Cord Blood Banking. The Birth of a New Field

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1 FEATURE Karen Titus Cord Blood Banking The Birth of a New Field iven the great availability of cord blood every new birth represents a possible source it's little wonder this blood product has drawn intense interest from patients and physicians. Cord blood, collected from the placenta and umbilical cord, is rich in stem cells and may be an effective, less costly alternative to bone marrow transplants for treating diseases ranging from leukemia and lymphoma to sickle cell anemia. G This is a rapidly evolving field. In many ways it's more exciting than anything we've seen in a long time. Sandy Mulligan, MT(ASCP) As research continues to yield encouraging results with cord blood transplants, laboratory professionals will need to stay abreast of the information flow as well. Already a handful of public and private blood banks specialize in collecting, processing, and storing cord blood, and the number is sure to grow as collections increase. Indeed, the National Heart, Lung, and Blood Institute recently authorized a $30 million, five-year study designed to add approximately 15,000 units of cord blood to the current public inventory. Also under development is a worldwide cord blood registry for matching donated units and potential recipients. "This is a rapidly evolving field. In many ways it's more exciting than anything we've seen in a long time," observes Sandy Mulligan, MT(ASCP), a former blood banker and currently manager of the cord blood business unit at Pall Medsep (Covina, Calif), which manufactures a sterile collection and storage system for processing cord blood stem cells. "In a way, medical technologists may feel like they're working on the frontier, because it's all so new." LABORATORY MEDICINE VOLUME 29, NUMBER 2 As with any frontier, the emerging cord blood field is fraught with potential trouble as well as great promise. Cord blood transplantation remains an experimental technology, with many questions still to be answered about its effectiveness and methods of use. It also raises a host of ethical issues and controversies. "The field is constantly evolving," says David T. Harris, PhD, director of the cord blood bank and professor of immunology at the University of Arizona in Tucson. "We're moving forward, but there's still much work that needs to be done." Biological Basics Among the basic challenges is unlocking the biological secrets of cord blood. Cord blood first became the object of broad attention nearly 10 years ago, when a 5-year-old boy with Fanconi's anemia, a rare and potentially fatal hereditary blood disorder whose sufferers are at high risk for leukemia, was successfully transplanted with cord blood harvested from his newborn sister. Despite nearly 600 transplants since then a number that is growing at a rate of about 30 a month, according to some estimates the intricacies of cord blood transplantation remain an enticing puzzle for researchers. In several important ways, cord blood stem cells are similar to stem cells gleaned from bone marrow and peripheral blood. Also known as hematopoietic progenitor cells, stem cells can replicate and differentiate into any type of blood cell, including erythrocytes, leukocytes, and megakaryocytes. Stem cells can be a valuable tool in treating numerous disorders. In many kinds of cancer, for example, chemotherapy or radiation treatments destroy not only the cancer but the patient's marrow, wiping out the immune system in the process. Transplanting healthy stem cells, however, enables the patient's hematopoietic system bone marrow to produce healthy cells. In other cases, an individual may be born with defective bone marrow, whose diseased stem cells need to

2 be replaced with healthy ones. Among the diseases shown to be effectively treated by cord blood transplants are a number of leukemias, including acute lymphocytic leukemia, acute myelogenous leukemia, chronic myelocytic leukemia, and juvenile myelogenous leukemia; neuroblastoma; sickle cell anemia; thalassemia; congenital cytopenia; Wiskott-Aldrich syndrome; and aplastic anemia. For many years the most common source of stem cells has been bone marrow. In some cases, physicians perform autologous bone marrow transplants, removing the patient's own bone marrow, storing it during chemotherapy, then replacing it after treatment is completed. Allogeneic transplants, on the other hand, involve using bone marrow from a donor rather than the patient. Often this donor is a sibling whose bone marrow demonstrates highly compatible or even identical HLA types. Such close HLA matches reduce the chance that the bone marrow recipient will reject the transplanted marrow. In other cases, a match may be found from an unrelated donor through registries such as the National Marrow Donor Program. Though the successes of autologous and allogeneic transplants are impressive, the procedure is far from ideal. Because a patient's bone marrow may contain residual tumor cells, for example, or stem cells of poor quality, autologous transplants are not always a viable option. Transplants from unrelated bone marrow, on the other hand, have an increased risk of graft- vs-host disease, in which a donor's T lymphocytes recognize the recipient's tissues as foreign