Prenatal identification of potential donors for umbilical cord blood transplantation for Fanconi anemia A.D. AUERBACH, Q.
Liu,
R. GHOSH,M.S. POLLACK,G.W. DOUGLAS,AND H.E. BROXMEYER
Reported here are studies of Fanconi anemia fetal cells that led to the first use of umbilical cord blood for hematopoietic reconstitution in a clinical trial. Prenatal diagnosis and HLA typing were performed in fetuses at risk for Fanconi anemia (FA) to identify, prior to birth, those that were unaffected with the syndrome and were HLA-identical to affected siblings. Umbilical cord blood was harvested at the delivery of these infants; assays of progenitor cells indicated the presence of colony-forming units-granulocyte-macrophage (CFU-GM) in numbers similar to those of bone marrow CFU-GM that are associated with successful engraftment in HLA-matched allogeneic bone marrow transplantation. The possibility that umbilical cord blood from a single individual can be used as an alternative to bone marrow for hematopoietic reconstitution has now been demonstrated by the successful engraftment of two patients with FA. Progenitor cell assays of umbilical cord blood collected at the birth of a child affected with FA, who had been misdiagnosed on the basis of chorionic villus sampling (CVS) studies, indicated a profound deficiency in colony formation, consistent with previously reported abnormalities in the growth of FA cells in vitro. These results suggest that the hematopoietic disorder in FA is related to an underlying problem with cell proliferation. TRANSFUSION 1990;30:682-687.
HUMANHEMATOPOIESIS IS SUSTAINED by a pool of stem and progenitor cells.' In vitro colony assays have demonstrated the presence of multipotential cells with replating ability, which are termed stem cells, as well as multipotential colony-forming units-granulocyteerythroid-macrophage-megakaryocyte(CFU-GEMM), burst-forming units-erythroid (BFU-E), and colonyforming units-granulocyte-macrophage (CFU-GM) progenitor cells in bone marrow, adult peripheral blood, and umbilical cord blood of humans.'-7 The possibility of using umbilical cord blood in place of bone marrow as a source of transplantable cells for the treatment of patients with certain hematopoietic diseases is raised by two circumstances. The first is the presence of hema-
topoietic stem or progenitor cells that are detectable in vitro in cord blood in frequencies (number of colonies/ number of cells plated) equal to or exceeding the fre~ - ' second is the presquency in adult bone m a r r o ~ . ~ .The ence in cord blood of CFU-GM in absolute numbers7 that are in the range of those in adult bone marrow that are associated with successful engraftment in autologous and HLA-matched allogeneic bone marrow transplantation.8-'0To test whether umbilical cord blood could be used as a source of stem or progenitor cells for transplantation, one would first need to harvest the cord blood at the birth of an individual known before birth to be histocompatible with a sibling with bone marrow failure and not to be affected with hematopoietic disease. Patients with Fanconi anemia (FA) provide such an opportunity. FA, a rare autosomal recessive syndrome, is characterized clinically by progressive pancytopenia, diverse congenital abnormalities, and increased predisposition to FA patients show a marked decrease in colony growth of bone marrow CFU-GM, colony-forming units-erthroid (CFU-E), and BFU-E in the preanemic,14 anemic,14-17and leukemic'*~'9phases of the syndrome. Although the molecular basis for the disease is unknown, hypersensitivity to the clastogenic effect of DNA-crosslinking agents such as diepoxybutane (DEB) Our provides a unique marker for the FA studies have shown that FA can be diagnosed prenatally by the study of cells obtained by chorionic villus sampling (CVS) or a m n i o ~ e n t e s i s , ~and ~ - ~that ~ these cell types can be tested by modified serologic techniques for HLA class I and classes I and I1 allotypes,
From the Laboratory for Investigativc Dermatology, The Rockefeller University, New York, Ncw York; the Histocompatibility Laboratory, The Methodist Hospital and Baylor College of Medicine, Houston, Texas; the Department of Obstetrics and Gynecology, New York University, New York; and the Departments of Medicine, Microbiology, and Immunology and the Walther Oncology Center, Indiana University School of Mcdicine, Indianapolis, Indiana. Supported in part by Public Health Service Grants Nos. HL 32987 (ADA), CA36464, and CA36740 (HEB), and CA40552 (MSP) from the National Institutes of Health; Clinical Research Grant No. 6-521 from the March of Dimes Birth Defects Foundation (ADA); General Clinical Rcscarch Ccnter Grant No. RR00102 from the National Institutes of Health to The Rockefeller University Hospital; The Pew Memorial Trust to the Laboratory for Investigative Dermatology; and Biocyte Corporation, New York, NY (HEB). Presented in part at thc Scientific Symposium at the 42nd Annual Meeting of the American Association of Blood Banks, New Orleans, Louisiana, October 1989. Received for publication January 4, 1990; revision received February 13, 1990, and acceptcd March 5, 1990.
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Individuals affected with FA usually die of hemorrhage, infection, or acute leukemia. Bone marrow transplantation offers the possibility of correction of the stem cell defect in this disease, but patients frequently lack a suitable marrow donor. Parents of children affected with FA often elect to have additional children, because of the 1-in-4 chance that a subsequent child will be HLAidentical to the affected sibling and thus will be a potential donor for bone marrow transplantation. Caring for a child affected with FA can be a considerable burden to families, and parents usually request prenatal monitoring of subsequent fetuses to rule out the presence of the syndrome. We report here studies in which the ability to determine prenatally whether fetuses monitored for FA were HLA-identical to affected siblings is demonstrated. In vitro assays of hematopoietic progenitor cells in umbilical cord blood obtained at birth in these cases indicated high numbers in unaffected individuals and a deficiency in the number of progenitor cells in a child affected with FA. These studies suggested that the ability to harvest umbilical cord blood at the birth of individuals who are known before birth to be unaffected with FA, karyotypically normal, and HLA identical to an affected sibling presented an opportunity to perform a cord blood transplant in certain cases of FA. This has led to the successful hematopoietic reconstitution of two FA patients with umbilical cord blood as the source of transplantable stem or progenitor cellsz7 (also, Gluckman E, Broxmeyer HE, Auerbach AD, et al., unpublished observations).
Materials and Methods
683
Cytogenetic studies Chorionic villus and amniotic fluid cell cultures were initiated in the prenatal diagnosis laboratory of the medical center at which the tissue was obtained. Two primary cultures from each patient were sent at ambient temperature by overnight carrier to The Rockefeller University Cytogenetics Laboratory. Details of the method used for prenatal diagnosis of FA by DEB testing have been We subcultured primary CVS or amniotic fluid cell cultures at a 1:3 ratio and plated them in Chang medium (Hana Biologics, Inc., Alameda, CA) at 37°C in an atmosphere of 5 percent CO,. After 24 hours, we added DEB (0.01 &mL) (Aldrich Chemical Co, Milwaukee, WI) to culture medium of treated cultures; we used untreated cultures for baseline breakage studies. After 72 hours, cells were subcultured at a 1:3 ratio into medium without DEB. We harvested cells after 24 to 48 hours and performed chromosome breakage studies. Umbilical cord blood samples obtained as described above were cultured for cytogenetic studies by a method previously used for peripheral b100d.2n~21 In summary, the culture unit consisted of 0.4 mL of heparinized blood added to 10 mL of medium (RPMI-1640, GIBCO, Grand Island, NY)supplemented with 15 percent fetal bovine serum (Hazelton Research Products, Denver, PA), 1 percent ~ - g l u tamine, 1 percent penicillin-streptomycin solution (GIBCO), and 1 percent phytohemagglutinin (PHA, Wellcome Diagnostics, Dartford, UK) and incubated for 72 or 96 hours at 37°C in a 5 percent CO, atmosphere at high humidity. We added DEB, at a final concentration in the medium of 0.1 pg per mL, to the treated cultures 24 hours after their initiation. Culture harvest and chromosome preparations follow standard methods. We analyzed chromosomal breakage in 100 Giemsa-stained metaphases from each preparation. To avoid bias in cell selection, we selected for study consecutive metaphases that appeared intact with sufficiently well defined chromosome morphology. Each cell was scored for chromosome number and for the numbers and types of structural abnormalities. We scored as gaps achromatic areas less than a chromatid in width and scored as rearrangements exchange configurations, translocations, and dicentric and ring chromosomes. Gaps were excluded from the calculations of chromosome breakage frequencies, and rearrangements were scored as two breaks.
