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104Cellular Therapy and Transplantation (CTT), Vol. 2, No. 7 doi: 10.3205/ctt-2010-en-000079.01
© 1989 Massachusetts Medical Society. All rights reserved. Originally published: N Engl J Med. 1989 Oct 26321(17)1174-8. pmid: 2571931
Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling
Eliane Gluckman1, Hal E. Broxmeyer2, Arleen D. Auerbach3, Henry S. Friedman4, Gordon W. Douglas5, Agnes Devergie1, Helene Esperou1, Dominique Thierry6, Gerard Socie1, Pierre Lehn1, Scott Cooper2, Denis English2, Joanne Kurtzberg4, Judith Bard7, and Edward A. Boyse7
1Bone Marrow Transplant Unit, Hôpital Saint-Louis, Paris, France; departments of Medicine (Hematology/Oncology), Microbiology and Immunology, Pathology, and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, USA; 3Laboratory for Investigative Dermatology, Rockefeller University, New York, USA; 4Department of Pediatrics (Hematology/Oncology), Duke University Medical Center, Durham, N.C., USA; 'Department of Obstetrics and Gynecology, New York University Medical Center, New York, USA; 6Central Nuclear Agency, Paris, France; 7Memorial Sloan-Kettering Cancer Center, New York, USA
Abstract
The clinical manifestations of Fanconi's anemia, an autosomal recessive disorder, include progressive pancytopenia, a predisposition to neoplasia, and nonhematopoietic developmental anomalies [1-3]. Hypersensitivity to the clastogenic effect of DNA-cross-linking agents such as diepoxybutane acts as a diagnostic indicator of the genotype of Fanconi's anemia, both prenatally and postnatally [3-6]. Prenatal HLA typing has made it possible to ascertain whether a fetus is HLA-identical to an affected sibling [7].
We report here on hematopoietic reconstitution in a boy with severe Fanconi's anemia who received cryo-pre-served umbilical-cord blood from a sister shown by prenatal testing to be unaffected by the disorder, to have a normal karyotype, and to be HLA-identical to the patient. We used a pretransplantation conditioning procedure developed specifically for the treatment of such patients [8]; this technique makes use of the hypersensitivity of the abnormal cells to alkylating agents that cross-link DNA [9,10] and to irradiation [11] In this case, the availability of cord blood obviated the need for obtaining bone marrow from the infant sibling.
This use of cord blood followed the suggestion of one of us that blood retrieved from umbilical cord at delivery, usually discarded, might restore hematopoiesis - a proposal supported by preparatory studies by some of us [12] and consistent with reports on the presence of hematopoietic stem and multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in human umbilical-cord blood (see the references cited by Broxmeyer et al. [12]).
Keywords: anemia, aplastic therapy, blood preservation, Fanconi anemia, female, fetal blood, HLA antigens, hematopoietic stem cell transplantation, histocompatibility testing, humans, male, pregnancy, prenatal diagnosis, preschool child
Case report
The patient, a five-year-old boy, was first noted to have pancytopenia at 24 months of age. An evaluation (Duke University Medical Center) showed the following hematologic values: hemoglobin concentration, 9.5 g per deciliter; white-cell count, 4.3 x 109 per liter, with 22 percent granulocytes, 62 percent lymphocytes, 3 percent monocytes, 1 percent eosinophils, and 8 percent baso-
phils; platelet count, 62 x 109 per liter; and reticulocyte level, 2.3 percent. The bone marrow was hypocellular. The patient had the classic malformations of Fanconi's anemia, including retarded growth (5th to 10th percentile), a rudimentary extra thumb on the left hand, the absence of a left kidney, and hypospadias. Testing of the patient's cells with diepoxybutane, performed at 24 months (Rockefeller University), confirmed the diagnosis of Fanconi's anemia. The parents were healthy, and there was no consanguini-
ty or past history of blood disorders in the family. The patient had been treated with danazol (300 mg per day for six months), with a progressive decrease in the blood-cell counts. Before admission, he had received three units of filtered and irradiated packed red cells.
The patient's mother became pregnant in June 1987. Cytogenetic analysis of cultured amniotic-fluid cells (Rockefeller University) showed that the values for chromosome breakage before and after the cells were exposed to diepoxybutane were within the normal range [5]. A girl was delivered vaginally without complications in February 1988. Studies of chromosome breakage in umbilical-cord blood cells indicated that the newborn had a normal karyotype and was not affected by Fanconi's anemia (Table 1).
