From www.bloodjournal.org by guest on December 29, 2014. For personal use only. Analysis of &Globin Mutations Shows Stable Mixed Chimerism in Patients With Thalassemia After Bone Marrow Transplantation By J. Kapelushnik, R. Or, D. Filon, A. Nagler, G. Cividalli, M. Aker, E. Naparstek, S.Savin, and A. Oppenheim The seventh patient hasless than 10%donorcells with, /?-thalassemia major(TM) is causedby any of approximately surprisingly, onlyminimal transfusion requirements. The de150 mutations within the /?-globin gene. To establish the tection of /?-globin gene point mutation,as used here, is a degreeofchimerism afterbonemarrowtransplantation highly specific and sensitive marker for engraftment MC and (BMT), we have performed molecular analysis of P-globin in patients with thalassemia. In light of its specificity, the mutations in14 patients with TM over a periodof 10 years. All patients underwent T cell-depleted allogeneic BMT from method is applicable in all cases ofTM, as it is independent of sex and othernon-globin-related DNAmarkers. The high HLA-identicalrelated donors,usingeither in vitroT-cell a incidence of MC found inour patients may be consequence depletion with CAMPATH 1M andcomplementor in vivo MC was associated depletion using CAMPATH 1Gthe in bone marrow collection of the pre-BMT T-cell depletion. Because with transfusion independence, complete eradication of rebag. To date, at different time periods after BMT, seven pasidual host cells for effective treatment of TM and possibly six of these patients, tients have some degree of chimerism; other genetic diseases may prove not to be essential. all blood transfusion-independent, have donor cells in the 0 7995 by The American Society of Hematology. range of 70% to 95%. with stable mixed chimerism (MC). H IGH-DOSE CHEMOTHERAPY followed by allogeneic bone marrow transplantation (BMT) is the treatment of choice for a wide range of nonmalignant hematologic diseases, including &thalassemia major ( T M > . l 2 * The pretransplant chemotherapy has a dual purpose: to kill hematopoietic host cells so as to gain bone marrow space and, at the same time, to induce adequate immunosuppression to prevent rejection of donor stem cells. Despite the significant and continuing advances in conditioning regimens, patients may ultimately develop either persistent host lymphohematopoietic cells or relapse with the basic disease. A number of studies have shown that patients in good clinical condition post-BMT and presenting with normal hematologic parameters may still harbor minimal amounts of residual host hematopoietic cells in their bone marrow and peripheral blood, as determined by highly sensitive technique^.^.^ The methods used thus far to detect chimerism after BMT for nonmalignant disorders include detection of sex chromosomes in cases of sex disparity between donor and recipient: analysis of hypervariable regions of the human genome by polymerase chain reaction (PCR) amplificat i ~ n , * . and ~ - ' ~AB0 typing." The present work focuses on the detection of point mutations in the &globin gene of patients with TM undergoing BMT. This innovative technique aIlows us to establish the degree of chimerism by measuring the level of residual host P-globin genes, using the point mutation as a marker, and permits correlation between the degree of chimerism and the clinical outcome after BMT. MATERIALS AND METHODS Patients. The study comprises 14 informative patients with TM (9 males and five females; age range, 1 to 7 years; median, 4 years at presentation) treated during the last decade at the Department of Bone Marrow Transplantation of the Hadassah University Hospital, Jerusalem, Israel. All patients received allogeneic BMT from HLAidentical, related donors. Six pairs were sex-mismatched. Pretransplant conditioning. All patients received oral busulfan (4 mgflrg/d X 4 days) and intravenous (IV) cyclophosphamide (50 mg/kg/d X 4 days). Thiotepa IV (5 m@g X 1 day) was added for four patients. Total lymphoid irradiation (TLI), 1O , OO cGy given in five daily fractions, was performed in 10 patients, and four patients received IV rat anti-human CDWS2 (IgG 2b) antibody (CAMPATH Blood, Vol86, No 8 (October 151, 1995: pp 3241-3246 1G) (0.2 mg/kg/d X 4 days) for in vivo depletion of hostlymphocytes before chemotherapy. Graft-versus-host disease (GVHD)prophylaxis. All patients received T cell-depleted bone marrow achieved by treatment either in vitro (nine patients) with CAMPATH IM, with donor serum as source of complement CAMPATH, or in vivo with CAMPATH 1G directly in thebone marrow collection bag (five patients). In the latter case, depletion was most likely achieved by antibody-dependent cellmediated cytotoxicity. CAMPATH 1Mand CAMPATH 1G were provided by Drs G . Hale and H. Waldmann (Department ofPathology, Dunn School of Pathology, Oxford, UK). The use of CAMPATH 1M in vitro and CAMPATH 1G in the marrow collecting bag has been described previ~usly.'