and then attack the recipient's cells. And despite the nearly 3 million donors registered with the National Marrow Donor Program, searches for an appropriate match are not always successful; even when they are, the match could take months a delay that may prove fatal to sicker patients. Finally, bone marrow extraction is costly and can be an arduous surgical procedure for donors. Peripheral blood stem cells basically are obtained from the bone marrow as well. Explains Mulligan: "Before harvesting peripheral blood stem cells, you administer a regimen of cytokines, often combined with other drugs, designed to flush stem cells in the bone marrow out into the peripheral blood." The stem cells are then harvested from the peripheral blood using the pheresis technique. As with bone marrow transplants, success with using peripheral blood stem cells depends largely on how closely the HLA antigenic makeup of the donor matches that of the recipient. That match appears to be less important when cord blood is used as the source of stem cells. "If you boil [the issue of HLA matching] down," says Mulligan, "it seems that umbilical cord stem cells may be more immature than those harvested from bone marrow or peripheral blood. So the match may not have to be quite as close." Researchers theorize that immature cells are less prone to demonstrating immunological features that could be rejected by the recipient's body. The first major question is, is cord blood a good source for bone marrow transplantation? Rebecca Haley, M D U n a n s w e r e d Questions Just how close a match is needed remains to be determined. It's just one of several unanswered research questions that need to be answered, according to Rebecca Haley, MD, senior medical officer for the American Red Cross Biomedical Services in Rosslyn, Va. "The first major question is, is cord blood a good source for bone marrow transplantation? More specifically, are there enough progenitor cells to repopulate a marrow? That's an important question," says Haley. For small children, she continues, using cord blood appears to be a "slam dunk." Generally there seems to be no problem repopulating the VOLUME 29, NUMBER 2 c o! About 25 ml of blood is drawn from the clamped and cut umbilical cord. The collection usually takes about 5 minutes. LABORATORY MEDICINE 91

3 marrow of patients weighing less than 40 kilograms, she says. "When you get into larger children and adults, however, there's some question that there may not be enough cells to repopulate the marrow." If you have a lot of w h i t e cells and a small patient, you're in good shape. Richard M o l d w i n, M D Most of the success to date has been in pediatric patients. Researchers are now studying the size of cell dose measured in number of cells per kilogram body weight of the recipient and how it relates to engraftment success. "With cord blood you're dealing with a limited amount of material" stem cells are rare to begin with, and cord blood volumes, by their very nature, are small "so if you have a fixed number of cells and a larger individual receiving them, then the [dose in terms of] cells per kilogram [in the recipient] is going to be less," Mulligan explains. According to Richard Moldwin, MD, PhD, medical director of the Chicago Community Christopher Goodman, MS, associate laboratory director at Cord Blood Registry, San Bruno, Calif, places samples of cord blood in a liquid nitrogen freeze tank. LABORATORY MEDICINE VOLUME 29, NUMBER 2 Cord Blood Bankin Offers Advantages... Cord blood stem cells are more abundant than stem cells obtained from bone marrow/peripheral blood. Cord blood transplantation costs less than bone marrow transplantation. Donating cord blood compared with donating bone marrow is less arduous for the donor. HLA match of cord blood stem cells may not need to be as close as that needed for bone marrow; there appears to be a reduced incidence of graft-vshost disease with unrelated cord blood transplantation compared with unrelated bone marrow transplantation. Cord blood stem cells may hold a potential for other clinical applications, including gene therapy. Cord Blood Bank, located at the University of Chicago Children's Hospital, the current best predictor of engraftment success seems to be the total nucleated cell count present in the original collection. "If you have a lot of white cells and a small patient, you're in good shape," says Moldwin, a pediatric hematologist and assistant professor of pediatrics at the University of Chicago. He notes that although the approximate number has been 107 nucleated white cells per kilogram, recent studies suggest that this cell dose may result in a problem with engraftment. "So what you probably [need in the collection] is 4 X 107 nucleated cells per kilogram, which makes it much more difficult [to obtain a dose adequate] to engraft a larger person." Because of the probable importance of the size of the cell dose, researchers are studying ways to expand populations of cord blood stem cells by manipulating cell division in vitro. Mere growth will not be enough, however. "Not only do you want the cells to divide, but they must maintain their ability to be naive to be able to differentiate down whatever cell line the body needs," Mulligan explains. "The key is to get them to expand while still remaining immature. You don't want to push them into the process of commitment to a particular cell line prior to transplantation." Another question, of course, is how much HLA disparity is acceptable. "This is what everyone wants to know," says Haley. While initial