Patients We identified potential subject families through an index of patients who showed clinical features of FA, including pancytopenia and various congenital malformations, as well as cellular hypersensitivity to the clastogenic effect of DEB. Chorionic villi, amniotic fluid, and cord blood were obtained by obstetricians at various medical centers, with the informed consent of patients. Cord blood was collected at the birth of histocompatible siblings of patients affected with FA as follows. Immediately after delivery, the cord was doubly clamped 5 to 7 cm from the infant umbilicus and transected between the clamps. After removal of the infant from the surgical field, the cord clamp was released to allow a free flow of blood from the placenta, which was facilitated by uterine contractions. Blood was also obtained from the removed placenta by needle aspiration of exposed, engorged vessels on the fetal surface. This was done without delay to avoid clotting and with care to avoid maternal blood contamination. Samples were collected into sterile wide-mouth glass bottles (Corning, Medfield, MA) containing ACD, 20 mL (Sigma Chemical Co., St Louis, MO) and penicillin (0.03 mghL)-streptomycin (0.05 mg/mL, Sigma) and sent to The Rockefeller University and the Indiana University School of Medicine for analysis.
HLA typing studies HLA class I typing was performed on subcultured cells sent to The Methodist Hospital's Histocompatibility Laboratory by a modification of methods for CVS or amniotic fluid cell typing described p r e v i ~ u s l y . ~We ~ . ~incubated ~ cells in flasks with 1000 U per mL human gamma interferon for 2 to 3 days, briefly trypsinized them, and adjusted the volume to approximately lo6 cells per mL. We added 1 JLL(1000 cells) to each well of several preplated typing trays containing 1 JLLof serum per well, which collectively defined all well-established HLAA,B,C specificities. After 45 minutes' incubation at room temperature, cells were incubated for an additional 45 minutes with 5 p L of rabbit complement per well. We simultaneously visualized living and complement-killed cells after staining them with a mixture of equal parts of fluorescein diacetate and ethidium bromide. We made antigen assignments by observing the staining patterns within 10 minutes via an inverted fluorescence microscope. In positive wells (complement-killed cells), membrane damage allowed most of the nuclei to become stained orange-red by the ethidium bromide; in negative wells (living cells), the cytoplasm was stained green by the fluorescein, which was retained in the cells.
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In vitro progenitor cell studies For CFU-GM assays, cord blood cells were plated in 0.3 percent agar culture medium in the presence of 10 percent (vol/ vol) medium conditioned by the human cell line 5637 (5637CM, a source of colony-stimulating factors) as described elsewhere.28 We counted colonies (>40 cells/aggregate) and clusters (3-40 cells/aggregate) containing granulocytes and/or macrophages after 14 days of incubation (5% C02, 5% 02, humidified conditions). We also plated cells to assay colonies deriving from CFU-GM, BFU-E, and CFU-GEMM in methylcellulose culture medium in the presence of 1 unit of erythropoietin (Toyoba New York, Inc., New York, NY)and 10 percent (vol/vol) 5637CM, as described elsewhere.29 Colonies forming in methylcellulose were also scored after 14 days of incubation under the same conditions as agar cultures. All cultures were set up to ensure optimal conditions for colony formation and detection of hematopoietic progenitor cells.
Results
Cytogenetic studies Chromosomal breakage studies to rule out a diagnosis of FA and HLA typing studies to determine whether the fetus was HLA-identical to an affected sibling were performed on 11 fetuses at risk for the syndrome. We did not include in this study 8 fetuses diagnosed as being unaffected with FA (prenatal HLA studies were not performed) or 5 fetuses diagnosed prenatally as being affected with FA. We studied 9 of the 11 fetuses by CVS; in 5 of these, we performed amniocentesis to confirm the diagnosis. We diagnosed the other 2 fetuses on the basis of amniocentesis only. Fetuses 3 and 4 made up a twin pregnancy. Results of prenatal diagnosis studies are shown in Table 1. In the fetuses studied by CVS, the baseline chromosomal breakage in the untreated cultures ranged from 0 to 0.35 breaks per cell (mean, 0.13), and that in cultures treated with DEB ranged from 0.02 to 0.29 breaks per cell (mean, 0.13). The ranges for normal control cultures obtained for advanced maternal age and cultures from affected fetuses were 0.02 to 0.10 and 0.30 to 0.46, respectively, in baseline studies and 0.02 to 0.14 and 1.0 to 1.4, respectively, in DEB-treated cultures.22
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The baseline breakage frequency in Fetus 3 was thus within the range found in cells from affected fetuses, but there was no significant increase in breakage frequency after exposure of the cells to DEB. We were unable to obtain amniotic fluid cells for retesting of this fetus, and intrauterine death at approximately 18 weeks' gestation prevented any follow-up for this diagnosis. Although the frequency of chromosomal breakage in some of the other cases was higher than that found in normal control cultures, analysis of both baseline and DEBtreated values indicated that these fetuses were not affected with FA. In amniotic fluid cell cultures, baseline chromosomal breakage in untreated cultures ranged from 0 to 0.19 breaks per cell (mean, 0.09) and that in cultures treated with DEB ranged from 0.01 to 0.18 breaks per cell (mean, 0.08). The r q e s for normal control cultures and cultures from affected fetuses were 0 to 0.02 and 0.18 to 0.45, respectively, in baseline studies, and 0 to 0.06 and 0.69 to 0.96, respectively, in DEB-treated cultures.23 In cases where amniotic fluid cells were tested in order to confirm previous results from CVS studies, the chromosomal breakage frequencies were similar. The results of chromosomal breakage studies of umbilical cord blood lymphocytes from newborn patients (Table 2) indicated that one fetus had been misdiagnosed as normal on the basis of CVS alone (Fetus 4, Table 1). Baseline and DEBinduced chromosomal breakage frequencies in cord blood and in two samples of peripheral blood from this individual are consistent with a diagnosis of FA. This child, who is now 2 years old, showed intrauterine as well as postnatal growth retardation. The other newborn infants listed in Table 2 are all phenotypically normal.