~"~ Rejection prevention. Cyclosporin A (3 mg/kg/d)was administered intravenously to all patients from day -1 until engraftment (polymorphonuclear cells, 2750 mmz). DNA preparation and PCR. DNA was prepared from peripheral blood according to standard procedures.16PCR was performed with Taq polymerase (Appligene, Strasbourg, France) using the conditions recommended by the manufacturer. The following primers, spanning the first and second exons of the 8-globin gene from 166 nucleotides (nt) upstream of the cap site to 138 nt in the IVS2 site, were used: PS (S' primer), CCAACTCCTAAGCCAGTGCC; and P12 (3' primer), CTGAGACTTCCACACTGATGC.The amplification cycle consisted of 1 minute at 92"C, 1.5 minutes at 62"C, and 1.5 minutes at 7 2 T , giving an amplification product of 799 bp. Testingfor mutant alleles. Before BMT, the B-thalassemia mutation(s) of the patient was identified: in parallel, the @-globingenotype of the donor was determined. Some of the donors were normal, while others were thalassemia carriers. After the transplantation, DNA samples obtained from peripheral blood were tested for chimeFrom the Departments of Pediatrics, Hematology, and Bone Marrow Transplantation, Hadassah University Hospital, Ein Kerem, Jerusalem, Israel. Submitted March 17, 1995; accepted June 19, 1995. Supported in part by a grant from the Social Security Institution in Israel and by the Robert A. Rosenblum Research Fund. Address reprint requests to Reuven Or, MD, Department of Bone Marrow Transplantation, Hadassah University Hospital, Jerusalem 91 120, Israel. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. seetion 1734 soleIy to indicate this fact. 0 1995 by The American Society of Hematology. oooS-4971/95/8608-04$3.00/0 3241 From www.bloodjournal.org by guest on December 29, 2014. For personal use only. KAPELUSHNIK ET AL 3242 A PCR cycles 15 20 18 23 25 30 B z a U 10’ Fig 1. Kinetics of the p-globin PCR. The reaction was set up withnormal control DNA in a total of 300 p L that was divided equally into six tubes. Tubes were withdrawn after different numbers of amplification cycles, as shown in panel A. Aliquots of the PCR products (10 pL) were analyzed by Southern blotting (B). For quantification, the membrane was cut, and the bands were counted in a scintillation counter. 1 10 15 20 25 30 Cycles rism as follows: equal aliquots of the PCR products (5 to 15 pL) were denatured in 0.4 molL NaOH125 mmolL EDTA for 20 minutes on ice and spotted onto two duplicate nylon membranes together with appropriate controls, namely, donor DNA controlandcontrolsfor each 8-globin mutation (homozygotes, heterozygotes, and normal). Hybridization was performed with excess allele-specific oligonucleotide probes (1 to 2 ng) labeled with [.‘’P]7-adenosine triphosphate (ATP).” For each patient, one membrane was hybridized with the mutantprobeandthe second, withthe respective normalprobe. Quantification of the autoradiograms was performed as follows. Until 1993, radioactive counts weredeterminedbycuttingthe spots from the membrane and measuring in a scintillation counter, since 1993, theradioactivity of the spots ismeasureddirectly using a phosphorimage analyzer. RESULTS The first objective was to establish the sensitivity and reliability of the method. For dependable quantification of the ratio of normal to mutant alleles, measurements must be performed within the linear range of detection. The PCR is expected to start at an exponential rate and to remain so until one of the reaction components becomes limiting. We, therefore, examined first the progress of the PCR under our conditions. Because the reaction was exponential up to 25 cycles (Fig l ) , 23 cycles were used for all subsequent tests. Linearity of the signal obtained by hybridization depends on the ratio between DNA and probe. The PCR products 35 were spotted onto