Source Of Cells Percent Donor Cells In Samplef Mean Chromosome Breaks/CellJ
before dab after dab before dab after dab
Patient's peripheral blood on day 0 0 0 0.18 10.6
Donor's cord blood on day 0 100 100 0.00 0.02
Patient's bone marrow on day 120 100 100 0.04 0.30
Patient's peripheral blood Day 50 30 52 0.7 4.0
Day 64 8 32 1.3 5.0
Day 120 12 32 1.9 6.4
Day 204 64 86 0.30 0.49
Table 1: Results of Cytogenetic Analysis.*
*DEB denotes diepoxybutane; its final concentration in the culture medium was 0.1 mg per milliliter in each analysis in this table.
tAnalysis performed on quinacrine-stained metaphase preparations. ^Analysis performed on Giemsa-stained metaphase preparations.
Prenatal HLA typing of amniotic-fluid cells (Dr. M.S. Pollack, Methodist Hospital and Baylor Medical Center, Houston) had shown that the fetus was HLA identical to the patient: the first ha-plotype was Al,B8,DR3, and the second haplotype A29,B44,DR3. Mixed lymphocyte cultures of cells from the patient with cells from the cord blood and peripheral blood of the donor were negative. The cord-blood cells were less reactive against a pool of allogeneic cells than were the peripheral-blood cells (incorporation of [3H]thymidine, 15,437 vs. 112,954 cpm). The ABO blood group of the donor was O Rh+, and that of the recipient B Rh+.
The patient was admitted in September 1988, with the following hematologic values: hemoglobin concentration, 6.8 g per deciliter; white-cell count, 3.1 x 109 per liter, with 7 percent granulocytes, 86 percent lymphocytes, and 5 percent monocytes; and platelet count, 18 x 109 per liter. The marrow was hypocellular, with 10 percent of normal cellularity, 24 percent myeloid cells, 2 percent erythroblast cells, 67 percent lymphocytes, and no megakaryocytes. There were no hemorrhagic or infectious complications of the patient's illness. His liver and kidney functions were normal. Serologic tests were positive for cytomegalovirus (on enzyme-linked immunosorbent assay) and negative for human immunodeficiency virus, hepatitis B, and toxoplasmosis.
The patient was isolated in a room with laminar airflow, and treatment with oral broad-spectrum nonabsorbable antibiotics (vanco-
mycin, tobramycin, and colistin [Colimycine]) was started nine days before transplantation (on day -8). Fluconazole (50 mg per day) was administered to prevent fungal infection, and oral acy-clovir (100 mg per day) to prevent herpes simplex virus infection. For pretransplantation conditioning, cyclophosphamide (5 mg per kilogram of body weight) was given intravenously for four consecutive days (from day -6 to day -3; total, 380 mg) along with hyperhydration. Irradiation was delivered to the thoracoabdominal region by a linear accelerator on day -1 (mean rate, 10.87 cGy per minute), for a total of 5 Gy over a period of 46 minutes; the abdomen received 500 cGy, and the lungs and the right liver lobe, which were shielded, received 67 cGy.
On the day of transplantation (day 0 - October 6, 1988), cryo-
preserved umbilical-cord blood was thawed and infused without further processing, according to predetermined optimal conditions12 (the surviving whole red cells that had not been hemolyzed belonged to group O). The patient received 0.4 x 108 nucleated cells per kilogram, of which a total of 4.37 x 105 were CFU-GM (as determined by assay in Paris). Two hours after the infusion, the patient had chills, fever, and hypotension. These symptoms soon resolved, while the patient was receiving the broad-spectrum antibiotics vancomycin and ceftazi-dime intravenously. For prophylaxis against graft-versus-host disease, cyclosporine was administered intravenously from day - 1 (4.5 mg per kilogram per day), according to a preliminary pharmacokinetic study. All blood products were irradiated (25 Gy).
Ethical and Regulatory Considerations
Written informed consent was obtained from the patient's parents for collecting the umbilical-cord blood and for the transplantation procedure. The treatment plan was reviewed and approved by the institutional review board for clinical investigation of the Duke University Medical Center and by the ethics committee of the Hôpital Saint-Louis. Approval for the receipt, cryopreservation, storage, and release of cord blood was received from the institutional review board of the Indiana University School of Medicine. The Food and Drug Administration considered the procedure equivalent to the storage and transplantation of bone marrow, which are currently not subject to its regulation.
