BRIEF REPORT Relocation of cryopreserved umbilical cord blood samples using a high-capacity dry shipper to a new laboratory: a cord blood banking experience Kalaivani Thiagarajah,1* Chee-Yin Wong,2* Vickneswary Veera Vijayan,1 Ghee-Chien Ooi,1 Mei-Theng Ng,3 Soon-Keng Cheong,4 and Kong-Yong Then1

BACKGROUND: Processed umbilical cord blood (UCB) must be stored at cryogenic temperature at all times to maintain the quality and viability of the cells. However, a challenge is presented in the form of moving a large number of cryopreserved UCB samples to a new location. In this report, we share our experience on relocating more than 100,000 units of cryopreserved UCB samples stored in 12 liquid nitrogen freezers (LNFs) to our new laboratory. STUDY DESIGN AND METHODS: For quality control purposes, 2 weeks before relocation, donor UCB samples were processed, cryopreserved, and stored in each LNF. On relocation day, half of the samples were retrieved to determine total nucleated cell count, percentage of CD34+ cells, and cell viability as controls for later comparison. UCB samples were transferred into dry shippers before being relocated to the new laboratory. Upon arrival, LNFs were serviced before transferring UCB samples back into its original location within the LNF. The remaining donor UCB samples were retrieved and analyzed for the same tests mentioned. RESULTS: We found no significant differences in preand postrelocation values of the tests performed. CONCLUSION: All UCB samples were successfully relocated into the new laboratory without affecting the quality.

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n 1988, Gluckman and colleagues1 led a transplantation team to perform the world’s first umbilical cord blood (UCB) transplantation for a child with Fanconi’s anemia. From that case onward, the use of UCB for transplantations increased tremendously.2 UCB, the source of hematopoietic stem cells, has been used in the treatment for a variety of hematologic disorders, selected hereditary immunodeficiency states, and metabolic diseases.3,4 Cryopreservation of UCB allows the immediate availability of UCB for transplantation whenever the need arises.5 However, for the maintenance of quality and viability of UCB cells, processed UCB samples must be stored in liquid nitrogen freezers (LNFs) or cryogenic storage tanks at −150°C and below at all times until the samples are required for transplantation, especially when the samples are intended to be stored for more than 1 year.6 The requirement to maintain processed UCB samples at cryogenic temperature posed several challenges when we were planning to relocate more than 100,000 units of cryopreserved UCB samples stored in 12 LNFs to our new laboratory, which is located 4.2 km away. ABBREVIATIONS: LNF(s) = liquid nitrogen freezer(s); TNC(s) = total nucleated cell(s); UCB = umbilical cord blood. From 1Cryocord and 2Cytopeutics, Cyberjaya, Selangor, Malaysia; the 3Krieger School of Arts & Sciences, Johns Hopkins University, Baltimore, Maryland; and the 4Faculty of Medicine and Health Sciences, Tunku Abdul Rahman University, Bandar Sungai Long, Selangor, Malaysia. Address reprint requests to: Kong-Yong Then, Suite 1-1, 1st Floor, Bio X Centre, Persiaran Cyberpoint Selantan, Cyber 8, 63000 Cyberjaya, Selangor, Malaysia; e-mail: [email protected] *Both authors contributed equally. Received for publication May 14, 2014; revision received October 13, 2014, and accepted October 14, 2014. doi: 10.1111/trf.12950 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **

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The first challenge was to choose a method to transfer the cryopreserved UCB samples at cryogenic temperature during the entire relocation process. A standard LNF, containing liquid nitrogen at the bottom, was designed for storing biologic samples at cryogenic temperature permanently at a static place only. It was not suitable for use in transporting samples because the excess weight of stored samples and liquid nitrogen can damage the device. Therefore, it was necessary to use dry shippers instead because these devices were specifically designed for shipping or transporting biologic samples. A dry shipper or vapor shipper is a high-quality insulated unit constructed of durable materials that is compatible with the divergent temperature extremes and has broad applications in cryobiology. To acquire the status of a “dry” shipper, the vessel is lined with absorbent material that is capable of adsorbing liquid nitrogen, ensuring no “free” liquid nitrogen in the vessel. Therefore, the risk of any liquid nitrogen spillage during the relocation process can be avoided completely.6 After considering the advantages of using dry shippers for the relocation process, we had chosen a dry shipper model, MVE 1536PD, because of its capacity to store more than 10,000 UCB samples in either 4.5-mL cryovials or cryobags at a time. The second challenge was to develop a step-by-step protocol for the relocation process to minimize human errors as much as possible. The protocol was developed by a team consisting of the laboratory management staffs, quality manager, stem cell scientists, and the technical team from the authorized local distributor of the dry shipper, who acted as our backup support for the relocation process. Each step in the protocol was carried out by a minimum of three laboratory staff members and monitored by the quality manager. The technical team provided their expertise gained from the manufacturer and was present during the relocation process with a dry shipper on stand-by, which we can immediately use, in the event that our dry shippers experienced any problem. Before the designated date to begin the relocation process, all personnel involved, including the truck driver, were briefed on the protocol. As an additional backup measure, our company had also purchased an insurance scheme that will provide monetary compensation to the customers, if any unfortunate incident occurs. The third challenge was to establish a quality control (QC) system to determine the quality of the cryopreserved UCB samples after the relocation and to validate the devices used in the relocation process, whichever necessary. Referring to the Standard for Cellular Therapy Services published by AABB, tests including total nucleated cell (TNC) count, percentage of CD34+ cells, and cell viability should be performed to determine the quality of the cryopreserved UCB samples.7 Therefore, we had included these tests as part of the QC system. The results from these tests are evidence on whether the quality of 2

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cryopreserved UCB was affected by the relocation process or not. A small team of biotechnologists were assigned to perform the steps as established in the QC system. Moreover, since the quality of the cryopreserved UCB samples is dependent on the storage temperature, it is important that the temperature, to which the samples will be exposed, is within specified control throughout the entire relocation process. At all times during relocation, all UCB samples must be maintained at below −150°C, which is less than a glass transition temperature of water at −132°C. At this temperature, all biologic and chemical activities cease because there is no water activity.8,9 Most researchers will quote −150°C as the critical temperature point for most cell products to allow sufficient safety margin for the normal working operation of a storage freezer. Hence, it is necessary to first validate the working temperature of the dry shippers that was used and then to constantly monitor the temperature within the dry shippers and LNFs to ensure that all devices are working properly throughout the entire process. In this report, we share our experience on relocating more than 100,000 units of cryopreserved UCB that were permanently stored in 12 LNFs into our new laboratory. Procedures including dry shipper validation, transference of cryopreserved UCB samples from LNF to dry shippers, and QC of UCB before and after relocation were presented in this report.

MATERIALS AND METHODS Validation of dry shipper and temperature loggers Dry shippers of Model MVE 1536PD (Chart, Ball Ground, GA), which can accommodate more than 10,000 4.5-mL cryovials, were chosen and used during the relocation of cryopreserved UCB to the new laboratory. Before the designated date, the dry shippers were charged with liquid nitrogen according to manufacturer’s instruction and were validated by monitoring the internal temperature for 20 days to ensure the temperature within each dry shipper was maintained at cryogenic temperature. As temperature change is an important variable that must be monitored closely during the relocation, two temperature loggers were installed inside the dry shipper: one temperature logger was placed on the same level as the lowest cryorack level (Probe 1) and another on the highest cryorack level (Probe 2). All temperature loggers were tested and validated before placing into the dry shippers.