duplicate membranes at different DNA concentrations; membranes were then hybridized to 1 ng/ mL (Fig 2B, closed circles) and 3 ng/mL probes (Fig 2B, open circles). Atboth probe concentrations, the reactions were linear over a wide range. Measurement of radioactivity by phosphorimage analysis showed quantification of the results to be highly reproducible. Replicate analyses of a sample, at the same or different DNA concentrations, demonstrated an experimental error of S 1%. Repeated analyses of the same sample at intervals of months were likewise reproducible. We, therefore, consider a 2% hybridization signal as significant. Hence, the method can detect 2% donor (or 2% residual host) cells when both donor and recipient are homozygous, or establish 4% donor cells when the donor is heterozygous (or the recipient is compound heterozygous). After BMT, peripheral blood samples were analyzed sequentially for detection of the P-globin gene point mutations for all14 patients. Table 1 lists the clinical parameters of the 14 patients and the outcome of their BMTs: Five patients (43%) were found to have full engraftment (lOO% donor cells) based on analysis of the P-globin alleles. These five patients are in excellent clinical condition, exhibiting normal growth and development; their hemoglobin levels are comparable with those of the respective donors. In seven patients (50%), mixed chimerism (MC) was detected by the P-globin From www.bloodjournal.org by guest on December 29, 2014. For personal use only. D-GLOBIN MUTATION IN THALESSEMIA POST-BMT 3243 A Relative DNA concentration B lo5 10' E P v Fig 2. Linear range of DNA concentration for allele-specific detection. /.?-globinPCR productsof normal control DNA were serially diluted (twofold each time) and blotted onto duplicate nylon membranes. The blots were hybridized to a '2P-labeled oligonucleotide probe. (A) Autoradiogram of hybridization to 1 nglmL probe. (B)Quantitative analysis: the membraneswere cut, and the spots were counted in a scintillation counter. Hybridizationwas performed with 1 nglml ( 0 )and 3 ng/rnL (0) probes. lo3 .03 .06 . l 2 2 5 .S 1 :c" I 0 lo2 0 10' gene point mutation method (and not by any of the other engraftment markers); six of these patients have 73% to 96% donor cells with stable MC, are independent of blood transfusions, and are in excellent clinical condition. The seventh patient has 4% to 7% donor cells with only a minimal transfusion requirement; ie, she presents clinically with thalassemia intermedia. The remaining 2 patients (7%) have no detectable donor genes (ie, 100% host cells) and suffer from TM. None of the 14 patients showed signs of acute or chronic GVHD. To date, the period of follow up of the entire group of patients ranges between 6 months and 11 years (median, 4 years). In the seven patients withMC, only one was sex-mismatched: a male was transplanted from a female donor (UPN 338, Table 1). For this recipient, PCR for the detection of Y chromosome was positive. The other five sex-mismatched recipients did not have MC. In the seven patients with MC,the degree of chimerism, as expressed by the host-to-donor DNA ratio, remains relatively stable post-BMT (follow up of 3 to 68 months; median, 18 months). Despite the presence of MC, the six patients with greater than 70% donor cells have redblood cell counts similar to those of their donors; the stable MC in this group of patients, therefore, indicates sustained engraftment and certainly not recurrence of the basic disease. Thus far, no correlation was found to exist between the 01 I I .l 1 Relative DSA 10 concentration pretransplant conditioning regimen or mode of T-cell depletion (TCD) and the incidence of chimerism. DISCUSSION In this study, we present the detection of-the P-globin gene point mutation as an innovative method to confirm engraftment, detect minimal residual disease, and establish degree of chimerism in patients with thalassemia after BMT. Whereas other methods depend on neutral, nonrelated markers for the evaluation of chimerism, the strategy described here is based on the direct measurement of the diseasecausing gene, albeit in irrelevant cell lineages. BMT for thalassemia is usually performed with stem cells from closely related, HLA-matched donors, prohibiting the use of HLA as a marker. The AB0 antigens as engraftment markers are of value only in cases of A B 0 mismatch