iv. PROCESSING, CRYOPRESERVATION AND INFUSION Page 3

17. A simple and reliable procedure for cord blood banking, processing, and freezing: St Louis and Ohio Cord Blood Bank experiences. Alonso JM, III, Regan DM, Johnson CE, Oliver DA, Fegan R, Lasky LC et al. Cytotherapy 2001; 3:429-433.

This report provides details of a modification of the hetastarch sedimentation and volume reduction approach of Rubinstein et al (Rubinstein et al PNAS USA 1995;92:10119-22). Cord blood is mixed with a 1:5 v/v ratio of hetastarch. The product is incubated for 45 minutes in an inverted position in a refrigerated centrifuge and then is spun for 5’ at 50g. RBC concentrate is drained from the bottom. The volume drained is calculated to remove 80% of RBCs. The cord blood unit is then resuspended and spun for 13 min at 420 g. Plasma is expressed from the top. A final product volume of 27 mL (range 16-58 mL) was obtained from an original 50-200 mL. The average yield of TNC pre and post-processing was 90% for the first 4,055 units banked. Pre- and post-processing CFU and CD34 yields were tested in a cohort and were similarly conserved. With a processing time of 3 hours for a single cord, this process is time efficient and lends itself well to processing several units at the same time. The technique has been exported to other laboratories with similar yields.

18. Predictive utility of the attached segment in the quality control of a cord blood graft. Rodriguez L, Garcia J, Querol S. Biol Blood Marrow Transplant. 2005;11:247-51.

A reliable method to assess the cell content and viability of a graft before transplantation is crucial. Although it has been shown that the number of total nucleated cells (TNCs), CD34+ cells, and colony-forming cells (CFCs) per kilogram body weight of the recipient in the graft are good predictors of patient survival, all of these variables are normally defined before cryopreservation. However, the quality of a cord blood graft might be affected during the freezing process, storage, and transportation to the transplant unit, as well as during the thawing process.

To validate the use of a segment attached to the umbilical cord blood (UCB) unit as a quality-control tool for the final product, UCB units (n = 20) stored in liquid nitrogen were analyzed. The UCB units and their attached segments were thawed, and the number and viability of total nucleated cells, mononucleated cells, CD45+ cells, and CD34+ cells were determined, as were colony-forming cell counts. There was no significant difference between UCB units and segments for any of the parameters assessed. Additionally, the linear correlation coefficient (R²) in these paired samples was 0.85 and 0.78 for CD34+ cells and colony-forming cells, respectively.

On the basis of these data, the authors concluded that the cell sample in the tube segment physically linked to the transplant UCB bag predicts the total cell content and functionality of the unit and may serve as a source for final quality control of the UCB unit before transplantation.

19. Use of nonvolume-reduced (unmanipulated after thawing) umbilical cord blood stem cells for allogeneic transplantation results in safe engraftment. Hahn T, Bunworasate U, George MC, Bir AS, Chinratanalab W, Alam AR et al. Bone Marrow Transplant 2003; 32:145-150.

Twenty-six 26 patients underwent transplantation with unmanipulated (n = 18) or volume-reduced (n = 8) cord blood units. Engraftment was similar in the two groups, and similar to reported series and to the authors’ series using volume-reduced units for cord blood transplantation. The results of this small study indicated that there was no serious toxicity from cord blood infusion using unmanipulated cord blood units. The authors concluded that unmanipulated cord blood units may be infused safely with adequate engraftment and survival.

20.Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34+ cells to dimethyl sulphoxide.  Hunt CJ, Armitage SE, Pegg DE.  Cryobiology 2003;46:61-75.

21. Cryopreservation of umbilical cord blood: 2. Tolerance of CD34+ cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing. Hunt CJ, Armitage SE, Pegg DE.  Cryobiology 2003;46:76-87.

Cryopreservation protocols for umbilical cord blood (UCB) have been based on methods established for bone marrow and peripheral blood stem cells. The a priori assumption that these methods are optimal for progenitor cells from UCB has not been investigated systematically. Studies of UCB stem cells have largely been directed to establishing that such methods provide an adequate level of post-thaw survival rather than attempting to derive optimal cryopreservation protocols from first principles. The authors established addition and elution protocols that prevent osmotic damage and they used these to investigate the effect of multimolar concentrations of Me₂SO on membrane integrity and functional recovery. They determined that the optimal recovery of CD34+ cells requires serial addition of Me₂SO, slow cooling at rates between 1^oC and 2.5^oC/min and serial elution of the cryoprotectant after thawing.

22. Assessment of cell viability and apoptosis in human umbilical cord blood following storage.  Xiao M, Dooley DC.  J Hematother Stem Cell Res. 2003 Feb;12:115-22.

