cells, thus allowing reduction of the overall tumour cell number in the final autograft. However, its major interest is that, in diseases where tumour cells do not express the CD34 antigen, it is theoretically able to completely eliminate the tumour contamination of the graft.Based on previous data showing that lymphoma, myeloma, neuroblastoma and breast cancer cells are not CD34+, pilot clinical trials for the separation and transplantation of CD34+cells selected from PBSC of patients with these diseases have recently been conducted. The efficacy of CD34 selection in reducing the tumour load of the PBSC of patients with these diseases has been reported. However, the efficacy of purging may greatly differ between individual patients, and complete eradication of contaminating cells from PBSC grafts was not always reached. There is now evidence that purified CD34+cells are capable of supporting haematopoietic reconstitution in autologous transplantation. However, until now no study has demonstrated clear evidence that the reduction of tumour cells from PBSC of patients by CD34+cell selection resulted in a lower relapse rate post-transplant, as compared to unselected PBSC infusion.