and, furthermore, have no significance with respect to minimal residual disease. DNA-based gender determination is limited to situations of donor-host sex disparity. The specificity of the 0-globin gene point mutation test, on the other hand, makes it applicable for every patient with TM receiving BMT. The method, based on allele-specific oligonucleotide hybridization, is highly sensitive and capable of detecting an allele even at a level as low as 1%. Thus, in the case of a normal donor, even 1% to 2%residual host cells are detected. From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 3244 KAPELUSHNIK ET AL Table 1. Chimerism and Outcome of BMT Chimerism UPN Age at BMT (yrs) P-Globin Genotype Conditioning RegimenflCD Patient Donor 50 152 170 184 1 2 1 1 BuCy, TLI/lM BuCy, TLI/lM BuCy, TLI/l M BuCy, TLI/l M FS44/FS5 IVS1, l/IVSl, 1 IVS2, l/lVS2, 1 IVS1, llO/IVSl, 110 FS44/A IVS1, l / A IVS2, 1/A IVS1, 11O/A 223 338 1.5 1 BuCy, TLI/lM BuCy, TLI/lM IVS1, llO/IVSl, 110 IVS2, l/lVS2, 1 IVS1, llO/A 364 1 BuCy, TLI/lM N37/A N37/N37 450 0.8 BuCy, TLI/l M IVS1, llO/IVSl, 110 IVSl, 11O/A 465 476 5 2 BuCy, TLI/l M BuCv, TLI/lG -30/N39 IVS1, 6/FS106-7 N39/A A/A 510 567 598 706 7 5 6 6 lG, BuTCy/lG 1G. BuTCy/lG 1G. BuTCy/lG l G , BuTCy/lG IVS1, 6/IVS1, 110 IVS1, 6/IVS1, 1 N39/N39 IVS1, 6/IVS1, 6 A/A A/A N39/A N.4 N.4 Months Post-BMT % Donor 85 68 20 24 29 45 51 36 25 41 3 20 3 26 43 2 3 7 10 20 2 5 3 1 2 3 100 90 100 5 4 7 6 100 73 83 Trace 0 90 100 100 0 89 84 84 81 100 94 96 96 96 97 Clinical Outcome Alive and well Alive and well Alive and well Thalassemia intermedia Alive and well Alive and well TM Alive and well TM Alive Alive Alive Alive and and and and well well well well Alive and well Abbreviations: BuCy, busulfan cyclophosphamide;TLI, total lymphoid irradiation; BuTCy, busulfan thiotepa cyclophosphamide; lM, CAMPATH 1M; 1G. CAMPATH 1G. If the donor is a carrier, the detection of residual host cells is less sensitive, because the donor DNA also carries the mutant allele. In such situations, the level of the mutant allele in the absence of residual host cells is already 50%; therefore, the level of detection begins only at 53% to 55% mutant alleles, corresponding to 5% to 10% host cells. Where other engraftment marker techniques have failed, the new method enabled us to demonstrate an incidence of 50% (7 of 14 patients) MC in the recipients of T cell-depleted allografts. Furthermore, the technique demonstrated that MC in the seven patients was stable. In malignant hematologic diseases using a variety of engraftment marker methods, an MC incidence of 10% to 20% has been reported after unmodified BMT.'s-20It should be emphasized that malignant cells may persist among the donor cells in a tumor dormancy-like state. This may be misleading when interpreting data, because MC in these situations is extremely unstable. In the nonmalignant disorder TM, MC has been evaluated recently in 72 patients by Manna et a13 using the analysis of the variable number of tandem repeats (VNTR). In that study, the incidence of MC decreased with time (ie, unstable chimerism), reaching 8% at 14 months post-BMT. However, as also noted by these investigators, the VNTR markers do not provide quantitative information on residual host cells. Some investigators have reported that either high-dose irradiation or additional chemotherapy may reduce the incidence of MC2' in a variety of diseases, whereas others found the development of MC to be independent of the conditioning regimen In the above-cited study by Manna et al.' the conditioning regimen was shown to influence the frequency of MC post-BMT. The present study indicates that the high incidence of MC may be related to TCD before BMT, despite increasing the conditioning regimen by either TLI or monoclonal anti-human lymphocyte antibody.23Indeed, TCD has been reported to increase relapse and rejection rates in malignant diseases because of a lack of donor anti-host lymphocyte-mediated reaction.22324 The fact that MC has remained stable to date (longest follow up, 68 months) suggests that MC in patients with thalassemia need not lead to rejection of the graft or recurrence of the disease. It should be emphasized that in our patients with stable MC, hematopoiesis is normal, even in those patients with only 70% donor cells. In this context, it should also be noted that the patient with approximately 5% residual donor cells