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CE Update Collection, Processing, and Banking of Umbilical Cord Blood Stem Cells for Clinical Use in Transplantation and Regenerative Medicine David T. Harris, PhD (Department of Immunobiology, University of Arizona, Tucson, AZ) DOI: 10.1309/64QG394K1M639L8A Abstract Cord blood banking has been the focus of many medical centers as it can provide a virtually unlimited source of ethnically-diverse stem-cell donors. In addition, its recent use in several regenerative medicine clinical trials has further spurred the motivation to collect and bank these stem cells. In the current study, we review the latest developments in cord blood banking. We have banked over 195,000 collections at our facility. Collections were processed by either Ficoll or AXP methodologies. An average 95% processing efficiency was obtained. The overall failure After reading this article, readers should be able to discuss the collection, processing, and preservation of cord blood samples by the Cord Blood Registry and explain their current uses and possible uses in the future. Bone marrow transplantation (BMT) is an often-used means of therapy for a variety of diseases, including chemotherapyresistant malignancies and genetic blood disorders (Table 1). For certain diseases (eg, some leukemias, a variety of aplastic anemias, and certain immunodeficiencies), it is the only proven treatment that can achieve long-term patient survival. For other diseases (eg, autoimmune diseases and some genetic blood disorders), it could offer the hope of a long-term cure via stem-cell transplantation or gene therapy. If a family member cannot provide a bone marrow donation, then a search must be performed for a human leukocyte antigen (HLA)-matched, unrelated volunteer stem-cell donor. The difficulty of finding suitably HLA-matched donors for patients needing stem-cell transplant (especially minority patients) has left many patients without treatment.1,2 Furthermore, graft-versus-host disease (GVHD) occurs more than 50% of the time in unrelated BMT,3 with approximately half being severe and life-threatening. Due to the problems of a lack of donors and Table 1_Current Stem Cell Transplant Uses Malignancies Blood disorders Immunodeficiency Leukemia Lymphoma Multiple myeloma Hodgkin’s disease Retinoblastoma Histiocytosis Sickle cell anemia Thalassemia Aplastic anemia Thrombocytopenia Diamond-Blackfan Amegakaryocytosis SCID Ataxia telangiectasia Wiskott-Aldrich syndrome DiGeorge syndrome Kostmann syndrome Omenn syndrome Autoimmune diseases Metabolic disorders Other inherited disorders Multiple sclerosis Leukodystrophy Osteopetrosis Systemic lupus Gaucher disease Osteogenesis imperfecta Rheumatoid arthritis Krabbe disease Lesch-Nyhan syndrome Evan syndrome Hunter syndrome Tay-Sachs disease Crohn’s disease Hurler syndrome Niemann-Pick disease SCID, severe combined immunodeficiency labmedicine.com rate was less than 4% in terms of samples containing too few cells to be clinically useful. Processed samples were frozen in cryovials or bags and banked in a liquid nitrogen Dewar. In conclusion, it is possible to simply and reproducibly harvest, process, and bank cord blood samples using current technology. Hematology exam 50801 questions and corresponding answer form are located after this CE Update article on page 179. the high incidence of GVHD, researchers have looked to alternate sources of stem cells for transplant. Work that was begun in the early 1980s revealed that cord blood (ie, the leftover blood in the umbilical cord and placenta after the birth of a child) was comparable to bone marrow in terms of its utility in stem cell transplantation.4-11 Cord blood offers a number of advantages over bone marrow,11,12 including a lower incidence of GVHD and less strict HLA-matching requirements, which could increase its availability to transplant patients. In addition, using related samples instead of unrelated donor samples can double survival rates for patients.11,13 During the past 10 years, clinical use of cord blood (with more than 8,000 transplants worldwide14) has shown that it is a suitable alternative to bone marrow. In addition to its use as a substitute for bone marrow, cord blood has recently been used in a variety of regenerative medicine applications. Work done by McGuckin and colleagues,15,16 