HLA typing studies The results of HLA studies showed that five fetuses were class I HLA-identical to affected siblings, five were matched for one haplotype, and one was matched for neither haplotype (Table 1). HLA studies appear to rule out maternal cell contamination as a problem in all of the CVS cultures, including that from Fetus 4, an affected individual who did not exhibit chromosomal breakage prenatally. In the future, class I1 identity will be verified for all potential class I-identical siblings
Table 1 . Prenatal diagnosis and H L A testing in fetuses at risk for Fanconi anemia (FA) Fetus number 1
2 36 4§
5
6 7 8
Gender F F M
F F F F
Mean chromosome breaks per cell by CVS* Baseline DEB$ 0.04 0.19 0.03 0.10 0.35 0.00
0.08
Mean chromosome breaks per cell by A F t Baseline DEBS
0.27
0.02 0.07
0.03 0.00 0.08 0.17 0.19 0.1 1 0.08
0.03 0.03 0.01 0.08 0.13 0.18 0.08
0.29 0.1 0 9 M 0.04 0.13 10 M 0.19 0.14 11 M 0.10 0.29 Control range 0.02-0.10 0.02-0.1422 0.00-0.02 0.00-0.0623 FA range 0.30-0.46 1 .0-1.422 0.1 8-0.45 0.69-0.9623 Chorionic villus sampling. t Amniotic fluid. $: Cells were exposed to 0.01 kg per mL of diepoxybutane (DEB) for 72 hours. 5 Fetuses 3 and 4 were from a twin pregnancy. M
Prenatal diagnosis Normal Normal Unknown Normal Normal Normal Normal Normal Normal Normal Normal
HLA
match +/-/+/+
+/+ +I+ +/+ +/+/-
+I+ +/+/-
Pregnancy outcome Elective abortion Live birth Fetal death Live birth Live birth Live birth Live birth Live birth Live birth Live birth Live birth
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Table 2. Frequency of chromosomal breakage in umbilical cord blood Infant number 2 4 5
Gender F
6, these parameters were only approximately twice those in Infant 4.
Mean chromosome breaks per cell Baseline DEB'
0.00 0.04 8.2 0.30 0.02 0.02 0.02 6 0.00 0.02 7 0.00 0.00 8 0.00 0.16 9' 0.06 0.00 0.02 11 Control ranget 0.00-0.05 0.00-0.10 FA ranget 0.02-0.80 1.06-23.9 Cells were exposed to 0.1 kg per mL of diepoxybutane (DEB) for 48 to 72 hrs. t FA = Fanconi anemia: from ref. 20. F F F F M M M
Table 3. Total nucleated cellularity and numbers of CFUGM, * BFU-E,t and CFU-GEMMS progenitor cells in unfractionated human umbilical cord blood Case numbers Parameters evaluated 4 5 6 Blood collected (mL) 99 150 160 Nucleated cells (No. x lo8) 5.42 12.7 11.9 Hematopoietic progenitor cells (No. x lo5) Agar culture 0.22 1.8 1.5 CFU-GM' (colonies) 5.3 2.5 CFU-GM (colonies + 0.73 clusters) Methylcellulose culture 3.8 1.6 CFU-GM (colonies) 0.14 4.0 BFU-Et (colonies) 0.03 16.5 0.8 0.4 CFU-GEMMS 0.01 (colonies) Colony-forming units-granulocyte-macrophage. t Burst-forming units-erythroid.
*
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CORD BLOOD TRANSPLANT FOR FANCONI ANEMIA
9 282 46.7
21.9 49.3 108.5 38.1 12.8
CFU-granulocyte-erythroid-rnacrophage-megakaryocyte.
through now-standard typing of fetal DNA using oligomer probes.3n
In vitro progenitor cell studies The results of assays for progenitor cells in cord blood samples collected at the birth of infants identified prenatally as HLA-identical to siblings affected with FA are shown in Table 3. These bloods were collected so as to optimize the volume obtained; blood was also recovered from the placentas after delivery of the babies. Numbers of CFU-GM, BFU-E, and CFU-GEMM scored for Infants 5 , 6, and 9 span the range previously reported for normal umbilical cord blood samples.' We found much lower numbers of progenitor cells for the various lineages in the cord blood sample from Infant 4, who was diagnosed after birth as being affected with FA. The numbers of CFU-GM (colonies, agar), CFU-GM (colonies clusters, agar), CFU-GM (colonies, methylcellulose), BFU-E (colonies, methylcellulose), and CFU-GEMM (colonies, methylcellulose) scored for Infants 5 , 6, and 9 were, respectively, 7 to 100, 3 to 67, 11 to 775, 133 to 1270, and 40 to 1280 times greater than those scored for Infant 4. These differences are not merely a reflection of the volume of cord blood or of the numbers of nucleated cells collected, as in Infants 5 and
+
Discussion
We report the prenatal identification of three individuals who are unaffected with FA, karyotypically normal, and class I HLA-identical to their affected siblings. Prenatal testing has enabled us to collect umbilical cord blood in optimal volumes at the birth of these individuals. Progenitor cell numbers in the samples of cord blood obtained, as determined by in vitro assays, were within the reported range associated with successful allogeneic bone marrow transplants. Engraftment of two FA patients using umbilical cord blood described here (Infants 5 and 6 ) has now been achievedz7 (Gluckman E, Broxmeyer HE, Auerbach AD, et al. unpublished observations). The diagnosis of FA in patients with some of the clinical features of the syndrome was based on the unique hypersensitivity of cells from affected individuals to the clastogenic effect of DEB.20-24As the risk for FA in each pregnancy is high (1 in 4) and the risk of the CVS procedure appears to be we generally recommend a first-trimester prenatal diagnosis. The occurrence of a false-negative result in CVS studies of one fetus from a twin pregnancy at risk for FA remains to be explained. HLA studies of cultured trophoblast cells from this female fetus appeared to rule out maternal cell contamination, as only a single maternal haplotype was detected; the only antigens identified were identical to those of an affected sibling. Nevertheless, it is possible that the flask of cells used for HLA typing contained fetal cells, while those included in the cytogenetic study were maternal cells. As the chromosomal instability usually associated with FA was not detected in cells obtained during the first trimester of this pregnancy, and as we have not had any false-negative or false-positive diagnoses in our more extensive experience with amniocentesis for FA prenatal diagnosis,23 we recommend that a confirmatory amniocentesis be performed in cases diagnosed as normal by
cvs.