Methods
Cytogenetic Studies
Chromosome-breakage studies were performed as described elsewhere [4]. Diepoxybutane was added to bone marrow cultures when they were begun, and the cultured cells were harvested after 24 hours. Peripheral blood was cultured in the presence of phy-tohemagglutinin for 72 hours; diepoxybutane was present in the medium during the last 48 hours of culture. The frequency of chro-
mosome breakage before and after the addition of diepoxybutane was analyzed in Giemsa-stained metaphase preparations; the ratio of cells from the patient to cells from the donor (male:female) was determined on quinacrine-stained slides to facilitate the identification of the Y chromosome.
described [13]. The probe used was CRI-pS232 (DXS278) (Collaborative Research, Boston), which hybridizes with sequences from the X and Y chromosomes.
Collection, Storage, and Shipment of Neonatal Blood
DNA Studies: Restriction-Fragment-Length Polymorphism
DNA samples obtained for Southern blotting were digested with Taql (New England Biolabs, Beverly, Mass.), separated by gel electrophoresis, transferred to an Immobilon-N filter (Millipore Corp., Bedford, Mass.), hybridized, and washed as previously
Source of Cells Progenitor Cellsf
all densities low density (<1.077 g/ml)
Donor (cord and placental blood){ total cells x 10-5
Agar culture Day 14 CFU-GM (colonies) 1.52 ND
Day 14 CFU-GM (colonies and clusters) 2.46 ND
Methylcellulose culture (colonies) Day 14 CFU-GM 1.56 ND
BFU-E-2 3.95 ND
BFU-E-1 3.60 ND
CFU-GEMM 0.39 ND
Recipient (bone marrow) cells/105 cells plated§
Methylcellulose culture, day 0 Day 14 CFU-GM (colonies) ND 1
Day 14 CFU-GM (colonies and clusters) ND 8
BFU-E-1 ND 0
CFU-GEMM ND 0
Methylcellulose culture, day 30 ND 9
Day 14 CFU-GM (colonies)
BFU-E-1 ND 0
CFU-GEMM ND 0
Agar culture, day 120 22±2 140±12
Day 7 CFU-GM (colonies)
Day 7 CFU-GM (colonies and clusters) 118±16 416±20
Day 14 CFU-GM (colonies) 36±4 156±8
Day 14 CFU-GM (colonies and clusters) 152±4 228±12
Methylcellulose culture, day 120 324±16 416±25
Day 14 CFU-GM (colonies)
BFU-E-2 77±2 134±6
BFU-E-1 96±8 132±10
CFU-GEMM 4±1 9±1
Immediately after the birth of the patient's sister, blood was obtained from her umbilical cord and the placenta as described elsewhere [12] and transported at ambient temperature by overnight express service to a laboratory for cellular analysis, cryopreser-vation, and storage (Indiana University School of Medicine). A sample of cord blood was also sent elsewhere for cytogenetic analysis (Rockefeller University). After a small sample (<2 ml) had been set aside for laboratory tests, including the determination of the level of nucleated cells and the enumeration of progenitor cells, the blood was frozen without further treatment in dimethyl sulfoxide at a final concentration of 10 percent (vol/vol) as previously described [12] Two bags of cord blood and one bag of placental blood were frozen. Samples (about 1 ml each in Nunc tubes) were similarly frozen for testing of cell recovery after thawing and for confirmation of the HLA types. The volume of blood and the numbers of nucleated and progenitor cells collected are shown in Table 2. These values were within the range associated with successful transplantation of HLA-matched allogeneic bone marrow [12]. Testing of thawed cells, including those in one tube sent from Indiana University to Duke University Medical Center (where the HLA types were confirmed), demonstrated recovery of 79 percent to 90 percent of nucleated cells; on day 14, the mean (±1 SEM) rates of recovery were 100 percent ofCFU-GM, 63±18 percent of BFU-E, and 79±15 percent of CFU-GEMM.
Table 2. Hematopoietic Progenitor Cells in Blood from the Umbilical Cord and Placenta of the Donor and Bone Marrow from the Recipient.