Rogers and colleagues,17 Kucia and colleagues,18 and Harris and colleagues19,20 has shown that cord blood contains a mixture of pluripotent stem cells capable of giving rise to cells derived from the endodermal, mesodermal, and ectodermal lineages. Thus, cord blood appears to be a practical substitute for embryonic stem cells and readily available for use in tissue engineering and regenerative medicine. Recently, clinical trials have begun using cord blood stem cells to treat type 1 diabetes, cerebral palsy, and peripheral vascular disease among others (Figure 1).21,22 In 1989, this laboratory began a series of studies examining the use of cord blood for transplantation.23-27 These studies spurred interest in the establishment of a cord blood bank. During this and subsequent work, we established the methodologies that were needed for efficient and reproducible cord blood collection, processing, and banking for clinical use.28-32 In June 1992, this laboratory established the first cord blood bank in the world. This cord blood bank later became the Cord Blood Registry (CBR) in 1996. Currently, the CBR has more than 195,000 cord blood samples banked at our facility, and is the largest familial (related) cord blood bank in the world. The March 2008 j Volume 39 Number 3 j LABMEDICINE 173 CE Update Figure 1_Cord blood stem cells and regenerative medicine. The figure illustrates preclinical and clinical uses of cord blood stem cells in regenerative medicine. results presented in this paper will summarize our cord blood banking efforts, highlighting the success that has been made with regard to cord blood collection, processing, and storage. Also, analyses as to collections from surgical and multiple births will be discussed. Finally, an analysis of the clinical utility of the collections will be presented. Collection of Cord Blood All patients are required to sign informed consent forms prior to collection of cord blood. All mothers are tested for infectious diseases as is typically done with blood donors (ie, reactivity for HIV, hepatitis B and C, etc.). Furthermore, the collected samples are tested for microbial sterility using an automated system (bioMérieux, Hazelwood, MO).33 Cord blood (CB) samples are obtained under the auspices of the patient’s caregiver (ie, physician or midwife). In the majority of cases, the collections are made after delivery of the infant and ligation of the cord, prior to expulsion of the placenta. Prior to collection of CB, the cord is wiped with alcohol or betadine to ensure sterility of the collection.28 There are a variety of methods used to collect cord blood, although primarily either large syringes (60 cc) or small bags (approximately 400 cc) are used. We have found that syringe collections provide visual feedback to the collector, allowing them to control not only the rate and volume of the collection, but also to restart collections that have stopped for any reason. Unattended bag collections, albeit somewhat more facile to perform, are not able to provide this option. Furthermore, first-time or inexperienced collectors routinely are able to collect larger volumes using the syringe method, with greater sterility of 174 LABMEDICINE j Volume 39 Number 3 j March 2008 collection. Routinely, collections are completed within 5 minutes (prior to placental expulsion). Experienced collectors are able to collect comparable samples using bags or syringes, and many collectors have expressed the opinion that bag collections are simpler to perform (noting the above caveats). In our experience, less than 2% of all clients were unable to have CB collected, generally due to rapid birth of the child. Regardless, both types of collection kits are provided in a sterile condition for use during surgical deliveries, as well as being pre-anticoagulated and containing all necessary shipping materials. These kits meet all regulatory requirements for shipping blood, including double containment and a crush-resistant container. Furthermore, the collection kits are insulated and padded for safety during transport, and studies have shown that these kits protect the sample from temperature extremes during shipment.31 The collected cord blood is shipped overnight to our laboratory in Tucson, AZ, to be received within 28 to 34 hours. At the same time, or prior to the CB collection, blood