Hematopoietic progenitor cells (colony-forming unitsculture [CFU-C], CFU-E, and BFU-E) have been reported to be markedly decreased or absent in cultures of bone marrow cells from patients with FA,14-17 even in individuals who were not anemic at the time of study.14 The results of our study indicate a deficiency of progenitor cells for various lineages in cord blood obtained at the birth of an affected child, which suggests that the aplastic anemia that usually develops during the first decade of life in children with FA results from an intrinsic stem-cell disorder present at birth. A peripheral blood count obtained when this child was 2 months of age showed a hemoglobin level of 13.2 g per dL, a
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hematocrit of 37.2, and a white cell count of 10.8 x lo9 per L with a differential of 38 percent granulocytes, 59 percent lymphocytes, 3 percent monocytes, and 434 x lo9 per L platelets; the mean corpuscular volume was 99.0. The normal peripheral blood counts suggest that the deficiency in the colony-forming ability of FA bone marrows may be due to a profound inability of the progenitor cells to proliferate. FA fibroblasts and lymphocytes have been shown to exhibit poor growth in vitro, particularly at ambient oxygen tension rather than hypoxic (5% vol/vol oxygen) culture condition^.^^-^^ This defective growth has been shown to result specifically from a prolongation of, and arrest in, the G2 phase of the cell cycle. That the bone marrow failure in FA is not due to an abnormality in the bone marrow microenvironment is demonstrated by the successful engraftment of bone marrow transplanted to FA patients. A long-term actuarial survival rate of 70 percent has been reported37 when a modified conditioning regimen with a reduction in the dose of cyclophosphamide was used to lessen toxicity to this DNA-crosslinking agent in hypersensitive FA patients. The results of a recent study7 suggested that umbilical cord blood from a single donor could serve as a source of transplantable, hematopoietic, repopulating cells. In the present study, the numbers of progenitor cells collected from cord blood at the birth of normal siblings of FA patients is within the range noted by others8-10to be associated with successful engraftment in bone marrow transplantation. The advantages of using umbilical cord blood for transplantation, instead of using a small infant as a bone marrow donor, include avoidance of the risks of anesthesia and other potential complications associated with bone marrow donation. The successful use of umbilical cord blood in the treatment of FA prompts us to suggest that these methods will have application to other diseases treatable by bone marrow transplantation, including other genetic disorders such as thalassemia, severe combined immunodeficiency, and the mucopolysaccharidoses and nonconstitutional disorders such as idiopathic aplastic anemia and leukemia. The prenatal identification of a healthy HLA-identical sibling and the collection of umbilical cord blood at birth could lead to the treatment of such patients by cord blood transplantation. In addition, we suggest that umbilical cord blood, a material that is usually discarded at the birth of an infant, might be used to establish a bank of cryopreserved cells for unrelated allogeneic transplantation. It may be possible also to use cryopreserved umbilical cord blood for autologous transplantation. Further studies are needed to determine whether umbilical cord blood will have wider applicability as suggested here. A concern expressed by o t h e r P is the possibility of contamination of cord blood by maternal
Vol. 30, No. 8-1990
cells. Our initial laboratory investigation using I- and iantigen analysis did not detect any such contamination, but further tests are in progress. Questions regarding the immunoreactivity of cord blood also should be addressed. Although neonatal blood is considered to be as immunocompetent as adult blood in regard to the generation of cytotoxic T lymphocytes, and thus to have the potential for causing graft-versus-host disease (GVHD),39 the two FA patients who have received a transplantation of umbilical cord blood have had less difficulty with GVHD than is usually seen in these patients. A potential concern is whether there will be enough stem or progenitor cells from a single cord blood collection to repopulate the hematopoietic system of an adult. In this regard, we have collected samples of cord blood that contain 5 to 22 times the number of progenitor cells present in the collections used for successful engraftment in the two patients with FA. In another case, we collected cord blood that contained progenitor cells in numbers that exceeded those used in the first clinical transplant, even though the number of nucleated cells was less than one-half that used in the transplant. In conclusion, we have demonstrated a successful clinical application for the usually discarded umbilical cord blood, but questions regarding the broad applicability of this material as a source of transplantable hematopoietic cells must await further study. Acknowledgment The authors wish to thank Cynthia Callaway, Gloria Grant, and Sarra Sorkin for expert technical assistance.
References 1. Broxmeyer HE, Williams DE. The production of mycloid blood cells and their regulation during health and discase. Crit Rev Oncol Hematol 1988;8:173-226. 2. Nakahata T, Ogawa M. Hemopoictic colony-forming cells in umbilical cord blood with extensive capability to gcnerate monoand multipotential hemopoietic progenitors. J Clin Invest 1982;70:1324-8. 3. Broxmeyer HE. Colony assays of hernatopoietic progenitor cclls and correlations to clinical situations. Crit Rcv Oncol Hematol 1984; 1227-57. 4. Leary AG, Ogawa M. Blast cell colony assay for umbilical cord blood and adult bone marrow progenitors. Blood 1987;69:953-6. 5. Rowley SD, Sharkis SJ, Hattenburg C, Senscnbrcnner LL. Culture from human bone marrow of blast progenitor cells with an extensive proliferative capacity. Blood 1987;69:804-8. 6. Brandt J, Baird N, Lu L, Srour E, Hoffman R. Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro. J Clin Invcst 1988;82:1017-27. 7. Broxmeyer HE, Douglas GW, Hangoc G, et al. Human umbilical cord blood as a potential source of transplantablc hematopoictic stem/progenitor cclls. Proc Natl Acad Sci USA 1989;86:3828-32. 8. Douay L, Gorin NC, Mary JY, et al. Recovery of CFU-GM from cryopreserved marrow and in vivo evaluation after autologous bone marrow transplantation are prcdictive of engraftment. Exp Hematol 1986;14:358-65. 9. Faille A, Maraninchi D, Gluckman E, et al. Granulocyte progcnitor compartments after allogcneic bonc marrow grafts. Scand J Haematol 1981;26:202-14.
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10. Spitzcr G, Vcrma DS, Fishcr R, et al. The myeloid progenitor ccll-its valuc in prcdicting hcmatopoictic recovery after autologous bone marrow transplantation. Blood 1980;55:317-23. 11. Fanconi G. Familial constitutional panmyelocytopathy, Fanconi’s ancmia (F.A.). I. Clinical aspects. Semin Hematol 1967;4:23340. 12. Schroeder TM, Tilgcn D, Kruger J, Vogcl F. Formal genetics of Fanconi’s ancmia. Hum Gcnef 1976;32:257-88. 13. Swift M. Fanconi’s anaemia in thc gcncfics of neoplasia. Nature 1971;230:370-3. 14. Daneshbod-Skibba G, Martin J, Shahidi NT. Myeloid and erythroid colony growth in non-anaemic patients with Fanconi’s anaemia. Br J Haematol 1980;44:33-8. 15. Lui VK, Ragab AH, Findley HS, Fraucn BJ. Bonc marrow cultures in children with Fanconi anemia and the TAR syndrome. J Pediatr 1977;91:952-4. 16. Saunders EF, Freedman MH. Constitutional aplastic anaemia: defective haematopoietic stem cell growth in vitro. Br J Haematol 1978;40:277-87. 17. Chu JY. Granulopoicsis in Fanconi’s aplastic anemia. Proc SOC Exp BioI Med 1979;161:609-12. 18. Prindull G, Jcntsch E, Hansmann I. Fanconi’s anaemia developing erythroleukaemia. Scand J Haematol 1979;23:59-63. 19. Aucrbach AD, Weiner MA, Warburton D, Yeboa K, Lu L, Broxmcycr HE. Acute myeloid leukemia as the first hematologic manifcstation of Fanconi anemia. Am J Hematol 1982;12:289-300. 20. Auerbach AD, Adler B, Chaganti RSK. Prenatal and postnatal diagnosis and carrier detection of Fanconi ancmia by a cytogenetic method. Pediatrics 1981;67:128-35. 21. Auerbach AD, Rogatko A, Schroeder-Kurth TM. International Fanconi Anemia Registry: relation of clinical symptoms to diepoxybutanc sensitivity. Blood 1989;73:391-6. 22. Aucrbach AD, Zhang M, Ghosh R, et al. Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi ancmia. Hum Genet 1986;73:86-8. 23. Auerbach AD, Sagi M, Adler B. Fanconi anemia: prenatal diagnosis in 30 fetuses at risk. Pediatrics 1985;76:794-800. 24. Aucrbach AD, Ghosh R, Pollio PC, Zhang M. Dicpoxybutane (DEB) test for prcnatal and postnatal diagnosis of Fanconi ancmia. In: Schroeder-Kurth TM, Auerbach AD, Obe G, eds. Fanconi anemia: clinical, cytogenetic, and expcrimental aspects. Heidelbcrg: Springcr-Vcrlag, 1989:71-82. 25. Callaway C, Falcon C, Grant G, ct al. HLA typing used with culturcd amniotic and chorionic villus cells for early prenatal diagnosis or parentage tcsting without one parent’s availability. Hum lmmunol 1986;16:200-4. 26. Maurer DH, Callaway C, Sorkin S, Pollack MS. Gamma interferon induces detectable serological and functional expression of DR and DP but not DQ antigcns on cultured amniotic fluid cells. Tissue Antigens 1988;31:174-82. 27. Gluckman E, Broxmeyer HE, 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. 28. Broxmeyer HE, Bognacki J, Ralph P, Dorner MH, Lu L, CastroMalaspina H. Monocyte-macrophage-derived acidic isoferritins: normal feedback regulators of granulocyte-macrophage progenitor cells in vitro. Blood 1982;60:595-607.
