Viral infection has been shown to induce aplastic anemia, unidentified types of hepatitis being the most common cause for aplastic anemia-associated viral hepatitis. The survival rate for this group of patients after bone marrow transplantation with stem cells from an HLA-matched sibling is not well known. The aim of this study was to determine the prevalence of hepatitis G virus (HGV) and transfusion transmitted virus (TTV) infection in non-A, non-B, non-C hepatitis associated-aplastic anemia (HAAA) patients, and to define the role of bone marrow transplantation (BMT) as a therapeutic modality for this disease. Sixty-eight patients (43 males and 25 females) with aplastic anemia, underwent allogeneic BMT at the Hadassah University Hospital between 1981 and 1997. Onset of hepatitis was defined as jaundice and elevated alanine aminotransaminase (ALT) levels. Onset of aplastic anemia was defined as the first date on which varying degrees of pancytopenia occurred: hemoglobin level below 10 g/dl, WBC below 2 × 109/l and low platelet count 10 × 1010/l. Serial serum samples from HAAA patients were assayed for virological and/or serological markers of hepatitis A, B, C, D, E, G viruses, TTV and parvovirus B19. Seventeen of the 68 patients with aplastic anemia (25%) suffered from hepatitis, 12 males and five females, ages 5 to 36 years. The mean interval between onset of hepatitis and first indication of aplastic anemia was 62 days (range 14–225 days). The development of aplastic anemia was unrelated to age, sex or severity of hepatitis. Ten of the 17 patients (59%) achieved complete ALT recovery prior to the diagnosis of aplastic anemia. Serum samples were available for 15 patients; none had evidence of acute or active hepatitis A, B, C, D, E, G and TTV virus infection at the time of diagnosis. Parvovirus B19 DNA sequences were not detectable in 10 of 12 tested cases; two positive results were detected in serum samples obtained after blood transfusion, making the analysis of these positive results difficult. All 17 patients underwent BMT. The mean post-BMT follow-up period was 38 months (range 1 day–123 months), five patients (30%) died 1 to 160 days post BMT, and 12 (70%) are alive 31 to 123 months after BMT. Relapsing hepatitis was not observed in any of the patients. In conclusion, HAAA is a disease of the young and the etiologic agent associated with HAAA remains unknown. HGV, TTV and parvovirus B19 sequences were not detected in any of the HAAA cases. The survival rate after BMT with stem cells from an HLA-matched sibling is similar to that for patients with non-hepatitis-associated aplastic anemia. Bone Marrow Transplantation (2001) 27, 183–190.
Aplastic anemia (AA) is a disease of low incidence1 and high fatality,2 the cause of which is unknown. It has been proposed that patients with AA have a genetic predisposition due to a high incidence of HLA class II antigens DR23 and DPw3.4 Several pathogenetic abnormalities have been found in these patients;56789 however, it is unclear whether they are inciting events or represent epiphenomena secondary to the disease. In many AA patients, an increase in the number and percentage of activated circulating suppressor T lymphocytes has been detected, which is consistent with response to a viral infection.10 In fact, several reports have implied that the viral illness was a precipitating event in some cases of AA.111213 The relationship between hepatitis and the subsequent development of AA has been documented in over 200 cases.11141516 Although HAV and HBV have been associated with AA in a small number of patients, most cases are related to non-A, non-B viral hepatitis.1718 The median survival of untreated patients with AA is 3 to 6 months, with only 20% surviving beyond 1 year.1920 Immediate and aggressive therapy is usually indicated for most patients with severe AA, including allogeneic BMT.1920
The aim of this study was to determine the causative viral hepatitis agents, possibly associated with HAAA, to describe the natural history of the disease, and to assess the effect of BMT on survival of patients with HAAA.
Patients and methods
Sixty-eight patients underwent BMT for AA at the Hadassah University Hospital between 1981 and 1997. HAAA was defined as AA, meeting the International Agranulocytosis and Aplastic Anemia Study Group definition,1 occurring in a patient who sought medical attention for hepatitis with documented ALT elevation, or in patients with a concurrent diagnosis of the two diseases, but who sought attention for anemia, bleeding or fever. Onset of hepatitis was defined as the first transaminase level greater than 2.5 times normal, or the first medical visit for a complaint of jaundice. Criteria for defining severe AA included two out of three peripheral blood findings, ie neutrophils <0.5 × 109/l, platelet count <20 × 109/l and reticulocytes less than 1% corrected for the hematocrit. In addition to peripheral blood findings, bone marrow hypocellularity (below 50%) was required.
Medical history, physical examination and laboratory tests were revised from medical files at the time of hepatitis, AA and BMT course. Serum samples were analyzed for potential etiological causes including hepatitis A, B, C, D, E, G viruses and parvovirus B19.
Sera of patients were collected, stored at −20°C, thawed and analyzed for serological markers. Anti-HAV IgM antibodies were detected by HAVAB-M (Abbott Laboratories, North Chicago, IL, USA). Anti-HAV Ig-total antibodies were detected by HAVAB RIA (Abbott Laboratories). HBV surface antigen (HBsAg) was detected by a radio immunoassay kit (AUSRIA; Abbott Laboratories). Anti-HBs IgG antibodies were detected by using a non-competitive ‘sandwich’ immunoradiometric test (AB-AUK-3; Sorin Biomedica, Saluggia, Italy). For anti-HCV detection we used third generation kits (either HCV EIA or IMX HCV, both from Abbott Laboratories). HCV serology was confirmed by a third generation recombinant immunoblot assay (RIBA HCV 3.0 SIA; Chiron Corporation, Emeryville, CA, USA and Ortho Diagnostic Systems, Raritan, NJ, USA). This assay uses five recombinant HCV encoded antigens, the two recombinant antigens (c33c which corresponds to NS3 and NS5) and a mix of two of the synthetic peptides (c100p and 5–1-1p both correspond to the NS4) are derived from putative nonstructural regions of the virus, while the third peptide (c22p) corresponds to the putative nucleocapsid (core) viral protein. Anti-HIV antibodies were tested using an in vitro enzyme immunoassay (Abbott HIV-1/HIV-2 3RD generation Plus EIA; Abbott Laboratories).
HCV RNA sequences were qualitatively tested using the AMPLICOR HCV assay (Roche Diagnostic System, Branchburg, NJ, USA).
HDV RNA was extracted from 200 μl serum in STAT-60 (Tel-Test ‘B’, Friendswood, TX, USA) and disolved in 20 μl sterile DDW. cDNA was synthesized using 5 μl RNA in 50 μl reaction volume containing 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 10 mM DTT, 0.5 mM dNTPs, 1 μM antisense primer 214 (nt 1331–1311): 5′-CTC AGG GGA GGG TTC TCC GAC-3′ 20 U RNasin, 20 U AMV RT (Life Sciences, Bethesda, MD, USA). The RT reaction was performed at 42°C for 2 h. PCR was performed in a reaction mixture volume of 50 μl containing 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100, 2.5 mM MgCl2, 0.2 mM dNTPs, 1 μM sense primer 120 (nt 885–909): 5′-ATG CCA TGC CGA CCC GAA GAG GAA-3′, 0.5 μM antisense primer 214, 2.5 U Taq polymerase and 10 μl cDNA. The reaction was carried out by 40 cycles of PCR consisting of 95°C for 1 min, 42°C for 1 min, 72°C for 1.5 min. PCR products (10 μl) were analyzed on a 2% agarose gel.
HEV-RNA was extracted from 200 μl serum in STAT-60 (RNA isolation reagent) and dissolved in 20 μl sterile DDW. cDNA was synthesized using 10 μl of RNA and 2.5 μM of antisense primer R1 (nt 4876–4858) 5′-CAG GGC CCC AA (G)T TCT TCT C-3′. The reaction mixture contained 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100, 2.5 mM MgCl2, 10 mM DTT, 0.5 mM dNTPs, 10 U RNasin, 10 U AMV RT (Life Sciences) for 120 min at 42°C. PCR was performed in a reaction mixture volume of 100 μl containing 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100, 3 mM MgCl2, 0.35 mM dNTPs, 1 μM sense primer F1 120 (nt 4459–4478): 5′-GCT ATT ATG GAF (A) GAG TGT GG-3′, 5 U Taq polymerase (Promega, Madison, WI, USA) and 20 μl cDNA. The reaction was carried out by 25 cycles of PCR consisting of 94°C for 1.5 min, 50°C for 1.5 min, 72°C for 1.5 min. The second PCR reaction was performed as before, with 10 μl of the first PCR reaction mixture and 10 μM of each of the nested primers, sense F2 (nt 4522–4539): 5′-GCG TGG ATC TTG CAG GCC-3′, and antisense R2 (nt 4760–4743): 5′-TTC AAC TTC AAG (A) CCA CAG CC-3′. PCR products (10 μl) were analyzed on a 2% agarose gel.
The detection of HGV-RNA sequences was similar to the recently reported method21 with some modifications. RNA was extracted from 200 l of human serum by using TrisolvTM reagent (Biotecx Laboratories, Houston, TX, USA). RT step was conducted as described employing the 5′-NCR primers, and the PCR of the targeted sequences was performed with Taq polymerase. Detection of the amplification products was performed by the Enzymun-Test DNA detection system (Boehringer Mannheim, Mannheim, Germany).