4 ...But Some Questions and Problems Remain. Cord blood volumes are small, which makes stem cell processing difficult.. Success of cord blood transplantation appears to be limited by the size of the recipient larger patients (weighing more than 40 kg) may require larger cell doses that currently are not available. The acceptable level of HLA disparity for cord blood stem cell transplantation is not known.. Long-term viability of stored cord blood remains to be seen. Regulatory oversight issues are still evolving, as are optimum collection, processing, storage, and reconstitution techniques.. The medical history is not sufficient for infant donors, who may later develop a disease transmittable through the donated unit stem cells. In cases where the cord blood has not been banked for private use, linking the identity of the donor with his or her cord blood may compromise privacy. The expense of private donation and storage may not be covered by insurance. indications appear to be strong that less-thanperfect HLA matches are fairly well tolerated by cord blood recipients, Haley suggests that the picture is far from clear. "If you're transplanting cord blood from one family member to another, and you transplant the noninherited maternal [HLA] allele, then the children receiving it seem to tolerate it extremely well. [However], if you transplant the noninherited paternal allele, then the likelihood of having problems with... graft-vs-host disease appears to be greater," says Haley. On a practical level, cord blood banks also are still working out the best ways to collect, process, and store this product (see "Just How Difficult Is It To Process Umbilical Cord Blood?"), as well as address regulatory issues. Before any regulatory oversight existed and before there was much scientific interest in the field, Harris says, the methodologies were rather loose in terms of documentation. "But they have evolved, more or less, over the last five years, to the point where I think everyone is aware that they need to have in place the necessary... documentation, standard operating procedures, [and] validation of lab methods." If you're banking cord blood for your family, the issues of identity are very different than if you were donating it to a bank for use by someone else. Jeremy Sugarman, M D, M P H, M A A Tangled Web Beyond the basic scientific and blood-handling issues lies a thicket of social and ethical dilemmas, not to mention the occasionally testy debate about whether private or public cord blood banks best serve patients and the medical community. Of great concern to many is protecting the identities of cord blood donors. This is particularly important for public cord blood banks, or any program where the intent is to donate the cord blood for use by someone other than a family member. Private banks, on the other hand, collect and store cord blood for the purpose of making the blood available to the newborn or a family member; thus, ensuring linkage between the newborn and the cord blood is of primary importance at private banks. "If you're banking cord blood for your family, the issues of identity are very different than if you were donating it to a bank for use by someone else," says Jeremy Sugarman, MD, MPH, MA, associate professor of medicine at Duke University (Durham, NC) and chair of the Working Group VOLUME 29, NUMBER 2 I After processing a newborn's cord blood sample, Marie B. McGrath, Viacord's director of operations, inspects a research freezer where cord blood cells are stored at -196'C. LABORATORY MEDICINE 93

5 on Ethical Issues in Umbilical Cord Blood Banking, whose members include blood banks, physicians, ethicists, legal experts, and social scientists. "Banking for related use is different from donating for public use, and each brings up a different set of practices and questions." Several factors complicate the issue of privacy in cord blood donation. Because the donor is an infant, not enough medical history exists to help determine the likelihood that the donated unit is free of infection. A donor eventually may develop a genetic or infectious disease that is transmittable through the donated unit stem cells but not identifiable at time of birth. Linkage between the donor's identity and his or her cord blood may ensure greater blood safety but at the risk of compromising privacy. Banking for related use is different from donating for public use, and each brings up a different set of practices and questions. Jeremy Sugarman, MD, MPH, MA Maintaining linkages also opens up the possibility that donors whose cord blood is well-matched with the recipient will be targeted with a request to donate bone marrow as well, to be used, for example, to supplement a small cord blood collection in the bank. Informed consent is yet another concern. Ideally, says Sugarman, expectant parents should be informed about the opportunity to donate or bank cord blood well before delivery. What they should be or are being told by those responsible for harvesting the blood, however, has been the