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29. Lu L, Walker D, Graham CD, Waheed A, Shadduck RK, Ralph P, Broxmeycr HE. Enhancement of release from MHC class II antigen-positive monocytes of hematopoictic colony stimulating factors CSF-1 and G-CSF by recombinant human tumor necrosis factor-alpha: synergism with recombinant human interfcrongamma. Blood 1988;72:34-41. 30. Pollack MS, Auerbach AD, Broxmeyer HE, Zaafran A, Erlich HA. The use of DNA amplification for DQ typing as an adjunct to serological prenatal HLA typing for the identificafion of potcntial donors for umbilical cord blood transplantation. Hum Immunol 1990; in press. 31. Rhoads GG, Jackson LG, Schlcsselman SE, et al. The safety and efficacy of chorionic villus sampling for early prenatal diagnosis of cytogenetic abnormalities. N Engl J Med 1989;320:609-17. 32. Weksberg R, Buchwald M, Sargcnt P, Thompson MW, Siminovitch L. Specific cellular defects in patients with Fanconi ancmia. J Cell Physiol 1979;101:311-23. 33. Dutrillaux B, Aurias A, Dutrillaux AM, Buriot D, Prieur M. The cell cycle of lymphocytes in Fanconi ancmia. Hum Genet 1982;62:327-32. 34. Kubbies M, Schindler D, Hochn H, Schinzel A, Rabinovitch PS. Endogenous blockage and delay of the chromosome cyclc dcspitc normal recruitment and growth phase explain poor prolifcralion and frequent endomitosis in Fanconi anemia cells. Am J Hum Genet 1985;37:1022-30. 35. Schindler D, Hoehn H. Fanconi anemia mutation causcs ccllular susceptibility to ambient oxygen. Am J Hum Genet 1988;43:42935. 36. Hoehn H, Kubbies M, Schindler D, Poot M, Rabinovitch PS. BrdU-Hoechst flow cytometry links the cell kinetic defect of Fanconi anemia to oxygen hypersensitivity. In: Schrocdcr-Kurth TM, Auerbach AD, Obe G, eds. Fanconi anemia: clinical, cytogcnctic, and experimental aspects. Heidelberg: Springcr-Verlag, 1989:16173. 37. Gluckman E, Devergie A, Dutrcix J. Bone marrow transplantation for Fanconi’s anemia. In: Schroeder-Kurth TM, Auerbach AD, Obc G, eds. Fanconi anemia: clinical, cytogenetic, and experimental aspects. Heidelberg: Springer-Verlag, 1989:60-8. 38. Linch DC, Brent L. Marrow transplanfations. Can cord blood be uscd? (ncws) Nature 1989;340:676. 39. Rayfield LS,Brent L, Rodeck CH. Development of cell-mediated lympholysis in human foetal blood lymphocytes. Clin Exp Immunol 1980;42:561-70. A.D. Auerbach, PhD, Laboratory for Invcstigativc Dcrmatology,
The Rockefeller Univcrsity, 1230 York Avcnue, Ncw York, NY 100216399. [Reprint requcsts] Q. Liu, MD, Laboratory for Investigative Dcrmatology, Thc Rockefeller University. R. Ghosh, MS, Laboratory for Investigativc Dcrmafology, Thc Rockefeller Univcrsity. M.S. Pollack, PhD, Histocompatibility Laboratory, The Mcthodisf Hospital and Baylor College of Medicine, Houston, TX. G.W. Douglas, MD, Department of Obstetrics and Gynecology, New York Univcrsity. H.E. Broxmeyer, PhD, Departments of Medicine. Microbiology, and Immunology, and the Walther Oncology Centcr, Indiana University School of Medicine, Indianapolis, IN.
Liu,
R. GHOSH,M.S. POLLACK,G.W. DOUGLAS,AND H.E. BROXMEYER
Reported here are studies of Fanconi anemia fetal cells that led to the first use of umbilical cord blood for hematopoietic reconstitution in a clinical trial. Prenatal diagnosis and HLA typing were performed in fetuses at risk for Fanconi anemia (FA) to identify, prior to birth, those that were unaffected with the syndrome and were HLA-identical to affected siblings. Umbilical cord blood was harvested at the delivery of these infants; assays of progenitor cells indicated the presence of colony-forming units-granulocyte-macrophage (CFU-GM) in numbers similar to those of bone marrow CFU-GM that are associated with successful engraftment in HLA-matched allogeneic bone marrow transplantation. The possibility that umbilical cord blood from a single individual can be used as an alternative to bone marrow for hematopoietic reconstitution has now been demonstrated by the successful engraftment of two patients with FA. Progenitor cell assays of umbilical cord blood collected at the birth of a child affected with FA, who had been misdiagnosed on the basis of chorionic villus sampling (CVS) studies, indicated a profound deficiency in colony formation, consistent with previously reported abnormalities in the growth of FA cells in vitro. These results suggest that the hematopoietic disorder in FA is related to an underlying problem with cell proliferation. TRANSFUSION 1990;30:682-687.
HUMANHEMATOPOIESIS IS SUSTAINED by a pool of stem and progenitor cells.' In vitro colony assays have demonstrated the presence of multipotential cells with replating ability, which are termed stem cells, as well as multipotential colony-forming units-granulocyteerythroid-macrophage-megakaryocyte(CFU-GEMM), burst-forming units-erythroid (BFU-E), and colonyforming units-granulocyte-macrophage (CFU-GM) progenitor cells in bone marrow, adult peripheral blood, and umbilical cord blood of humans.'-7 The possibility of using umbilical cord blood in place of bone marrow as a source of transplantable cells for the treatment of patients with certain hematopoietic diseases is raised by two circumstances. The first is the presence of hema-
topoietic stem or progenitor cells that are detectable in vitro in cord blood in frequencies (number of colonies/ number of cells plated) equal to or exceeding the fre~ - ' second is the presquency in adult bone m a r r o ~ . ~ .The ence in cord blood of CFU-GM in absolute numbers7 that are in the range of those in adult bone marrow that are associated with successful engraftment in autologous and HLA-matched allogeneic bone marrow transplantation.8-'0To test whether umbilical cord blood could be used as a source of stem or progenitor cells for transplantation, one would first need to harvest the cord blood at the birth of an individual known before birth to be histocompatible with a sibling with bone marrow failure and not to be affected with hematopoietic disease. Patients with Fanconi anemia (FA) provide such an opportunity. FA, a rare autosomal recessive syndrome, is characterized clinically by progressive pancytopenia, diverse congenital abnormalities, and increased predisposition to FA patients show a marked decrease in colony growth of bone marrow CFU-GM, colony-forming units-erthroid (CFU-E), and BFU-E in the preanemic,14 anemic,14-17and leukemic'*~'9phases of the syndrome. Although the molecular basis for the disease is unknown, hypersensitivity to the clastogenic effect of DNA-crosslinking agents such as diepoxybutane (DEB) Our provides a unique marker for the FA studies have shown that FA can be diagnosed prenatally by the study of cells obtained by chorionic villus sampling (CVS) or a m n i o ~ e n t e s i s , ~and ~ - ~that ~ these cell types can be tested by modified serologic techniques for HLA class I and classes I and I1 allotypes,
From the Laboratory for Investigativc Dermatology, The Rockefeller University, New York, Ncw York; the Histocompatibility Laboratory, The Methodist Hospital and Baylor College of Medicine, Houston, Texas; the Department of Obstetrics and Gynecology, New York University, New York; and the Departments of Medicine, Microbiology, and Immunology and the Walther Oncology Center, Indiana University School of Mcdicine, Indianapolis, Indiana. Supported in part by Public Health Service Grants Nos. HL 32987 (ADA), CA36464, and CA36740 (HEB), and CA40552 (MSP) from the National Institutes of Health; Clinical Research Grant No. 6-521 from the March of Dimes Birth Defects Foundation (ADA); General Clinical Rcscarch Ccnter Grant No. RR00102 from the National Institutes of Health to The Rockefeller University Hospital; The Pew Memorial Trust to the Laboratory for Investigative Dermatology; and Biocyte Corporation, New York, NY (HEB). Presented in part at thc Scientific Symposium at the 42nd Annual Meeting of the American Association of Blood Banks, New Orleans, Louisiana, October 1989. Received for publication January 4, 1990; revision received February 13, 1990, and acceptcd March 5, 1990.