samples are obtained from the mother for infectious disease marker (IDM) testing, a regulatory requirement, using the provided vacutainers. Analysis of the last 195,000 CB collections made by more than 41,000 physicians at 3,128 birthing sites has shown that cord blood collection is simple and reproducible. Analysis of the last 100,000 collections made by CBR revealed that more than 96% of all samples shipped to the cord blood bank arrive (well) within 36 hours of collection, with more than 75% arriving within 24 hours of collection. The average arrival time of all samples has been 19.7 hours after collection. Thus, CB collection anywhere in North America (as well as most places worldwide) can be easily accomplished with overnight transport to the cord blood bank. For all samples, the average range is 70 to 80 labmedicine.com CE Update cc in size, regardless of the birthing situation (vaginal or surgical deliveries; Table 2). Cord blood collections from twin and triplet births are smaller, as expected (as the newborns are generally smaller), but are routinely large enough for clinical use (Table 3). Further, as more than 99% of all samples are sterile upon testing, the collection method invariably allows for a CB collection free of microbial and fungal contamination (as assessed by MacConkey blood agar plate cultures and by an automated BacT Alert system, bioMérieux). Finally, analysis of these collections for clinical utility revealed that approximately 10% of all collections did not meet the laboratory’s volume criteria for sample acceptance (ie, greater than 30 cc); however, volume is not an accurate measure of clinical utility as will be discussed later, as a high yield of stem cells can be obtained from a relatively low volume of blood (Table 4). Thus, all collections, regardless of size, are processed, frozen, and banked as the majority will be clinically useful for both transplant and regenerative medicine applications. 12.6 × 106 nucleated cells/cc of cord blood collected and processed. This number of cells is more than 4 times the minimum number of CB cells that has been successfully used for clinical transplant. Thus, the failure rate of cord blood collection based on cell numbers is less than 4% (Table 4). As CD34+ cells constitute approximately 1% of CB mononuclear cells,23 an average of 6 × 106 CD34+ cells are obtained from a single CB sample (an average of 5 × 104 CD34+ cells/kg patient body weight is routinely used for transplant), implying that the average CB sample contains enough cells to transplant both a child as well as a full-sized adult recipient. Samples of all sizes and obtained from all types of birthing/ delivery modes can be easily processed with these methodologies with high recoveries (Table 1). As expected, samples obtained from multiple births provide smaller numbers of cells after processing (due to the initial smaller starting volumes) but are well above minimum thresholds needed for clinical use. Processing does not introduce a significant risk of contamination when Processing of Cord Blood Presently, the vast majority of CB collections are red blood cell (RBC) reduced prior to cryopreservation. Several methods are in use to accomplish this goal, including Hespan sedimentation to obtain a modified buffy coat,12 density gradient centrifugation to obtain enriched mononuclear cells (MNC),28 and 2 automated processes (Sepax and automated processing platform [AXP])34,35 that result in a buffy coat product. The Hespan, Sepax, and AXP processing methods result in cord blood products composed of all nucleated cell populations found in the original collection (MNC, neutrophils, some RBCs), while the Ficoll method enriches for the stem-cell-containing MNC subpopulation (generally greater than 85% MNC with a few contaminating neutrophils and nucleated RBC). Cell counts obtained in the final Ficoll product are generally half the cell counts found in the other processes for this reason, although the stem cell recovery may be similar. Our facility has extensive experience with the use of density gradient centrifugation and more recently with the automated AXP process. Both methods reproducibly recover greater than 95% of the cord blood stem cells in a typical collection and result in a reduced final volume of approximately 20 cc for final storage (Figure 2). The latter