Preparation of UCB samples for QC Two weeks before the designated day, three fresh donor UCB samples were processed, cryopreserved, and stored in each LNF. All UCB donors had signed informed consent. Donors’ UCB samples were processed according to our standard cord blood processing procedure with

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all UCB samples were transferred into the dry shippers, the lids were closed and kept secured with cable ties. Liquid nitrogen was discarded from the now-empty LNF and each unit was thawed completely before transporting together with the UCB samples containing dry shippers to a new laboratory that is located 4.2 km away. All dry shippers and permanent LNFs were moved into a truck with a forklift. The truck was driven at a slow speed to minimize any impact from movement or vibration from the vehicle. Upon arrival at the new laboratory, UCB samples were either transferred into temporary-storage LNFs or kept in dry shippers overnight while waiting for permanent-storage LNFs to be Fig. 1. A summary of the workflow for relocation process with time frames for speserviced and commissioned. Once the cific activities shown in parentheses. One day before and after relocation, donor UCB permanent-storage LNFs were ready for samples (n = 3), which were previously aliquoted into 24 vials each and two vials use, liquid nitrogen was filled and tested from each donor were placed into each permanent LNF, were retrieved for QC activifor proper operation. The temperature ties including TNC count, percentage of CD34+ cells, and cell viability. was monitored to ensure that cryogenic temperature was maintained before hetastarch method. Briefly, UCB were mixed with transferring UCB samples back in its original position hydroxyethyl starch before double spun at 50 × g for 6 within the permanent LNFs. Racks of UCB samples were minutes at 20°C and 400 × g for 13 minutes at 20°C to verified and temperatures inside the LNFs and dry shipper recover the TNC count. Each donor UCB sample was proor temporary LNFs were monitored during this step. cessed and aliquoted into 24 vials giving a total of 72 vials. Six vials of donor UCB samples, or two vials from each UCB QC after relocation donor, were then stored in each of the 12 LNFs used for permanent cryogenic storage. One vial from each donor and from each freezer was One day before relocation, one vial from each donor retrieved and sent for QC. TNC count was determined and from each LNF were retrieved and analyzed for QC. with hematology analyzers, percentage of CD34+ cells in TNC count was determined with hematology analyzers UCB was analyzed by flow cytometry, and cell viability was (Beckman Coulter, Pasadena, CA), percentage of CD34+ determined by trypan blue. cells was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ), and cell viability was determined by Statistical analysis trypan blue method (Gibco, Grand Island, NY). A workflow of entire relocation procedure was summarized in Parametric data is expressed as mean ± 1 SD. Comparison Fig. 1. data between before and after relocation were calculated using paired t test. Data are stored and analyzed using computer software (SPSS 14.0, SPSS, Inc., Chicago, IL) and Transference of UCB samples significance was set at a p value of less than 0.05. On the designated day, UCB samples were transferred from permanent LNF to dry shippers, one cryorack at a RESULTS time. The entire process was monitored by a quality Dry shipper validation and LNF manager. The specific rack number were identified and temperature monitoring verified by two personnel before a third person recorded the numbers onto a transfer form. Immediately after, the Before relocation, the dry shipper was validated and the cryoracks were transferred into dry shippers (vapor temperature was maintained below −150°C (Probe 1 and phase). During transfer of these racks, the temperature Probe 2) for at least 20 days. During the entire relocation values inside the permanent LNF and the dry shippers process, the temperature of the dry shipper was recorded were monitored for every 5 minutes and recorded. Once at −157.2 ± 4.6°C (Probe 1) and −159.6 ± 5.1°C (Probe 2). Volume **, ** **

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Fig. 2. Prerelocation (■) and postrelocation (□) values of (A) TNC count, (B) cell viability, and (C) percentage of CD34+ of donor UCB samples. No significant differences were found in all three variables.

Besides monitoring the temperature of the dry shipper, the permanent and temporary LNFs were monitored as well. Before relocation, temperatures for permanent-storage LNFs were recorded at −185.2 ± 5.9°C (Probe 1) and −180.6 ± 0.3°C (Probe 2). Upon arrival at the new laboratory, the LNFs were serviced, filled with liquid nitrogen, and tested for proper operation. When the samples were transferred back to its original position in the LNF, the recorded temperatures were −192.4 ± 3.0°C (Probe 1) and −187.6 ± 4.8°C (Probe 2). In temporarystorage LNFs, the temperature was recorded at −187.3 ± 6.6°C (Probe 1) and −183.6 ± 7.1°C (Probe 2) during the entire relocation process.