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CORD BLOOD TRANSPLANT FOR FANCONI ANEMIA
Individuals affected with FA usually die of hemorrhage, infection, or acute leukemia. Bone marrow transplantation offers the possibility of correction of the stem cell defect in this disease, but patients frequently lack a suitable marrow donor. Parents of children affected with FA often elect to have additional children, because of the 1-in-4 chance that a subsequent child will be HLAidentical to the affected sibling and thus will be a potential donor for bone marrow transplantation. Caring for a child affected with FA can be a considerable burden to families, and parents usually request prenatal monitoring of subsequent fetuses to rule out the presence of the syndrome. We report here studies in which the ability to determine prenatally whether fetuses monitored for FA were HLA-identical to affected siblings is demonstrated. In vitro assays of hematopoietic progenitor cells in umbilical cord blood obtained at birth in these cases indicated high numbers in unaffected individuals and a deficiency in the number of progenitor cells in a child affected with FA. These studies suggested that the ability to harvest umbilical cord blood at the birth of individuals who are known before birth to be unaffected with FA, karyotypically normal, and HLA identical to an affected sibling presented an opportunity to perform a cord blood transplant in certain cases of FA. This has led to the successful hematopoietic reconstitution of two FA patients with umbilical cord blood as the source of transplantable stem or progenitor cellsz7 (also, Gluckman E, Broxmeyer HE, Auerbach AD, et al., unpublished observations).
Materials and Methods
683
Cytogenetic studies Chorionic villus and amniotic fluid cell cultures were initiated in the prenatal diagnosis laboratory of the medical center at which the tissue was obtained. Two primary cultures from each patient were sent at ambient temperature by overnight carrier to The Rockefeller University Cytogenetics Laboratory. Details of the method used for prenatal diagnosis of FA by DEB testing have been We subcultured primary CVS or amniotic fluid cell cultures at a 1:3 ratio and plated them in Chang medium (Hana Biologics, Inc., Alameda, CA) at 37°C in an atmosphere of 5 percent CO,. After 24 hours, we added DEB (0.01 &mL) (Aldrich Chemical Co, Milwaukee, WI) to culture medium of treated cultures; we used untreated cultures for baseline breakage studies. After 72 hours, cells were subcultured at a 1:3 ratio into medium without DEB. We harvested cells after 24 to 48 hours and performed chromosome breakage studies. Umbilical cord blood samples obtained as described above were cultured for cytogenetic studies by a method previously used for peripheral b100d.2n~21 In summary, the culture unit consisted of 0.4 mL of heparinized blood added to 10 mL of medium (RPMI-1640, GIBCO, Grand Island, NY)supplemented with 15 percent fetal bovine serum (Hazelton Research Products, Denver, PA), 1 percent ~ - g l u tamine, 1 percent penicillin-streptomycin solution (GIBCO), and 1 percent phytohemagglutinin (PHA, Wellcome Diagnostics, Dartford, UK) and incubated for 72 or 96 hours at 37°C in a 5 percent CO, atmosphere at high humidity. We added DEB, at a final concentration in the medium of 0.1 pg per mL, to the treated cultures 24 hours after their initiation. Culture harvest and chromosome preparations follow standard methods. We analyzed chromosomal breakage in 100 Giemsa-stained metaphases from each preparation. To avoid bias in cell selection, we selected for study consecutive metaphases that appeared intact with sufficiently well defined chromosome morphology. Each cell was scored for chromosome number and for the numbers and types of structural abnormalities. We scored as gaps achromatic areas less than a chromatid in width and scored as rearrangements exchange configurations, translocations, and dicentric and ring chromosomes. Gaps were excluded from the calculations of chromosome breakage frequencies, and rearrangements were scored as two breaks.
Patients We identified potential subject families through an index of patients who showed clinical features of FA, including pancytopenia and various congenital malformations, as well as cellular hypersensitivity to the clastogenic effect of DEB. Chorionic villi, amniotic fluid, and cord blood were obtained by obstetricians at various medical centers, with the informed consent of patients. Cord blood was collected at the birth of histocompatible siblings of patients affected with FA as follows. Immediately after delivery, the cord was doubly clamped 5 to 7 cm from the infant umbilicus and transected between the clamps. After removal of the infant from the surgical field, the cord clamp was released to allow a free flow of blood from the placenta, which was facilitated by uterine contractions. Blood was also obtained from the removed placenta by needle aspiration of exposed, engorged vessels on the fetal surface. This was done without delay to avoid clotting and with care to avoid maternal blood contamination. Samples were collected into sterile wide-mouth glass bottles (Corning, Medfield, MA) containing ACD, 20 mL (Sigma Chemical Co., St Louis, MO) and penicillin (0.03 mghL)-streptomycin (0.05 mg/mL, Sigma) and sent to The Rockefeller University and the Indiana University School of Medicine for analysis.
HLA typing studies HLA class I typing was performed on subcultured cells sent to The Methodist Hospital's Histocompatibility Laboratory by a modification of methods for CVS or amniotic fluid cell typing described p r e v i ~ u s l y . ~We ~ . ~incubated ~ cells in flasks with 1000 U per mL human gamma interferon for 2 to 3 days, briefly trypsinized them, and adjusted the volume to approximately lo6 cells per mL. We added 1 JLL(1000 cells) to each well of several preplated typing trays containing 1 JLLof serum per well, which collectively defined all well-established HLAA,B,C specificities. After 45 minutes' incubation at room temperature, cells were incubated for an additional 45 minutes with 5 p L of rabbit complement per well. We simultaneously visualized living and complement-killed cells after staining them with a mixture of equal parts of fluorescein diacetate and ethidium bromide. We made antigen assignments by observing the staining patterns within 10 minutes via an inverted fluorescence microscope. In positive wells (complement-killed cells), membrane damage allowed most of the nuclei to become stained orange-red by the ethidium bromide; in negative wells (living cells), the cytoplasm was stained green by the fluorescein, which was retained in the cells.