method (AXP) allows for greater throughput with fixed personnel numbers (increasing the economy of operations) and is an FDA-cleared, functionally closed system (which is recommended under the current regulatory guidelines36) and is capable of processing cord blood collections of any volume. Red blood cell reduction not only facilitates the banking procedure, but it also helps to eliminate concerns about ABO or Rh incompatibility between donor and recipient upon clinical use of the sample. Further, much higher recoveries, as well as higher cell viabilities, are obtained upon thawing such RBC-depleted products. Also, by performing RBC reduction, the sample is amenable to immediate use in gene therapy, cell expansion, as well as storage in multiple aliquots for later multiple uses. Using either the Ficoll or AXP method, we routinely recover more than 95% of the original mononuclear cell population in the sample, as well as more than 95% of the CD34+ cells and colony-forming units granulocyte-macrophage (CFU-GM) progenitors (both surrogate measures for stem cell recovery). Upon analysis of the last 15,320 samples processed by the AXP method (the preferred method for the reasons stated above), we obtained a final product encompassing 882 × 106 total nucleated cells, or labmedicine.com Table 2_Comparative Cellular Harvests by Delivery Route Type of Collection N Volume (cc) MNC MNC/cc Vaginal C-section 124,057 67,258 71 72 574 528 8.1 7.3 Familial cord blood collections were analyzed according to type of birthing situation: vaginal deliveries versus surgical deliveries (C-section). Volume is presented as cc of blood minus anticoagulant. MNC and MNC/cc are presented as x106. No significant differences were observed between groups. Data is the compilation of products obtained with both Ficoll and AXP processing methods. MNC, mononuclear cells. Table 3_Effect of Multiple Births on Cord Blood Banking Cellular Harvest Sample N Volume (cc) MNC Singletons 157,623 71 512 Twins 8,090 53 335 Triplets 507 36 193 Quadruplets 24 37 204 MNC/cc 7.3 6.3 5.4 5.5 Familial cord blood collections were analyzed by number of infants delivered for volume (minus anticoagulant, presented in cc), total MNC post-processing (x106), and MNC/cc blood post-processing (x106). Singleton collections were significantly different from all other collections in terms of volume and total MNC obtained (P<0.05). Twins and triplets collections were not significantly different from one another. The quadruplet collection was significantly different from twins and triplets collections (P<0.05) in terms of volume and total MNC obtained. The MNC/cc was not significantly different between any of the groups. Data is the compilation of products obtained with both Ficoll and AXP processing methods. MNC, mononuclear cells. Table 4_Samples Below Predicted Clinically Usable Limits Number of samples below Number of samples below 30 cc: 19,802 out of 190,000 (10.4%) 1 x 108 TNC: 6,890 out of 190,000 (3.6%) Familial cord blood collections were analyzed for estimated clinical sufficiency as determined by 2 criteria: absolute volume of blood obtained and total numbers of TNC obtained post-processing. The numbers of samples below the indicated threshold levels are the percent failure rates for the collections according to each criterion. TNC, total nucleated cells. March 2008 j Volume 39 Number 3 j LABMEDICINE 175 CE Update Figure 2_Comparison of processing methods. A comparison of the most commonly-used cord blood processing methods. The percentages are the MNC/CD34 recovery rates after processing. TNC, total nucleated cells; MNC, mononuclear cells; AXP, automated processing platform. performed properly, as shown by an overall microbial contamination rate due to processing of <1%. During the past 14 years we have provided 61 cord blood samples for use in transplant to 26 different transplant centers (Figure 3). Although most of our early uses were for typical transplants (ie, to treat malignant and inherited blood disorders), recently increased use for regenerative medicine applications has occurred. Notably, when the samples were thawed at the respective transplant centers, an average post-thaw viability of 90% was obtained, validating the banking methodology. Significantly, a sample almost 10 years in storage was thawed with a 99% recovery, indicating that