* The preparation for the assays has been described elsewhere [12]. The assays of the cord blood and the bone marrow on day 120 were performed in Indianapolis, and those of the bone marrow at day 0 and day 30 in Paris.
t Plus-minus values are means ± 1 SEM; ND denotes not done.
t The total volume of cord and placental blood collected was 160 ml and contained 1.19 x 109 nucleated cells.
§ Normal range (Indianapolis laboratory) for low-density cells. 10 to 70 for CFU-GM and 15 to 80 for BFU-E-1 and BFU-E-2.
These were mainly microclusters, with less than 20 cells per cluster.
The two bags of frozen cord blood and the bag of placental blood were sent with an escort by air (with the approval of the airline) to the Hôpital Saint-Louis from Indiana University two weeks before transplantation. The bags were sent in a Dry Shipper container (CMC-3200 wide mouth with platform; Cryomed, New Baltimore, Mich.) maintained at -175°C, a temperature optimal for cryopreservation.
Hematopoietic Progenitor Cells in Vitro
Assays were prepared as previously described [12] In the CFU-GM assay, colonies (>40 cells per aggregate) and clusters (3 to 40 cells per aggregate) were scored after 7 days and 14 days of incubation in agar culture medium. Large-sized colonies formed (>1000 cells). Cell counts were expressed also as colonies plus clusters, for a more accurate estimate of the total CFU-GM compartment. In the assays for BFU-E, CFU-GEMM, and CFU-GM, colonies were scored after 14 days of incubation in methylcellulose culture medium. In the BFU-E-1, CFU-GEMM,
and CFU-GM assays, colonies were scored from the same plates, which included erythropoietin (1 unit per milliliter), hemin (0.1 mM), and 5637 conditioned medium (10 percent vol/vol). BFU-E-2 cells were cultured as BFU-E-1, but without 5637 conditioned medium. Each BFU-E-2 colony contained at least 50 cells or comprised at least three subcolonies, each of which contained at least 10 cells, but were usually much larger. Colonies derived from BFU-E-1 were much larger than those derived from BFU-E-2.
Thawing and Recovery of Cells
Eighty-two percent of nucleated cells of the thawed blood transfused into the patient were viable. Progenitor-cell assays performed in Paris (which differed slightly in technique from assays performed in Indianapolis) indicated that the rate of cell recovery was equal to or greater than the count of progenitor cells recorded before freezing.
Results
Clinical Findings
The clinical course of the patient was uneventful and without complications. He tolerated the pretransplantation conditioning without evident toxic effects. On day 15, a transient skin rash and fever resolved with methylprednisolone treatment (2 mg per kilogram). A skin biopsy revealed few vacuolar basal epidermal cells with a mild lymphoid infiltrate, indicative of grade I graft-versus-host disease as defined according to the Seattle classification [14]. Liver-function tests showed that the serum levels of aspartate and alanine aminotransferases were twice normal, probably as a result of the graft-versus-host disease; values returned to normal on day 47. Cytomegalovirus was repeatedly isolated from the urine, but the patient never had any signs of active infection, and all tests for
viremia were negative. Five months after transplantation the patient was discharged with normal clinical and laboratory findings. The doses of cyclosporine and corticosteroids were progressively reduced, and the drugs were discontinued at six months. At present (nine months after transplantation), the patient has no chronic graft-versus-host disease and leads a normal life.
Hematologic Findings
The blood-cell counts began to return to normal (Table 3).
The reticulocyte and granulocyte counts had begun to rise by day 22 after transplantation. The patient received eight transfusions of packed red cells (O Rh+) and 48 units of random platelets. Red cells were last transfused on day 54, and platelets on day 43. Bone marrow aspirated on day 17 was aplastic: an evaluation on day 28 showed 20 percent cellularity, with 19 percent myeloid cells, 73 percent erythroid cells, and few megakaryocytes. On day 120, the marrow had normal cellularity, with 40 percent myeloid cells, 44 percent erythroid cells, and a normal level of megakaryocytes.
Hematopoietic Progenitor Cells
Before transplantation, the marrow contained few or no detectable progenitor cells (Table 2). At 30 days after transplantation, CFU-GM cells were detected but not BFU-E or CFU-GEMM; at 120 days, progenitors were apparent at normal to supranormal frequencies (colonies or clusters per number of cells plated).