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In vitro progenitor cell studies For CFU-GM assays, cord blood cells were plated in 0.3 percent agar culture medium in the presence of 10 percent (vol/ vol) medium conditioned by the human cell line 5637 (5637CM, a source of colony-stimulating factors) as described elsewhere.28 We counted colonies (>40 cells/aggregate) and clusters (3-40 cells/aggregate) containing granulocytes and/or macrophages after 14 days of incubation (5% C02, 5% 02, humidified conditions). We also plated cells to assay colonies deriving from CFU-GM, BFU-E, and CFU-GEMM in methylcellulose culture medium in the presence of 1 unit of erythropoietin (Toyoba New York, Inc., New York, NY)and 10 percent (vol/vol) 5637CM, as described elsewhere.29 Colonies forming in methylcellulose were also scored after 14 days of incubation under the same conditions as agar cultures. All cultures were set up to ensure optimal conditions for colony formation and detection of hematopoietic progenitor cells.
Results
Cytogenetic studies Chromosomal breakage studies to rule out a diagnosis of FA and HLA typing studies to determine whether the fetus was HLA-identical to an affected sibling were performed on 11 fetuses at risk for the syndrome. We did not include in this study 8 fetuses diagnosed as being unaffected with FA (prenatal HLA studies were not performed) or 5 fetuses diagnosed prenatally as being affected with FA. We studied 9 of the 11 fetuses by CVS; in 5 of these, we performed amniocentesis to confirm the diagnosis. We diagnosed the other 2 fetuses on the basis of amniocentesis only. Fetuses 3 and 4 made up a twin pregnancy. Results of prenatal diagnosis studies are shown in Table 1. In the fetuses studied by CVS, the baseline chromosomal breakage in the untreated cultures ranged from 0 to 0.35 breaks per cell (mean, 0.13), and that in cultures treated with DEB ranged from 0.02 to 0.29 breaks per cell (mean, 0.13). The ranges for normal control cultures obtained for advanced maternal age and cultures from affected fetuses were 0.02 to 0.10 and 0.30 to 0.46, respectively, in baseline studies and 0.02 to 0.14 and 1.0 to 1.4, respectively, in DEB-treated cultures.22
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The baseline breakage frequency in Fetus 3 was thus within the range found in cells from affected fetuses, but there was no significant increase in breakage frequency after exposure of the cells to DEB. We were unable to obtain amniotic fluid cells for retesting of this fetus, and intrauterine death at approximately 18 weeks' gestation prevented any follow-up for this diagnosis. Although the frequency of chromosomal breakage in some of the other cases was higher than that found in normal control cultures, analysis of both baseline and DEBtreated values indicated that these fetuses were not affected with FA. In amniotic fluid cell cultures, baseline chromosomal breakage in untreated cultures ranged from 0 to 0.19 breaks per cell (mean, 0.09) and that in cultures treated with DEB ranged from 0.01 to 0.18 breaks per cell (mean, 0.08). The r q e s for normal control cultures and cultures from affected fetuses were 0 to 0.02 and 0.18 to 0.45, respectively, in baseline studies, and 0 to 0.06 and 0.69 to 0.96, respectively, in DEB-treated cultures.23 In cases where amniotic fluid cells were tested in order to confirm previous results from CVS studies, the chromosomal breakage frequencies were similar. The results of chromosomal breakage studies of umbilical cord blood lymphocytes from newborn patients (Table 2) indicated that one fetus had been misdiagnosed as normal on the basis of CVS alone (Fetus 4, Table 1). Baseline and DEBinduced chromosomal breakage frequencies in cord blood and in two samples of peripheral blood from this individual are consistent with a diagnosis of FA. This child, who is now 2 years old, showed intrauterine as well as postnatal growth retardation. The other newborn infants listed in Table 2 are all phenotypically normal.
HLA typing studies The results of HLA studies showed that five fetuses were class I HLA-identical to affected siblings, five were matched for one haplotype, and one was matched for neither haplotype (Table 1). HLA studies appear to rule out maternal cell contamination as a problem in all of the CVS cultures, including that from Fetus 4, an affected individual who did not exhibit chromosomal breakage prenatally. In the future, class I1 identity will be verified for all potential class I-identical siblings
Table 1 . Prenatal diagnosis and H L A testing in fetuses at risk for Fanconi anemia (FA) Fetus number 1
2 36 4§
5
6 7 8
Gender F F M
F F F F
Mean chromosome breaks per cell by CVS* Baseline DEB$ 0.04 0.19 0.03 0.10 0.35 0.00
0.08
Mean chromosome breaks per cell by A F t Baseline DEBS
0.27
0.02 0.07
0.03 0.00 0.08 0.17 0.19 0.1 1 0.08
0.03 0.03 0.01 0.08 0.13 0.18 0.08
0.29 0.1 0 9 M 0.04 0.13 10 M 0.19 0.14 11 M 0.10 0.29 Control range 0.02-0.10 0.02-0.1422 0.00-0.02 0.00-0.0623 FA range 0.30-0.46 1 .0-1.422 0.1 8-0.45 0.69-0.9623 Chorionic villus sampling. t Amniotic fluid. $: Cells were exposed to 0.01 kg per mL of diepoxybutane (DEB) for 72 hours. 5 Fetuses 3 and 4 were from a twin pregnancy. M
Prenatal diagnosis Normal Normal Unknown Normal Normal Normal Normal Normal Normal Normal Normal
HLA
match +/-/+/+
+/+ +I+ +/+ +/+/-
+I+ +/+/-
Pregnancy outcome Elective abortion Live birth Fetal death Live birth Live birth Live birth Live birth Live birth Live birth Live birth Live birth
TRANSFUSION 1990-Vnl. 30. Nu. 8
Table 2. Frequency of chromosomal breakage in umbilical cord blood Infant number 2 4 5
Gender F
6, these parameters were only approximately twice those in Infant 4.
Mean chromosome breaks per cell Baseline DEB'
0.00 0.04 8.2 0.30 0.02 0.02 0.02 6 0.00 0.02 7 0.00 0.00 8 0.00 0.16 9' 0.06 0.00 0.02 11 Control ranget 0.00-0.05 0.00-0.10 FA ranget 0.02-0.80 1.06-23.9 Cells were exposed to 0.1 kg per mL of diepoxybutane (DEB) for 48 to 72 hrs. t FA = Fanconi anemia: from ref. 20. F F F F M M M
Table 3. Total nucleated cellularity and numbers of CFUGM, * BFU-E,t and CFU-GEMMS progenitor cells in unfractionated human umbilical cord blood Case numbers Parameters evaluated 4 5 6 Blood collected (mL) 99 150 160 Nucleated cells (No. x lo8) 5.42 12.7 11.9 Hematopoietic progenitor cells (No. x lo5) Agar culture 0.22 1.8 1.5 CFU-GM' (colonies) 5.3 2.5 CFU-GM (colonies + 0.73 clusters) Methylcellulose culture 3.8 1.6 CFU-GM (colonies) 0.14 4.0 BFU-Et (colonies) 0.03 16.5 0.8 0.4 CFU-GEMMS 0.01 (colonies) Colony-forming units-granulocyte-macrophage. t Burst-forming units-erythroid.