the samples can probably be stored indefinitely. Cryopreservation of Cord Blood Cord blood samples are cryopreserved using an automated, microprocessor-controlled cell freezer. Briefly, CB cells are resuspended in ice-cold autologous plasma at densities up to 300 × 106 cells/mL. An equal volume of cryopreservative solution containing autologous plasma and the cryoprotectant dimethylsufoxide (DMSO) is added slowly over the course of approximately 20 minutes. The cryopreservation protocol uses a controlled-rate freezing process to slowly reduce the temperature to –180°C. At the end of the freezing procedure the cells are stored in a specially constructed liquid nitrogen freezer (MVE, Inc, Laguna Beach, CA) that allows for vapor storage at liquid nitrogen temperatures.37 Autologous plasma is used for cryopreservation to avoid exposure to non-self and/or animal proteins (and the inherent infectious disease risks associated with such use). A controlled-rate freezer, rather than other methods, such as methanol immersion, was chosen so that each and every sample frozen would have a controlled and documented cryopreservation run, ideal for meeting regulatory guidelines. Furthermore, this approach produces better results upon thawing as our own experience documents (>90% cell recovery and viability as determined by independent transplant centers using the samples for transplant). Vapor phase storage prevents cross-sample contamination. Certain viruses, such as hepatitis and papilloma viruses, are known to survive exposure to liquid nitrogen and have been 176 LABMEDICINE j Volume 39 Number 3 j March 2008 shown to cross-contaminate bags of bone marrow during liquid nitrogen storage.38 If samples are to be banked for indefinite periods of time then this aspect becomes a major concern, and efforts must be made to minimize if not eliminate this potential problem. We have chosen to cryopreserve our CB samples in multiple aliquots for several reasons. Multiple aliquots allow for future use of the stem cells in cell expansion, in gene therapy, or for regenerative medicine uses that may only require a fraction of the frozen sample. Thus, it is not necessary to thaw the entire sample unless absolutely needed, avoiding the damaging effects of repeated episodes of freezing/thawing. Multiple aliquots also allow for repeated testing of the sample if needed to resolve any issues of misidentification or sample potency. Banking of Cord Blood Our CB samples are stored in the vapor phase of the largest liquid nitrogen Dewars commercially available (MVE, Inc, Figure 3_Categories of cord blood stem cell use. An illustration of the types of applications for cord blood samples banked by the Cord Blood Registry. Data is shown as the percentage of uses for each application along with the absolute numbers of samples. labmedicine.com CE Update model 1830). Even in the absence of continuous liquid nitrogen availability, these devices will maintain their –196°C temperatures for more than 1 week. Other facilities may use liquid phase liquid nitrogen storage, but then cross-contamination becomes an issue unless other measures are implemented (eg, storage container overwraps composed of impermeable materials). As a matter of precaution (and to meet regulatory requirements), the Dewars are hard-piped to a 9,000-gallon liquid nitrogen container on-site at the facility, with the liquid nitrogen container connected via telemetry directly to the liquid nitrogen supplier, eliminating the concern of running out of liquid nitrogen. Each Dewar is monitored continuously for liquid nitrogen levels and Dewar temperature. There are multiple backup and fail-safe systems in place, including the immediate availability of spare Dewars. The entire facility is alarmed and monitored, and it requires 2 individuals with separate access codes to access the Dewars. Overall, the bank is extremely secure against any unforeseen events. Conclusions As described above, the collection, processing, and banking of CB for immediate or future clinical use can be reproducibly performed with the proper methodology. It is important to note that all procedures used in the cord blood banking endeavor described herein have met and passed regulatory scrutiny (including AABB accreditation). Such regulatory compliance comes at a price but is essential in providing the assurance