The authors attempted to identify a simple and rapid technique for assessing the quality and recovery of umbilical cord blood (UCB) cells following laboratory manipulation. They determined that UCB held for 72 hours showed higher levels of cell deterioration than were present in UCB <48 hours old. Staining with 7-AAD was more sensitive to cellular damage than was uptake of Trypan Blue, and correlated with retention of hematopoietic function (progenitor assays). The authors concluded that 7-AAD staining of UCB mononuclear cells provides a rapid and simple technique for assessing the viability, recovery, and hematopoietic functionality of stored UCB.

23. Patient care during infusion of hematopoietic progenitor cells. Sauer-Heilborn A, Kadidlo D, McCullough J. Transfusion. 2004;44:907-16.

The authors provide a detailed review of the factors which should be taken into consideration and the adverse reactions that may occur in relation to the process of hematopoietic progenitor cell (HPC) infusion. Most published data refer to autologous or allogeneic BMT or PBPC infusions, and adverse reactions associated with cord blood infusions are less frequent and appear to be less severe. The thawed HPC product contains granulocyte debris, RBC stroma, and free hemoglobin which may cause side effects when infused. Washing of the cells is an option but must be weighed against the loss of viable cells. In the largest study reported, severe reactions occurred in only 0.4 percent of 1412 patients. The most frequent symptoms were nausea, vomiting, hypertension, hypotension and bradycardia. Some reports describe more side effects with a higher dose of DMSO. Hemoglobinuria has been reported in a high percentage of patients in relation to the RBC content of the transfused product. Side effects are usually mild to moderate; however, life-threatening cardiologic, neurologic, pulmonary, anaphylactic, and renal events have been reported. Patients receiving autologous BM containing an average of 1.55 ml of RBCs per kg experienced significantly more arrhythmias than patients receiving PBPC with a median RBC content of 0.23 ml per kg. There are reports of severe bradycardia and even two case reports of a cardiac arrest immediately after the infusion of autologous BM. To minimize the time of contact, there is an incentive to infuse the HPCs as rapidly as possible. However, in the study with the lowest rate of bradycardia and heart blocks, the authors suggested that that might be due to a slow infusion rate of 5.5 to 6 ml per minute. One study used an infusion rate of 20 to 50 ml per minute and found cardiac arrhythmias and hypertension in a high percentage of the patients (82 and 41%, respectively). Patients should be carefully monitored during and after the infusion for 6-24 hours.

24. Microbial contamination of hematopoietic progenitor cell grafts-incidence, clinical outcome, and cost-effectiveness: an analysis of 735 grafts. Kamble R, Pant S, Selby GB, Kharfan-Dabaja MA, Sethi S, Kratochvil K, Kohrt N, Ozer H. Transfusion. 2005;45:874-8.

The authors point out that the current guidelines of AABB recommend bacterial and fungal microbial surveillance of HPC grafts. Microbial cultures are therefore obtained immediately after HPC collection and also after HPC processing. However, the clinical significance of microbial contamination on HPC engraftment and transplant outcome is unclear. It is the general experience of the transplant community that positive cultures are not associated with adverse events. Unfortunately, there are few published data available to support this impression.

In this study, a retrospective analysis of 735 consecutive marrow and peripheral blood progenitor cell harvests between 1998 and 2003 was performed. Analysis included incidence, clinical outcome, and cost outcomes of positive blood cultures and antibiotic therapy.

Thirty-three of 735 (4.5%) harvests were contaminated. The incidence of microbial contamination varied with the source of the graft: 4 of 26 (15%) were cord blood, 8 of 177 (4.5%) were marrow, and 21 of 532 (3.9%) were peripheral blood. Coagulase-negative Staphylococcus (n=22) and Propionibacterium acnes (n=8) were most frequently isolated. Potentially pathogenic organisms were isolated in 6 of 735 (0.81%) grafts. The estimated total cost of surveillance was approximately $81,585. The cost of vancomycin therapy in 4 patients who received prophylactic antibiotic therapy was approximately $10,000.

No adverse sequelae followed infusion of contaminated grafts.

The authors concluded that clinical sequelae following infusion of microbially contaminated progenitor cells is extremely rare. Prophylactic empiric antibiotics may be unnecessary. Routine microbial surveillance of progenitor cell grafts is a low-yield procedure.

(Present requirements for cord blood units by accrediting agencies (FACT/NETCORD and AABB) require that, if positive cultures are obtained, antibiotic sensitivities must be provided to the transplant center. Some investigators (See v. Quality Issues, Citation #1) recommend that cord blood units with a positive culture should not be placed into useable inventory and available for transplant. The data presented in this article suggest that transplant physicians should continue to have the option to accept or refuse units with positive cultures.)

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