Reconstitution by Donor Cells
Blood- Type Studies
The patient's red cells (B Rh+) disappeared progressively and became undetectable on day 90 after transplantation; 46 days after the last transfusion, 100 percent of red cells were of the donor's
Day Hemoglobin Leukocytes Granulocytes Lymphocytes Platelets Reticuloytes
g/dl no. of cells x 10-9 per liter
-20 6.8 3.1 0.25 2.8 18 10
0 9.7 0.8 0.0 0.8 120 0
8 10.9 0.4 0.0 0.4 80 0
15 11.6 0.4 0.0 0.4 39 0
22 7.8 0.9 0.3 0.6 50 5
29 8.5 1.0 0.3 0.5 105 17
36 9.4 1.7 0.6 0.5 55 36
43 11.3 5.1 2.4 1.9 31 90
50 8.9 3.4 1.5 0.7 62 162
57 8.9 5.6 3.2 1.0 174 63
90 11.3 5.1 4.0 1.1 296 50
120 13.0 3.9 2.3 1.1 265 40
160 12.0 3.7 1.4 1.6 293 45
240 12.3 5.2 2.7 1.6 354 50
282 12.2 4.8 2.3 1.2 315 ---
Table 3. Blood-Cell Counts before and after Transplantation. 4 www.ctt-journal.com 2010;2(7)
blood type (O Rh+) and were found to have remained so at 240 days after transplantation.
Cytogenetic Studies
Table 1 shows the results of cytogenetic studies of bone marrow aspirated on day 120 and peripheral blood obtained on days 50, 64, 120, and 204. The chromosomal complement of the bone marrow was 46,XX. No cells of the patient (male cells) in metaphase were seen among 50 quinacrine-stained or 100 Giemsa-stained cells analyzed. The chromosomebreakage frequencies were 0.04 and 0.30 breaks per cell in the base-line and diepoxybutane-treated cultures, respectively.
Through day 120 after transplantation, more than half the me-taphases seen in cytogenetic preparations of phytohemaggluti-nin-stimulated peripheral-blood cultures were of host origin, as indicated by the presence of a Y chromosome. These cells, of lymphoid origin, had greatly elevated levels of chromosome breakage at base line and severe radiation damage, manifested by the presence of multiple dicentrics, rings, and chromosomal fragments; they were also hypersensitive to the clastogenic effect of diepoxy-butane, showing multiple chromatid breaks and exchanges typical of the cells characterizing Fanconi's anemia. The cells of donor origin (female) did not have elevated levels of chromosome breakage. By day 204 (6"2 months after transplantation), the majority of peripheral-blood lymphocytes were of donor origin.
These findings reflected engraftment with the donor cells and survival of a minor population of radiationdamaged host cells in the blood - an outcome similar to that in reports of bone marrow transplantation by others [15].
DNA Studies
The CRI-pS232 probe recognizes a complex set of fragments at a highly polymorphic locus on the X chromosome, as well as a polymorphic locus on the Y chromosome [16]. In the present case, all the variable bands present in the DNA of the donor were seen in the DNA extracted from the peripheral blood of the recipient after transplantation (Fig. 1). In addition, the X and Y alleles of the recipient were seen as faint bands, indicating some chimerism in the peripheral blood, with primarily donor cells present.
DNA was extracted from the peripheral blood of the father (lane 1), the mother (lane 2), the patient before transplantation (lane 3), the patient's HI.A-identical sister (lane 4), the patient's HA non-identical sister (lane 5), and the patient on post-transplantation day 50 (lane 6), day 64 (lane 7), and day 120 (lane 8).
The DNA obtained from the patient after transplantation was primarily of donor origin. Each DNA sample was digested with the restriction enzyme Taq1 and examined with the probe CRI-pS232, which recognizes a complex set of fragments at a highly polymorphic locus on the X chromosome as well as a locus on the Y chromosome. The thick arrows indicate X-specific bands from the donor, and the thin arrows denote those from the recipient. Y-specific bands are seen at 4.9 and 3.8 kb. A constant band at 3.0 kb is seen in the DNA of all the subjects.
Discussion
Bone marrow and, under certain circumstances, the blood of adults (see references cited by Broxmeyer et al [12]) and the liver of fe-
1 2 3 4 5 6 7 8
^ÊQUÊÊ flM^
Figure 1. Southern Blot Analysis of DNA from the Patient and His Family.