to clients and the transplant physician that each sample is banked under optimal conditions and will continue to be in optimal condition years later, if needed. Although many individuals elect to bank cord blood for its potential use in the treatment of cancer and genetic blood disorders, more and more clients are banking upon uses that are only now coming to light. Such uses include autologous stem-cell transplants for the treatment of autoimmune diseases, stem-cell applications in tissue engineering and regenerative medicine, and stem-cell gene therapy (Tables 2 and 3). Already, stem cells have been shown to be able to repair damaged heart and nervous tissues, to treat type 1 diabetes, to give rise to liver and corneas, to treat children born with neurological defects, and to cure severe combined immunodeficiency disease via gene therapy. In the next decade, there will undoubtedly be additional uses that are not yet anticipated. If so, then the odds of use for a family-banked CB sample will increase from 3 in 1,000 for the treatment of cancer, to upwards of 1 in 5 for use in regenerative medicine (Figure 4).39 As cord blood stem cells come Figure 5_The Cord Blood Registry Facility. Top panel: the CBR facility from the outside. Bottom panel: the cord blood storage facility. to be realized as the next-best alternative to embryonic stem cells, then the routine banking of CB stem cells for use by the family or newborn should become a routine and standard part of the birthing process. LM Acknowledgments: I would like to acknowledge the invaluable technical assistance of the Cord Blood Bank personnel that have made this study possible, particularly Heather Brown, Jody Gorton, and Aaron McGaffey. I would also like to acknowledge the numerous physicians, midwives, and nurses that have participated in the collection of the cord blood samples. 1. Hows JM. Histocompatible unrelated donors for bone marrow transplantation. Bone Marrow Transplant. 1987;1:259–263. 2. Bradley BA, Gilks WR, Gore SM, et al. How many HLA typed volunteer donors for bone marrow transplantation (BMT) are needed to provide an effective service? Bone Marrow Transplant. 1987;2:79. 3. Sullivan KM, Weiden PL, Storb R, et al. Influence of acute and chronic graftversus-host disease on relapse and survival after bone marrow transplantation from HLA-identical siblings as treatment of acute and chronic leukemia. Blood. 1989;73:1720–1728. 4. Broxmeyer HE, Gluckman E, Auerbach A, et al. Human umbilical cord blood: A clinically useful source of transplantable hematopoietic stem/ progenitor cells. Intl J Cell Cloning. 1990;8:76. 5. Gluckman E, Broxmeyer HE, Auerbach A, et al. Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical cord blood from an HLA-identical sibling. N Eng J Med. 1989;321:1174–1178. 6. Gluckman E. Stem cell harvesting from cord blood: A new perspective. In: Peripheral Blood Stem Cell Autografts. Ed. Henon and Wunder, Springer Verlag, 1990. 7. Broxmeyer HE, Kurtzburg J, Gluckman E, et al. Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation: An expanded role for cord blood transplantation. Blood Cells. 1991;17:330–337. Figure 4_Comparison of stem cell sources. A comparison of the characteristics of cord blood, adult, and embryonic stem cell sources. labmedicine.com 8. Broxmeyer HE, Kurtzburg J, Gluckman E, et al. Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation. Blood Cells. 1991;17:313–330. March 2008 j Volume 39 Number 3 j LABMEDICINE 177 CE Update 9. Broxmeyer HE, Douglas GW, Hangoc G, et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA. 1989;86:3828–3832. 20. Sunkomat JNE, Goldman S, Harris DT, et al. Cord blood-derived MNCs delivered intracoronary contribute differently to vascularization compared to CD34+ cells in the rat model of acute ischemia. Stem Cells. 2007; In press. 10. Vilmer E, Sterkers G, Rahimy C, et al. HLA-mismatched cord blood transplantation in a patient with advanced leukemia. Transplantation. 1992; 53:1155–1157. 21. Harris DT, Badowski M, Ahmad N, et al. The potential of cord blood stem cells for use in regenerative medicine. Expert Opinion on Biological Therapy. 2007;7:1311–1322. 11. Wagner JE, Kernan NA, Steinbuch M, et al. Allogeneic sibling umbilical cord blood transplantation in children with malignant and nonmalignant disease. Lancet. 1995;346:214–219. 