displays clinical symptoms of thalassemia intermedia at more than 4 years post-BMT. Judged by other engraftment assays, such a patient might be considered to harbor 100% host cells. A similar observation, ie, that stable MC permits functional donor hematopoiesis, has recently been described in a murine model, in which correction of &thalassemia was achieved in mice transplanted with normal congenic bone m a r r ~ w . ~The . ~ ' mice were conditioned by sublethal total From www.bloodjournal.org by guest on December 29, 2014. For personal use only. D-GLOBIN MUTATION IN THALESSEMIAPOST-BMT body irradiation, and, notwithstanding the resulting mixed red blood cell chimerism, correction of anemia was recorded. Quantitation of donor-type early hemapoietic progenitor cells (CFU-S) demonstrated that the correction of the anemia arose from a minority of normal immature bone marrow cells. The investigators concluded that successful BMT for @thalassemia does not necessarily require total ablation of endogenous host cells. Because the &globin gene point mutation marker is completely independent of HLA, sex, blood type, and VNTR, it constitutes a sensitive and accurate tool for determination of chimerism in patients with TM after BMT, as evidenced by the high proportion of patients with MC detected in the present study. In conclusion, our study suggests that the &globin gene point mutation assay is ideal for early detection of engraftment, rejection, or recurrence of Th4 after BMT. The incidence of MC in this report was higher than might have been expected from the studies published to date, which leads us to surmise that posttransplant MC with respect to nonmalignant diseases is underreported. This may be attributed to the fact that the methods currently in use to detect residual host cells and MC are of limited sensitivity. One should also consider that TCD may play a critical role in the occurrence of MC. In light of this possibility, the mode of GVHD prophylaxis as practiced today should be reconsidered. Future studies will be required to address the issue of cell-mediated immunotherapy to eliminate residual host cells in patients with TM after BMT. Finally, a crucial point of this report concerns the stable MC exhibited by our patients, as detected by the &globin mutation assay. These findings imply that total eradication of the host hematopoietic system to ensure sustained engraftment in patients with TM is not an absolute necessity. ACKNOWLEDGMENT We thank Merav Siani for assistance in some of the experiments and Dr T. Pugatsch for the Y chromosome analyses. REFERENCES 1. Lucarelli G, Galimberti M, Polchi P, Angelucci E, Giardini C, Politi P, Durazzi SM, Muretto P, Albertini F Bone marrow transplantation in patients with thalassemia. N Engl J Med 322:417, 1990 2. Lucarelli G, Galimberti M, Polchi P, Angelucci E, Baronciani D, Giardini C, Andreani M, Agostinelli F, Albertini F: Marrow transplantation in patients with thalassemia responsive to iron chelation therapy. N Engl J Med 329:840, 1993 3. Manna M, Nesci S , Andreani M, Tonucci P, Lucarelli G: Influence of the conditioning regimens on the incidence of mixed chimerism in thalassemic transplanted patients. Bone Marrow Transplant 12:70, 1993 (suppl 1) 4. Nesci S , Manna M, Andreani M, Fattorini P, Graziosi G, Lucarelli G: Mixed chimerism in thalassemic patients after bone marrow transplantation. Bone Marrow Transplant 10:143, 1992 5. Lawler M, Humphries P, McCann SR: Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction. Blood 77:2504, 1991 6. Durnam DM, Anders KR, Fisher L, O’Quigley J, Bryant EM, Thomas ED: Analysis of the origin of marrow cells in bone marrow transplant recipients using a Y-chromosome-specific in situ hybridization assay. Blood 74:2220, 1989 3245 7. Blazar BR, Orr HT, Arthur DC, Kersey JH, Fillipovich AH: Restriction fragment length polymorphism as markers of engraftment in allogeneic marrow transplantation. Blood 66:1436, 1985 8. Roth MS, Antin JH, Bingham EL, Ginsburg D: Use of polymerase chain reaction-detected sequence polymorphisms to document engraftment following allogeneic bone marrow transplantation. Transplantation 49:714, 1990 9. Uggozoli L,Yam P, Petz LD, Ferrara GB, Champlin RE, Forman SJ, Koyal D, Wallace RB: Amplification by the polymerase chain reaction of hypervariable regions of the human genome for evaluation of chimerism after bone marrow