DNA was extracted from the peripheral blood of the father (lane 1), the mother (lane 2), the patient before transplantation (lane 3), the patient's HI.A-identical sister (lane 4), the patient's HA non-identical sister (lane 5), and the patient on post-transplantation day 50 (lane 6), day 64 (lane 7), and day 120 (lane 8).
The DNA obtained from the patient after transplantation was primarily of donor origin. Each DNA sample was digested with the restriction enzyme Taq1 and examined with the probe CRI-pS232, which recognizes a complex set of fragments at a highly polymorphic locus on the X chromosome as well as a locus on the Y chromosome. The thick arrows indicate X-specific bands from the donor, and the thin arrows denote those from the recipient. Y-specific bands are seen at 4.9 and 3.8 kb. A constant band at 3.0 kb is seen in the DNA of all the subjects.
tuses [17] have been used in the transplantation of hematopoietic cells. The clinical and biologic data presented above show that the umbilical-cord blood of a single newborn is sufficient to induce hematopoietic reconstitution. The virtually complete occupation of the patient's myeloid system by cells from his sibling was indicated by the cytogenetic studies, blood typing, and studies of DNA polymorphisms and by the absence of undue chromosomal fragility. At present, a small percentage of lymphoid cells with chromosome damage remains in the circulation, but there has been a trend toward the elimination of these damaged cells.
There may be several reasons why cellular repopulation was somewhat delayed in our patient as compared with patients who have undergone transplantation with HLA-matched allogeneic (sibling) bone marrow. For instance, cells that have been frozen and thawed may take longer than fresh cells to seed, respond, and differentiate in their respective inducing microenvironments. Also, consistent with the relative paucity of CFU-GM on day 7 as compared with the more immature CFU-GM on day 14 [12], umbilical-cord blood may typically have a profile with immaturity and less differentiation of hematopoietic stem and progenitor cells. The modified conditioning regimen was adequate to permit complete engraftment with 0.4 x 108 total nucleated cord-blood cells per kilogram, as compared with the mean of 3 x 108 total nucleated bone marrow cells per kilogram that is usually given for bone marrow transplantation.
The use of cord blood in the treatment of Fanconi's anemia may be extended to the treatment of other conditions for which bone marrow transplantation is indicated, and demonstrates the potential of cryopreservation for prospective use of autologous blood.
In the latter respect, our success with allogeneic cord blood is particularly important, because studies in animals have shown that fewer reconstituting cells are required to restore hematopoietic function when these are syngeneic rather than allogeneic [18].
Thus, umbilical-cord blood can be considered an efficacious source of sufficient cells for clinical hematopoietic reconstitution.
Acknowledgements
We are indebted to Dr. Bo Dupont (Sloan-Kettering Institute for Cancer Research) for suggesting the clinical application in Fanconi's anemia, to Dr. Marilyn Pollack (Baylor Medical Center) for HLA typing of the amniotic cells, to Dr. Rebecca H. Buckley (Duke University) for the data from the mixed lymphocyte cultures, to Rita Ghosh, Lui Qian, and Drew Olsen (Rockefeller University) for technical help with the cytogenetic studies, to Dr. William Mann and V.S. Venkatraj (Rockeleller University) for DNA analysis, to Françoise Varrin (Paris) for technical assistance in the assessment of hematopoietic progenitors, to Ms. Linda Cheung (Indiana University) for help in preparing the manuscript, and to Collaborative Research for the probe pS232.
Supported in part by grants from the Biocyte Corporation, by grants (CA36464 and CA-36740 to Dr. Broxmeyer and CA-39827 to Dr. Boyse) from the National Cancer Institute, by a grant (HL-32987 to Dr. Auerbach) from the National Institutes of Health, and by a grant from the Pew Memorial Trust to Rockefeller University. Dr. Boyse holds a Research Professorship from the American Cancer Society, and Dr. Kurtzberg is a Special Fellow of the Leukemia Society of America. In accordance with the Journal's policy, the authors have provided the following information: Dr. Broxmeyer, Dr. Douglas, Ms. Bard, and Dr. Boyse are shareholders in Biocyte Corporation (New York), and Dr. Douglas and Dr. Boyse are members of its board of directors.
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© 1989 Massachusetts Medical Society. All rights reserved. Originally published: N Engl J Med. 1989 Oct 26;321(17):1174-8. PMID: 2571931