22. Harris DT, Rogers I. Umbilical cord blood: A unique source of pluripotent stem cells for regenerative medicine. Current Stem Cell Research & Therapy. 2007; In press. 12. Rubinstein P, Rosenfield RE, Adamson JW, et al. Stored placental blood for unrelated bone marrow reconstitution. Blood. 1993;81:1679–1690. 23. Harris DT, Schumacher MJ, LoCascio J, et al. Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes. Proc Natl Acad Sci USA. 1992;89:10006–10010. 13. Gluckman E, Rocha V, Boyer-Chammard A. Outcome of cord-blood transplantation from related and unrelated donors. N Eng J Med. 1997;337: 373–381. 14. Rubinstein P. Why cord blood? Hum Immunol. 2006;67:398–404. 24. Harris DT, Schumacher MJ, LoCascio J, et al. Immunoreactivity of umbilical cord blood and post-partum maternal peripheral blood with regard to HLAhaploidentical transplantation. Bone Marrow Transplant. 1994;14:63–68. 15. McGuckin C, Forraz N, Baradez MO, et al. Production of stem cells with embryonic characteristics from human umbilical cord blood. Cell Prolif. 2005; 38:245–255. 25. Harris DT, LoCascio J, Besencon FJ. Analysis of the alloreactive capacity of human umbilical cord blood: Implications for graft-versus-host disease. Bone Marrow Transplant. 1994;14:545–553. 16. McGuckin CP, Forraz N, Allouard Q, et al. Umbilical cord blood stem cells can expand hematopoietic and neuroglial progenitors in vitro. Exp Cell Res. 2004;295:350–359. 26. Harris DT. In vitro and in vivo assessment of the graft-versus-leukemia activity of cord blood. Bone Marrow Transplant. 1995;15:17–23. 17. Rogers I, Yamanaka N, Bielecki R, et al. Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation. Exp Cell Res. 2007;313:1839–1852. 18. Kucia M, Halasa M, Wysoczynski M, et al. Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human umbilical cord blood-preliminary report. Leukemia. 2007;21:297–303. 19. Harris DT, He X, Badowski M, et al. Regenerative Medicine of the Eye: A Short Review. Stem Cell Repair & Regeneration, Vol. 3, Levicar N, Habib NA, Dimarakis I, Gordon MY (Eds.), Imperial College Press (2007), In press. 27. Harris DT. GVL and GVHD Implications of cord blood. Proceedings of the International Conference/Workshop on Cord Blood Transplantation and Biology/Immunology; Blood Cells. 1994;20:560–565. 28. Harris DT, Schumacher MJ, Rychlik S, et al. Collection, separation and cryopreservation of umbilical cord blood for use in transplantation. Bone Marrow Transplant. 1994;13:135–143. 29. Harris DT. What Every Physician Needs to Know About Cord Blood Banking, Round-Up (Maricopa County Medical Society News), December, 1994. 30. Harris DT. Experience in autologous and allogeneic cord blood banking. J Hematotherapy. 1996;5:123–128. 31. Harris DT. Cord blood banking for transplantation. The Canadian Journal of Clinical Medicine-Medical Scope Monthly. 1997;4:1–8. 32. Harris DT. Cord blood banking. The University of Arizona experience: Successes, problems and cautions. Cancer Research Therapy and Control. 1998;7:63–67. 33. Kielpinski G, Prinzi S, Duguid J, et al. Roadmap to approval: Use of an automated sterility test method as a lot release test for Carticel, autologous cultured chondrocytes. Cytotherapy. 2005;7:531–541. 34. Papassavas AC, Goika V, Chatzistamatiou T, et al. A strategy of splitting individual high volume cord blood units into two half subunits prior to processing increases the recovery of cells and facilitates ex vivo expansion of the infused hematopoietic progenitor cells in adults. Intl J Lab Hemat. 2007 (epub). 35. Harris DT, McGaffey AP, Schwarz RH, et al. Comparing the mononuclear cell (MNC) recovery of AXP and Hespan. Obstet Gynecol. 2007;109:93S. 36. Standards for Cellular Therapy Product Services, 2nd Ed. AABB Press; 2007. 37. Lane TA, Plunkett M, Buenviaje J, et al. Recovery of leukocytes in cord blood units after cryopreservation by controlled rate freeze in DMSO and storage in vapor phase liquid nitrogen. Poster, ISCT Conference, 2002. 38. Harris DT, Mapother M, Goodman C. Prevention of cross-sample and infectious contamination during cord blood banking by use of cryovials for storage in liquid nitrogen. Transfusion. 2000;40:111S. 39. 2020: A new vision—A future for regenerative medicine. Available at http:// www.dhhs.gov/reference/newfuture.shtml. Accessed on 12/04/2007. t 178 LABMEDICINE j Volume 39 Number 3 j March 2008 labmedicine.com