F. Landi(1,2), F. Zinno(1,2), M.C. Scerpa(2), N. Daniele(1,2), M. Caniglia(2), M.J. Miele(2), F. Locatelli(2,3), G. Isacchi(1,2) (1)Immunohematology Section, “Tor Vergata” University Rome, Italy; (2) IRCCS Bambino Gesù Pediatric Hospital Rome, Italy; (3) University of Pavia, Italy.
OBJECTIVES: This study aims to highlight the effectiveness of an automated technique, for volume reduction of stem cells from bone marrow. This system allows to manipulate blood units in a closed loop in order to comply with the GMP criteria. Also, purchase a considerable importance, especially in the case of transplantation with ABO incompatibility. The critical importance of cell dose in the clinical outcome has motivated the need to develop techniques aimed at reducing cell losses and increasing data reproducibility. The aim of this study is to evaluate the efficacy of the Sepax S-100 (Biosafe Group, Eysins, Switzerland) device to process volume reduction HPC-M (Figure 1). In particular, we have analysed nucleated cells (NC), depletion red blood cells (RBC), volume reduction and CD34+ cells recovery. METHODS: 27 procedures were carried out on concentrated collected from 21 patients, undergoing autologous transplantation, and from 6 donors for allogeneic transplantation. The “Volume Reduction” protocol is designed to remove RBC and plasma, while maintaining a good recovery of mononuclear cells. The by-products of the blood separation (plasma and red-blood cells) are collected in standard blood bags while the output product (buffy coat) is collected in a cryogenic storage bag or in a standard blood bag (Figure 2).
VOLUME REDUCTION PROTOCOLS
A: Separation chamber 200 ml B: Line pressure monitor luer with microbial filter C: Stopcock manifold D: Bubble chamber CS-490 kit E: Spike to source bag F: 500 ml PVC collection bag Figure 2 G: Spike to buffy-coat collection bag
The processing protocol of sample from 30 to 880 ml, relies Figure 3 solely on the hematocrit value and the initial volume of the sample to be processed, to calculate the final volume. The system uses a rotating syringe technology that provides both separation through rotation of the syringe chamber (centrifugation) and component transfer through displacement of the syringe piston (Figure 3). To evaluate CD34+ cells the protocol ISHAGE was used. RESULTS: In the patients who underwent autologous transplant, the automated procedure resulted in a median recovery of Autologous transplants Allogeneic transplants 80% (58-109; SD 13.1) NC and 89% (59(n. of patients=21) (n. of donors=6) 134.9; SD 22) CD34+ cells, RBC depletion 73% (62-81; SD 5.41); volume reduction Average Range SD Average Range SD 88% (85-93; SD 2.2) (Table 1). NC recovery (%) 80 58-109 13.1 75.1 55-98 11.8 In the donors for allogeneic transplant, the CD34+ recovery (%) 89 59-134.9 22 79.6 59-158 24 automated procedure resulted in a median recovery of 75,1% (55-98; SD 11.8) NC RBC depletion (%) 73 62-81 5.41 75.4 62-82 5.5 and 79.6% (59-158; SD 24) CD34+ cells, RBC depletion 75.4% (62-82 SD 5.5); Volume reduction (%) 88 85-93 2.2 88 83-91 2.4 volume reduction 88% (83-91; SD 2.4) (Table 1). Table 1 CONCLUSION: The volume reduction of bone marrow using the Sepax S100 automated and closed system, does not affect the cell functionality assuring a satisfactory result in terms of nucleated cells, CD34+ cells recovery, RBC depletion and volume reduction. The results showed a recovery of NC and CD34+ equal to 80%, RBC depletion to 75%, without the use of HES, a reagent commonly used in the absence of an automated procedure, but relying solely on the hematocrit value and the initial volume of the sample to be processed.