Transference and relocation of UCB samples More than 100,000 UCB samples stored in 12 LNFs were relocated into the new laboratory. Each LNF contained between 5000 and 30,000 cryovials or cryobags of UCB samples. A total of 6 days were used for relocating the samples and every day, samples were relocated to the new laboratory from one to three permanent-storage LNFs. During each transfer step, cryoracks were transferred one at a time by a minimum of three personnel in the presence of a quality manager. One person retrieved a rack of samples from the LNF and passed it to another person who put the rack into the dry shipper, while a third person registered the information of the transferred rack. The transfer times from permanent LNFs to dry shippers were less than 30 seconds each time while it took 13.4 ± 4.4 minutes to transfer whole freezer samples to another freezer. Upon completion of the task, all personnel signed the transfer form. Location of the racks and samples ID were checked and matched with the original database. Once confirmed, the quality manager verified the transfer.

Pre- and postrelocation QC For QC purpose, three donor UCB samples were processed, cryopreserved, and stored in LNFs. On the day just before relocation, half of the samples were thawed and the TNC counts for these samples were 5.5 × 109 ± 0.6 × 109/L 4

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(Sample 1), 3.9 × 109 ± 0.8 × 109/L (Sample 2), and 3.9 × 109 ± 0.7 × 109/L (Sample 3). Cell viabilities for these samples were 92.8 ± 2.2, 92.0 ± 1.8, and 92.5 ± 1.6%, respectively. TNC counts for postrelocation samples were 5.2 × 109 ± 0.9 × 109/L (Sample 1), 3.7 × 109 ± 0.4 × 109/L (Sample 2), and 3.9 × 109 ± 0.7 × 109/L (Sample 3). The corresponding cell viabilities were 92.6 ± 2.1, 90.9 ± 3.1, and 92.3 ± 1.7%, respectively (Figs. 2A and 2B). The differences in TNC and cell viability values, before and after relocation, were insignificant. Meanwhile, the readings on percentage of CD34+ cells for Samples 1, 2, and 3 (before relocation) were 0.69 × 109 ± 0.50 × 109, 0.87 × 109 ± 0.51 × 109, and 0.51 × 109 ± 0.44 × 109/L, respectively. The readings, after relocation, were 0.52 × 109 ± 0.45 × 109/L (Sample 1), 0.86 × 109 ± 0.57 × 109/L (Sample 2), and 0.46 × 109 ± 0.54 × 109/L (Sample 3; Fig. 2C). No significant differences in percentage of CD34+ cells were found.

DISCUSSION Transferring or relocating a small quantity or volume of cryopreserved samples such as sperm or embryo at cryogenic temperature with a small dry shipper has already been reported.10-12 However, it was a challenge to relocate a large amount of cryopreserved samples to a new location. Therefore, proper planning was required to ensure that the whole relocation process runs smoothly without jeopardizing the quality of the cryopreserved samples. To the best of our knowledge, this is the first report on relocating a high volume of cryopreserved samples. Being our first experience in relocating a large number of cryopreserved samples, there were several limitations in this process, especially the QC system established for the relocation process. The first limitation was the small sample size for the QC system established specifically for this relocation process. Only UCB samples from three donors were used to determine the quality of cryopreserved UCB before and after relocation. The nature of our business is to provide storage facilities to customers who want to store their babies’ UCB at cryogenic stage until required for use in the future. Therefore,