source of debate. Just How Difficult Is It To Process Umbilical Cord Blood? "There's nothing magical about it," says J. Mario Alonso, MT(ASCP), of the St Louis (Mo) Cord Blood Bank. "Cord blood is essentially just a new source for stem cells, and processing it involves basically the same techniques used in processing bone marrow and peripheral blood stem cells. If you've worked in any cryopreservation lab at all, you can handle cord blood," he adds. "Those with good laboratory training in either processing blood or... processing stem cells, can do it without spending a lot of time having to be trained," adds David T Harris, PhD, director of the cord blood bank at the University of Arizona in Tucson. Medical technologists may be involved in processing cord blood at any step of the way, from collection to freezing to reconstitution. Here's a quick overview of some common methods of handling cord blood, based on the suggestions and experiences of sources interviewed for the accompanying article. (Consult "Further Reading" for articles providing more detailed information about processing.) Collection Collection methods vary considerably, but regardless of which method is used, collections are done after delivery of the infant and ligation of the cord and usually while the placenta is still in utero. Among the methods for collecting blood are: Umbilical venipuncture using anticoagulated syringes Cannulating the umbilical vein with a standard blood donor set Hanging the placenta (after it has been expelled) on a special apparatus and collecting the blood using a standard blood donor set Cutting the distal end of the umbilical cord and draining the blood into a sterile vessel At the Chicago Community Cord Blood Bank, reports its medical director, Richard Moldwin, MD, PhD, collectors use a two-syringe technique. One syringe is equipped with a piece of reusable extension tubing and check valve on one end and a needle on the other. Collectors aspirate placental blood through the needle and extension tubing into the syringe. "Then you cap the first syringe and attach the second syringe to the extension tubing," he says. Processing and Testing After it is collected, the stem cells need to be concentrated. "Space is at a premium. With a cord blood bank, you're going to need to store [many units] in order to have a usable blood bank," notes Alonso. Ten years ago, recalls Harris, cord blood banks often simply froze the unprocessed samples. While this simple method is less costly and is still in use in some facilities, he says, the disadvantages outweigh the advantages. "You can't store as many samples in a liquid nitrogen container as you could if you processed them. And it becomes more problematic when you try to thaw the samples and use them, because all the red cells and granulocytes lyse after thawing." Processing is aimed at ridding the sample of red blood cells and granulocytes and can be done through a variety of sedimentation techniques, using starches and centrifugation. Which method is best? 94 LABORATORY MEDICINE VOLUME 29, NUMBER 2 on 29 November 2017

6 "It depends on what you're good at," Harris offers. "What we found is if you have a method you prefer, and you are good at it and can document and validate your procedures, you should use it." While medical technologists may be familiar with the basic techniques of processing, Sandy Mulligan, MT(ASCP), a former blood banker now working for Pall Medsep (Covina, Calif), a manufacturer of a cord blood processing system, inserts a word of caution. "We're working with such small volumes of cells about 25 cc's of fluid at the time of cryopreservation. So you need to be careful to guard every one of those precious cells." Among the tests that may be performed on cord blood units are HLA typing, cell counts, CD34 analysis, and colony-forming units. Units also are cultured for bacterial contamination. Some banks store backup samples so that additional assays, such as genetic tests, can be performed on cord blood units as they become available. The mother must be tested for infectious diseases as well, including sexually transmitted diseases; some centers also test the infant. Freezing and Reconstitution Under currently accepted methods, cord blood is cryopreserved using a 10% dimethyl sulfoxide (DMSO) cryoprotectant, with cells commonly resuspended in autologous plasma prior to freezing. Units can be frozen using a controlled-rate freezing approach, such as automated, computercontrolled cell freezers, and stored in a liquid nitrogen freezer in a liquid phase. Other centers forgo controlled-rate freezing, eliminating the need for using the enormous quantities of liquid nitrogen this approach requires. Some suggest performing an immediate post-thaw wash with a solution of dextran and albumin to remove the DMSO and plasma. Doing so reduces the possibility of transfusing DMSO, donor plasma, or both into the patient. Furthermore, some note, failing to remove the cryoprotectant reduces the viability of leukocyte and stem cells. Many decry what they say are misleading statements and questionable marketing techniques of some commercial cord blood banks. Such statements suggest, for example, that a child's cord blood may be used in the future by that child should he or she develop a life-threatening illness such as