*
685
CORD BLOOD TRANSPLANT FOR FANCONI ANEMIA
9 282 46.7
21.9 49.3 108.5 38.1 12.8
CFU-granulocyte-erythroid-rnacrophage-megakaryocyte.
through now-standard typing of fetal DNA using oligomer probes.3n
In vitro progenitor cell studies The results of assays for progenitor cells in cord blood samples collected at the birth of infants identified prenatally as HLA-identical to siblings affected with FA are shown in Table 3. These bloods were collected so as to optimize the volume obtained; blood was also recovered from the placentas after delivery of the babies. Numbers of CFU-GM, BFU-E, and CFU-GEMM scored for Infants 5 , 6, and 9 span the range previously reported for normal umbilical cord blood samples.' We found much lower numbers of progenitor cells for the various lineages in the cord blood sample from Infant 4, who was diagnosed after birth as being affected with FA. The numbers of CFU-GM (colonies, agar), CFU-GM (colonies clusters, agar), CFU-GM (colonies, methylcellulose), BFU-E (colonies, methylcellulose), and CFU-GEMM (colonies, methylcellulose) scored for Infants 5 , 6, and 9 were, respectively, 7 to 100, 3 to 67, 11 to 775, 133 to 1270, and 40 to 1280 times greater than those scored for Infant 4. These differences are not merely a reflection of the volume of cord blood or of the numbers of nucleated cells collected, as in Infants 5 and
+
Discussion
We report the prenatal identification of three individuals who are unaffected with FA, karyotypically normal, and class I HLA-identical to their affected siblings. Prenatal testing has enabled us to collect umbilical cord blood in optimal volumes at the birth of these individuals. Progenitor cell numbers in the samples of cord blood obtained, as determined by in vitro assays, were within the reported range associated with successful allogeneic bone marrow transplants. Engraftment of two FA patients using umbilical cord blood described here (Infants 5 and 6 ) has now been achievedz7 (Gluckman E, Broxmeyer HE, Auerbach AD, et al. unpublished observations). The diagnosis of FA in patients with some of the clinical features of the syndrome was based on the unique hypersensitivity of cells from affected individuals to the clastogenic effect of DEB.20-24As the risk for FA in each pregnancy is high (1 in 4) and the risk of the CVS procedure appears to be we generally recommend a first-trimester prenatal diagnosis. The occurrence of a false-negative result in CVS studies of one fetus from a twin pregnancy at risk for FA remains to be explained. HLA studies of cultured trophoblast cells from this female fetus appeared to rule out maternal cell contamination, as only a single maternal haplotype was detected; the only antigens identified were identical to those of an affected sibling. Nevertheless, it is possible that the flask of cells used for HLA typing contained fetal cells, while those included in the cytogenetic study were maternal cells. As the chromosomal instability usually associated with FA was not detected in cells obtained during the first trimester of this pregnancy, and as we have not had any false-negative or false-positive diagnoses in our more extensive experience with amniocentesis for FA prenatal diagnosis,23 we recommend that a confirmatory amniocentesis be performed in cases diagnosed as normal by
cvs.
Hematopoietic progenitor cells (colony-forming unitsculture [CFU-C], CFU-E, and BFU-E) have been reported to be markedly decreased or absent in cultures of bone marrow cells from patients with FA,14-17 even in individuals who were not anemic at the time of study.14 The results of our study indicate a deficiency of progenitor cells for various lineages in cord blood obtained at the birth of an affected child, which suggests that the aplastic anemia that usually develops during the first decade of life in children with FA results from an intrinsic stem-cell disorder present at birth. A peripheral blood count obtained when this child was 2 months of age showed a hemoglobin level of 13.2 g per dL, a
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hematocrit of 37.2, and a white cell count of 10.8 x lo9 per L with a differential of 38 percent granulocytes, 59 percent lymphocytes, 3 percent monocytes, and 434 x lo9 per L platelets; the mean corpuscular volume was 99.0. The normal peripheral blood counts suggest that the deficiency in the colony-forming ability of FA bone marrows may be due to a profound inability of the progenitor cells to proliferate. FA fibroblasts and lymphocytes have been shown to exhibit poor growth in vitro, particularly at ambient oxygen tension rather than hypoxic (5% vol/vol oxygen) culture condition^.^^-^^ This defective growth has been shown to result specifically from a prolongation of, and arrest in, the G2 phase of the cell cycle. That the bone marrow failure in FA is not due to an abnormality in the bone marrow microenvironment is demonstrated by the successful engraftment of bone marrow transplanted to FA patients. A long-term actuarial survival rate of 70 percent has been reported37 when a modified conditioning regimen with a reduction in the dose of cyclophosphamide was used to lessen toxicity to this DNA-crosslinking agent in hypersensitive FA patients. The results of a recent study7 suggested that umbilical cord blood from a single donor could serve as a source of transplantable, hematopoietic, repopulating cells. In the present study, the numbers of progenitor cells collected from cord blood at the birth of normal siblings of FA patients is within the range noted by others8-10to be associated with successful engraftment in bone marrow transplantation. The advantages of using umbilical cord blood for transplantation, instead of using a small infant as a bone marrow donor, include avoidance of the risks of anesthesia and other potential complications associated with bone marrow donation. The successful use of umbilical cord blood in the treatment of FA prompts us to suggest that these methods will have application to other diseases treatable by bone marrow transplantation, including other genetic disorders such as thalassemia, severe combined immunodeficiency, and the mucopolysaccharidoses and nonconstitutional disorders such as idiopathic aplastic anemia and leukemia. The prenatal identification of a healthy HLA-identical sibling and the collection of umbilical cord blood at birth could lead to the treatment of such patients by cord blood transplantation. In addition, we suggest that umbilical cord blood, a material that is usually discarded at the birth of an infant, might be used to establish a bank of cryopreserved cells for unrelated allogeneic transplantation. It may be possible also to use cryopreserved umbilical cord blood for autologous transplantation. Further studies are needed to determine whether umbilical cord blood will have wider applicability as suggested here. A concern expressed by o t h e r P is the possibility of contamination of cord blood by maternal
Vol. 30, No. 8-1990
cells. Our initial laboratory investigation using I- and iantigen analysis did not detect any such contamination, but further tests are in progress. Questions regarding the immunoreactivity of cord blood also should be addressed. Although neonatal blood is considered to be as immunocompetent as adult blood in regard to the generation of cytotoxic T lymphocytes, and thus to have the potential for causing graft-versus-host disease (GVHD),39 the two FA patients who have received a transplantation of umbilical cord blood have had less difficulty with GVHD than is usually seen in these patients. A potential concern is whether there will be enough stem or progenitor cells from a single cord blood collection to repopulate the hematopoietic system of an adult. In this regard, we have collected samples of cord blood that contain 5 to 22 times the number of progenitor cells present in the collections used for successful engraftment in the two patients with FA. In another case, we collected cord blood that contained progenitor cells in numbers that exceeded those used in the first clinical transplant, even though the number of nucleated cells was less than one-half that used in the transplant. In conclusion, we have demonstrated a successful clinical application for the usually discarded umbilical cord blood, but questions regarding the broad applicability of this material as a source of transplantable hematopoietic cells must await further study. Acknowledgment The authors wish to thank Cynthia Callaway, Gloria Grant, and Sarra Sorkin for expert technical assistance.
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CORD BLOOD TRANSPLANT FOR FANCONI ANEMIA
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The Rockefeller Univcrsity, 1230 York Avcnue, Ncw York, NY 100216399. [Reprint requcsts] Q. Liu, MD, Laboratory for Investigative Dcrmatology, Thc Rockefeller University. R. Ghosh, MS, Laboratory for Investigativc Dcrmafology, Thc Rockefeller Univcrsity. M.S. Pollack, PhD, Histocompatibility Laboratory, The Mcthodisf Hospital and Baylor College of Medicine, Houston, TX. G.W. Douglas, MD, Department of Obstetrics and Gynecology, New York Univcrsity. H.E. Broxmeyer, PhD, Departments of Medicine. Microbiology, and Immunology, and the Walther Oncology Centcr, Indiana University School of Medicine, Indianapolis, IN.