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we were unable to use existing and new cryopreserved UCB samples of these customers in this relocation process because the consent forms signed disallow the company from using their samples for research purposes. At the same time, the relocation of cryopreserved samples was scheduled to occur at a specific time in the timeline to relocate the entire laboratory operation. Coincidently, despite our best efforts, we were only able to obtain consent from three donors to use their UCB samples for research purposes. As a team, we have discussed and decided that a sample size of 3 would be the minimal representation of all the cryopreserved samples we have at that time. We realized that the small sample size is not sufficient to determine the quality of the UCB. Thus, we planned to monitor the quality of the UCB samples involved in the relocation process when any of these UCB samples is thawed for transplantation purposes in the future. In the relocation process, the quality of relocated samples was only determined in UCB samples stored in cryovials. Customers who opted for storage package in cryobags represent approximately 10% of the total customers whose samples were relocated to the new laboratory. Moreover, having just three UCB samples for use in the QC system for the relocation process, we cannot run a sample in cryobags and only have two samples in cryovials. In the future, when we must relocate the laboratory, we should develop a QC system that can realistically represent the UCB samples we have in cryogenic storage. For the QC system, only three variables were analyzed to determine whether the quality of cryopreserved UCB is affected by the relocation process or not and the tests were TNC count, percentage of CD34+ cells, and cell viability. AABB did recommended determining the quality of cryopreserved UCB samples based on nucleated red blood cell count.7 However, since the UCB samples were kept at cryogenic stage throughout the relocation process and the distance between the old laboratory and the new laboratory is a short distance of 4.2 km, there will be an extremely low chance of the UCB cells undergoing apoptosis during the process. Therefore, we have decided that determining the quality based on a minimum of three variables is sufficient. However, for any future relocation of this kind or when the relocation is over a long distance, extra tests will be included in the QC system at that time. These extra tests will include counting colony-forming units to determine the UCB functionality, checking for aldehyde dehydrogenase levels and determining percentage of apoptotic cells by assessing annexin V and

caspase 3. In conclusion, all cryopreserved UCB units stored in 12 LNFs were successfully relocated into the new laboratory while maintaining the quality of UCB in terms of TNC count, percentage of CD34+ cells, and cell viability. CONFLICT OF INTEREST KYT is a shareholder of Cryocord and also a member of the Board of Directors. The other authors have disclosed no conflicts of interest.

REFERENCES 1. Gluckman E, Broxmeyer HA, Auerbach AD, et al. Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical cord blood from an HLA identical sibling. N Engl J Med 1989;321:1174-8. 2. Oran B, Shpall E. Umbilical cord blood transplantation: a maturing technology. Hematology Am Soc Hematol Educ Program 2012;2012:215-22. 3. Copelan EA. Hematopoietic stem-cell transplantation. N Engl J Med 2006;354:1813-26. 4. Ballen KK, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood 2013;122:491-8. 5. Meyer TP, Hofmann B, Zaisserer J, et al. Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood. Cytotherapy 2006;8:26576. 6. Geraghty RJ, Capes-Davis A, Davis JM, et al. Guidelines for the use of cell lines in biomedical research. Br J Cancer 2014;111:1021-46. 7. American Association of Blood Banks. Standard for cellular therapy services. 6th ed. Bethesda (MD): American Association of Blood Banks; 2013. 8. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984;247:C125-42. 9. Mortimer D. Current and future concepts and practices in human sperm cryobanking. Reprod Biomed Online 2004;9: 134-51. 10. Liu E, Kitajima S, Wiese E, et al. Re-establishment of complement C6-deficient rabbit colony by cryopreserved sperm transported from abroad. Exp Anim 2007;56:167-71. 11. Batista M, Santana M, Niño T, et al. Sperm viability of canine and caprine semen samples preserved in a dry shipper. Anim Reprod Sci 2012;130:105-10. 12. Abdel Hafez F, Xu J, Goldberg J, et al. Vitrification in open and closed carriers at different cell stages: assessment of embryo survival, development, DNA integrity and stability during vapor phase storage for transport. BMC Biotechnol 2011;11:29.

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Relocation of cryopreserved umbilical cord blood samples using a high-capacity dry shipper to a new laboratory: a cord blood banking experience.

Processed umbilical cord blood (UCB) must be stored at cryogenic temperature at all times to maintain the quality and viability of the cells. However,...
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