cancer. "Private companies try to sell [cord blood banking] to parents as biological insurance," says J. Mario Alonso, MT(ASCP), of St Louis Cord Blood Bank, a public bank affiliated with Cardinal Glennon Children's Hospital/Saint Louis University. "But it's not that simple. If your kid comes down with acute lymphocytic leukemia, for instance, you're not going to want that same, diseased [source of stem cells] back. If anything, you'd want to use stem cells from an unrelated donor." "Certainly no one has ever proven that [storing cord blood] will protect your child for life," adds the Red Cross's Haley. "But that's what some [private] companies are implying. They're suggesting cord blood can be used in ways that simply have not been shown to be true, such as to help treat a mother's breast or ovarian cancer. I'm not saying it won't be shown to be true down the road, but it's not fair to make such claims now." Patricia Parker, senior laboratory technician, performs one of 25 processing steps performed at the Cord Blood Registry, San Bruno, Calif. An additional 42 tests are performed for each cord blood sample to ensure viability, sterility, absence of infectious diseases, and maximum cell yield. VOLUME 29. NUMBER 2 LABORATORY MEDICINE u I (A

7 "Parents are extremely vulnerable to these messages," she adds. "Who doesn't want the best for their [children]? But even though cord blood is a highly promising source of stem cells, that needs to be balanced out by what we actually know to be true right now." In fact, note some observers, the Federal Trade Commission appears to be cracking down hard on some private cord blood banking enterprises that it considers to be misrepresenting the possible benefits of donation. We need to have a concerted effort b e t w e e n the public and private banks, versus fighting between the t w o sides which, unfortunately, happens quite often. David T. Harris, PhD Not surprisingly, private companies voice a different opinion. Says Cynthia Fisher, president and chief executive officer of Viacord, a private, Boston-based cord blood banking service: "The intent of the literature Viacord provides to expectant parents and physicians is to fully educate them about cord blood banking.... This is a growing field of medical technology, and researchers are investigating further applications for cord blood. It is important that families be aware that there is a great deal of promising research currently underway." The distinction between public and private banks is not always clear-cut. The public Chicago Community Cord Blood Bank, for example, which chiefly stores units donated for non-family use, permits private storage when it seems warranted. "If there is a sibling with leukemia and the mom is having a baby, for example, we will let the family store the cord blood free of charge here until we determine whether it's a match," says Moldwin. If it is, the blood bank continues to store the unit until it is needed; if it's not a match, the unit becomes part of the random donor bank. Most acknowledge public and private cord blood banks do not need to be at odds, however, and that both have a role to play. MEDICINE VOLUME 29, NUMBER 2 on 299 6November LABORATORY 2017 "I think we need to have a concerted effort between the public and private banks, versus fighting between the two sides which, unfortunately, happens quite often," says Harris. Concurs Viacord's Fisher: "There's no need for a rift between the two sides. It's not a matter of one replacing the other. Each can learn from the other, and each offers a different service." More important than any turf battle, says Harris, is the need to expand the current size of the donor pool, with an eye toward increasing ethnic diversity. A wide demographic range of donors will help improve availability of well-matched units for recipients of all races. "We need, I think, to make a concerted effort to attract donors to serve that patient pool, and to figure out just how big the banks need to be to serve a wide patient population." And beyond these major issues? "Eventually we'll be looking at cutting-edge therapies, perhaps using cord blood [in treating] metastatic cancers and in gene therapy," Harris says. "The field is wide open." Karen Titus is a freelance writer in Chicago. Further Reading Flake AW, Zanjani ED. In utero hematopoietic stem cell transplantion. JAMA. 1997;278: Gluckman E, Vanderson R, Boyer-Chammard A, et al. Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med. 1997;337: Haley R. Cord blood banking. Immunohematology. 1997;13: Harris DT. Cord blood banking for transplantation. Can J Clin Med. 1997;4:1-8. Kohli-Kumar M, Shahidi NT, Broxmeyer HE, et al. Haemopoietic stem/progenitor cell transplant in Fanconi anaemia using HLA-matched sibling umbilical cord blood cells. Br J Haematol. 1993;85: Menitove J, ed. Standards for Hematopoietic Progenitor Cells. Bethesda, Md: American Association of Blood Banks; Pelehach L. The story of the stem cell. Lab Med. 1996;27: Rubinstein P, Dobrila L, Rosenfield RE, et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci. 1995;92: Sugarman J, Kaalund V, Kodish E, et al. Ethical issues in umbilical cord blood banking. JAMA. 1997;278: Wagner JE, Kernan NA, Steinbuch M, et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet. 1995;346: