Predicting the CD34 content of mobilized peripheral blood leukapheresis products: single institution experience over 20 years.

BACKGROUND AIMS: Since the standardization of CD34 measurement by flow cytometry, predictors of leukapheresis CD34 yield have played a pivotal role in planning donor leukaphereses. We describe here a single institution's experience with a multivariate predictor that was used for 2,929 products without alteration for 20 years. METHODS: The ordinary least squares regression model variables included log peripheral CD34 count, collection duration (3- versus 4-hours), collection number, donor sex, and transplant type. RESULTS: During the study period we changed flow cytometers twice and leukapheresis instruments once. During the Cobe Spectra era the predictor explained 90% of the variability in CD34 collection yield for autologous transplants (r2 = 0.90), and 70% for allogeneic transplants with an overall sensitivity to predict a CD34 yield of ≥ 1 × 106/kg of 97.7%, and specificity of 81.4%. CONCLUSIONS: Implemented prospectively with real-time result reporting, the model allowed us to predict CD34 yield with both 3- and 4-hour collection scenarios. Given this guidance, 3-hour collections were selected by the clinical team 25% of the time, saving patient leukapheresis time and resources. When faced with a prediction of < 1 × 106 CD34/kg, the clinical team chose to defer collection 72% of the time. In instances where leukapheresis was performed despite a poor predicted outcome, 85% of patients collected on the Cobe Spectra, and 92% of patients collected on the Optia, failed to collect at least 1 × 106 CD34/kg. A revised model is tested retrospectively on Optia data, and suggestions for further improvements are discussed.

Optimizing interpretation of survival studies of fresh and aged transfused biotin-labeled RBCs.

BACKGROUND: Ex vivo labeling with 51 chromium represents the standard method to determine red blood cell (RBC) survival after transfusion. Limitations and safety concerns spurred the development of alternative methods, including biotinylated red blood cells (BioRBC). STUDY DESIGN AND METHODS: Autologous units of whole blood were divided equally into two bags and stored under standard blood bank conditions at 2 to 6°C (N = 4 healthy adult volunteers). One bag was biotinylated (15 µg/ml) on storage days 5 to 7 (fresh) and the other was biotinylated (3 µg/ml) on days 35 to 42 (aged). The proportion of circulating BioRBC was measured serially, and cell-surface biotin was quantified with reference to molecules of equivalent soluble fluorochrome. Clearance kinetics were modeled by RBC age distribution at infusion (Gaussian vs. uniform) and decay over time (constant vs. exponential). RESULTS: Data were consistent with biphasic exponential clearance of cells of uniform age. Our best estimate of BioRBC clearance (half-life [T1/2 ]) was 49.7 ± 1.2 days initially, followed by more rapid clearance 82 days after transfusion (T1/2  = 15.6 ± 0.6 days). As BioRBC aged in vivo, molecules of equivalent soluble fluorochrome declined with a T1/2 of 122 ± 9 days, suggesting gradual biotin cleavage. There were no significant differences between the clearance of fresh and aged BioRBC. CONCLUSION: Similar clearance kinetics of fresh and aged BioRBC may be due to the extensive washing required during biotinylation. Survival kinetics consistent with cells with uniform rather than Gaussian or other non-uniform age distributions suggest that washing, and potentially RBC culling, may extend the storage life of RBC products.

Current good manufacturing practices-compliant manufacture and measurement of biotin-labeled red blood cells.

BACKGROUND: Red blood cells (RBCs) can be labeled with N-hydroxysuccinimidobiotin (sulfo-NHS-biotin), which binds to cell surface proteins under aqueous conditions. Biotinylated RBCs can be safely infused and detected in peripheral blood samples using flow cytometry, using a fluorochrome-conjugated streptavidin (SA) detection reagent. Biotinylated RBCs have been used to track survival of transfused RBCs, and have applications in optimizing RBC storage and in understanding donor genetic, environmental and disease factors affecting RBC products. METHODS: We have developed a closed-system, current good manufacturing practices (cGMP)-compliant procedure for biotinylation of RBCs and a quantitative flow cytometric assay to estimate the dose of cell-bound biotin delivered to the patient. Resulting products were characterized for variability, sterility, endotoxin, hemolysis, total dose of cell-bound biotin and stability. RESULTS: The density of biotin-labeling increased as a log-linear function of sulfo-NHS-biotin-labeling concentration, with greater variability at lower concentrations. The upper estimates of biotin doses in the average product (mean RBC content = 5.55 × 1011) were 9.8 and 73.0 µg for products labeled at 3 and 15 µg sulfo-NHS-biotin/mL of total reaction mixture (27 and 135 nmol/mL packed RBCs), respectively. All products were negative for bacterial and fungal growth at 14 days and were below the limit of endotoxin detection. Biotinylated RBCs were stable in vitro for up to 50 days after labeling. DISCUSSION: We have validated a closed-system procedure for biotinylating RBCs for investigational use. A standard operating procedure is presented in sufficient detail for implementation in a cGMP-compliant cell-processing facility.

Human adipose stromal vascular cell delivery in a fibrin spray.

BACKGROUND AIMS: Adipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system. METHODS: SVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVage system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel fibrin spray system. SVF cell growth and differentiation were documented by immunofluorescence staining and photomicrography. RESULTS: SVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SVF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD146+ and α-smooth muscle actin+ perivascular/stromal cells. CONCLUSIONS: Human adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting.

BACKGROUND AIMS: Donor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs. METHODS: We performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4-T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler. RESULTS: The majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 µm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10(10) total cells, 6.2 ± 2.2 × 10(8) of which were CD45(+) CD34(+). Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34(+) CD90(+) CD117(dim) hematopoietic stem cells (15.5 ± 7.5% of the CD34 (+) cells) and CD45(-) CD73(+) CD105(+) mesenchymal stromal cells (0.04 ± 0.04% of the total cells). CONCLUSIONS: This procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.

Intra-operative preparation of autologous bone marrow-derived CD34-enriched cellular products for cardiac therapy.

BACKGROUND AND AIMS: With the advent of regenerative therapy, there is renewed interest in the use of bone marrow as a source of adult stem and progenitor cells, including cell subsets prepared by immunomagnetic selection. Cell selection must be rapid, efficient and performed according to current good manufacturing practices. In this report we present a methodology for intra-operative preparation of CD34(+) selected autologous bone marrow for autologous use in patients receiving coronary artery bypass grafts or left ventricular assist devices. METHODS AND RESULTS: We developed a rapid erythrocyte depletion method using hydroxyethyl starch and low-speed centrifugation to prepare large-scale (mean 359 mL) bone marrow aspirates for separation on a Baxter Isolex 300i immunomagnetic cell separation device. CD34 recovery after erythrocyte depletion was 68.3 ± 20.2%, with an average depletion of 91.2 ± 2.8% and an average CD34 content of 0.58 ± 0.27%. After separation, CD34 purity was 64.1 ± 17.2%, with 44.3 ± 26.1% recovery and an average dose of 5.0 ± 2.7 × 10(6) CD34(+) cells/product. In uncomplicated cases CD34-enriched cellular products could be accessioned, prepared, tested for release and administered within 6 h. Further analysis of CD34(+) bone marrow cells revealed a significant proportion of CD45(-) CD34(+) cells. CONCLUSIONS: Intra-operative immunomagnetic separation of CD34-enriched bone marrow is feasible using rapid low-speed Hetastarch sedimentation for erythrocyte depletion. The resulting CD34-enriched product contains CD45(-) cells that may represent non-hematopoietic or very early hematopoietic stem cells that participate in tissue regeneration.

Clinical implementation of a procedure to prepare bone marrow cells from cadaveric vertebral bodies.

AIMS: Vertebral bone marrow is a rich and easily accessible source of hematopoietic and mesenchymal stem cells that has been used to promote chimerism and transplantation tolerance in connection with cadaveric organ transplantation. The purpose of this study is to provide a detailed account of the procedure used to prepare the first five vertebral bone marrow products for infusion in conjunction with the first hand/hand-forelimb transplants performed at the University of Pittsburgh (PA, USA). MATERIALS & METHODS: The cell separation and release testing were performed at the University of Pittsburgh Cancer Institute's Hematopoietic Stem Cell Laboratory, a Good Manufacturing Practice-compliant facility accredited for clinical cell processing by the Foundation for Accreditation of Cellular Therapy (FACT) and for clinical flow cytometry by the College of American Pathologists (CAP).