transplantation. Blood 77:1607, 1991 10. Chalmers EA, Sproul A M , Mills KI, Gibson BE, Burnett AK: Use of the polymerase chain reaction to monitor engraftment following allogeneic bone marrow transplantation. Bone Marrow Transplant 6:399, 1990 1l . Baer BMAM, Schattenberg A, Van Dijk BA, De Man MM, Kunst VAJM, De Winter T: Host and donor erythrocyte repopulation patterns after allogeneic bone marrow transplantation analysed with antibody-coated fluorescent microspheres. Br J Haematol 72:239, 1989 12. Cobbold S , Martin G, Waldmann H: Monoclonal antibodies for the prevention of graft versus host disease and marrow graft rejection. The depletion of T cell subsets in vitro and in vivo. Transplantation 42:239, 1986 13. Waldmann H,Polliack A, Hale G, Or R, Cividalli G, Weiss L, Weshler Z, Samuel S , Manor D, Brautbar C, Rachmilewitz EA, Slavin S : Elimination of graft-versus-host disease by in vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (CAMPATH-l). Lancet 2:483, 1984 14. Slavin S , Waldmann H, Or R, Cividalli G, Naparstek E, Steiner-Salz D, Michaeli J, Galun E, Weiss L, Samuel S , Morecki S , Bar S , Brautbar C, Weshler Z, Hale G, Rachmilewitz EA, Reisner Y: Prevention of graft vs host disease in allogeneic bone marrow transplantation for leukemia by T cell depletion in vitro prior to transplantation. Transplant Proc 17:465, 1985 15. Naparstek E, Hardan I, Ben-Shahar H, Cohen P, Mumcuglu M, Samuel S , Weiss L, Hale G, Waldmann H, Slavin S : Anew method for prevention of graft versus host disease (GVHD). XVIII Annual Meeting of the International Society for Experimental Hematology, Paris, France, 1989, p 723 (abstr 17) 16. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 161215, 1988 17. Rund D, Cohen T, Filon D, Dowling CE, Warren TC, Bar& I, Rachmilewitz EA, Kazazian HH, Oppenheim A: Evolution of a genetic disease inan ethnic isolate: P-thalassemia in the Jews of Kurdistan. Proc Natl Acad Sci USA 88:310, 1991 18. Roux E, Abdi K, Speiser D, Helg C, Chapuis B, Jeannet M, Roosnek E: Characterization of mixed chimerism in patients with chronic myeloid leukemia transplanted with T-cell-depleted bone marrow: Involvement of different hematologic lineages before and after relapse. Blood 81:243, 1993 19. Knowlton RG, Brown VA, Braman JC, Barker D, Schumm J W , Murray C, Takvorian T, Ritz J, Donis-Keller H: Use of highly polymorphic DNA probes for genotypic analysis following bone marrow transplantation. Blood 68:378, 1986 20. Bertheas MF, Lafage M, Levy P,Blaise D, Stoppa A M , Viens P, Mannoni P, Maraninchi D: Influence of mixed chimerism on the results of allogeneic bone marrow transplantation for leukemia. Blood 78:3103, 1991 21. Frassoni F, Stmda P, Sessarego M, Miceli S , Corvo R, Scarpati D, Vitale V, Piaggio G, Raffo MR, Sogno G: Mixed chimerism after allogeneic marrow transplantation for leukaemia: Correla- From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 3246 tion with dose of total body irradiation and graft-versus-host disease. Bone Marrow Transplant 5:235, 1990 22. Roy DC,Tantravahi R, Murray C, Dear K, Gorgone B, Anderson KC, Freedman AS, Nadler LM, Ritz J: Natural history of mixed chimerism after bonemarrow transplantation with CD6-depleted allogeneic marrow: A stable equilibrium. Blood 75:296, 1990 23. Or R, Naparstek E, Aker M, Cividalli G , Engelhard D, Brautbar C, Weshler Z, Weiss L, Mumcuglu M, Rachmilewitz EA, Slavin S: Bone marrow transplantation with T-cell depleted allo- KAPELUSHNIK ET AL grafts for the treatment of severe beta thalassemia major. Prog Clin Biol Res 309:217, 1989 24. van den Bos C, Kieboom D, Wagemaker G: Correction of murine P-thalassemia by partial bone marrow chimerism: Selective advantage of normal erythropoiesis. Bone Marrow Transplant I2:9. 1993 25. Wagemaker G , Visser TP, van Bekkum DW: Cure of murine thalassemia by bone marrow transplantation without eradication of endogenous stem cells. Transplantation 42:248, 1986 From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 1995 86: 3241-3246 Analysis of beta-globin mutations shows stable mixed chimerism in patients with thalassemia after bone marrow transplantation J Kapelushnik, R Or, D Filon, A Nagler, G Cividalli, M Aker, E Naparstek, S Slavin and A Oppenheim Updated information and services can be found at: http://www.bloodjournal.org/content/86/8/3241.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
© Copyright 2024