B19 parvovirus-DNA detection
DNA was extracted from 200 μl of human serum by using the QIAamp Blood kit (Qiagen, Hilden, Germany). Nested PCR was carried out according to Musiani et al,22 using 10 μl DNA, 2.5 U Taq Polymerase (Promega), in reaction mixture containing Taq buffer, 200 mM dNTPs, 1.5 mM Mg and 300 ng of first round primers: 5′-CTTT AGGT ATAG CCAA CTGG-3′ and 5′-ACAC TGAG TTTA CTAG TGGC-3′, 35 cycles of 94°C for 1 min, 50°C for 0.5 min, and 72°C for 1 min. Second round PCR was performed at 10 μl of reaction product, using nested primers: 5′-CAAA AGCA TGTG GAGT GAGG-3′ and 5′-CCTT ATAA TGGT GCTC TGGG-3′, and using the same amplification protocol. PCR products were analyzed on 3% agarose gel.
DNA was extracted from 200 μl of serum using Qiagen Blood Kit (Qiagen) spin columns and resuspended in 50 μl of water. Ten μl of the DNA was subjected to nested PCR in a 50 μl reaction mixture containing 10 times PCR buffer, 2 mM MgCl2, 0.5 mM dNTPs and 0.5 U of Taq polymerase (Promega). For the first round, sense primer TT1 (5′-CAG ACA GAG GAG AAG GCA ACA TC-3′) and antisense primer TT2 (5′-TAC CA(T) TTA GCT CTC TAT TCT AT(A)-3′) were used for the amplification of the target sequence of 326 base pairs. For the second round, sense primer TT3 (5′-GG(AC) AA(CT)ATG (CT)T(AG) TGG ATA GAC TGG-3′) and antisense primer TT4 (5′-CTA CCT CCT GGC AAT TTA CCA-3′) were used for a 277 base pair target sequence. MgCl2 concentration was increased to 2.5 mM in the second round. The reaction was carried out by 35 cycles of PCR consisting of 94°C for 30 s, 60°C for 45 s, 72°C for 45 s and 5 min extension at 72°C. The amplicons were electrophoresed in 2% agarose gel, stained with ethidium bromide and observed under ultraviolet light.
Sixty-eight patients were diagnosed as suffering from severe AA (43 males and 25 females). The mean age of these 68 patients was 18.9 years (range 3–36 years). Seventeen of them (25%) fulfilled the criteria of HAAA (Table 1). The other 51 patients did not fulfill the criteria of HAAA.
In the study group of 17 patients with HAAA, 12 were males and five females (Table 1), with a mean age of 18.2 years (range 5–36 years). All cases were healthy prior to the hepatitis event, and took no medications prior to the development of hepatitis. Eight patients were Palestinian Arabs, four were Sephardic Jews, three were Ashkenazi Jews and two were Greek.
The onset of hepatitis was documented by jaundice and elevated ALT in all cases. The mean ALT level was 1462 IU (normal levels 6–53 IU), ranging from 260 to 2908 IU. One patient (case No. 11) had subfulminant hepatic failure, which resolved without the need of orthotopic liver transplantation (OLT). The mean interval between onset of hepatitis and development of AA was 62.3 days (range 14–225 days). At diagnosis of AA, ALT levels returned to normal in 10 patients and were elevated in seven, ranging from 260 to 2847 IU. The mean interval between onset of AA and BMT was 71.4 days (range 18–364 days).
Sixteen of 17 patients had a hemoglobin level below 10 g% at the initial presentation of AA, 16/17 had low WBC count although severe leukopenia (WBC count below 2 × 109/l) was detected in only seven cases. All patients had low platelet counts (10 × 1010/l); 14/17 had severe thrombocytopenia below 20 × 109/l. All 17 patients underwent BMT (Table 2).
All 51 AA patients without underlying hepatitis had positive serum anti-HAV Ig-total antibodies, while anti-HAV IgM and anti-HCV antibodies were undetectable at presentation of AA. However, seven of the 51 (7%) had positive serum anti-HBc-total antibodies, three of them also had anti-HBs antibodies but none of them had serum HBsAg. HDV, HEV, HGV, TTV Parvovirus B19 were not investigated in this group.
Serum samples were available for 16/17 patients with AAAH. Serological and virological parameters were negative for ongoing infection by hepatitis A, B, C, D, E and G at the time of clinical hepatitis (Table 3).
HAV: All serum samples collected at the time of diagnosis of hepatitis were negative for anti-HAV IgM antibodies, and 15 of 16 patients had anti-HAV IgG antibodies. Case number 12 was negative for both anti HAV IgG and IgM antibodies.
HBV: All 15 serum samples collected at the time of the diagnosis of hepatitis were negative for both HBsAg and anti-HBc IgM antibodies. However, five patients had anti-HBc IgG antibodies, and four of the five anti-HBc IgG cases also had anti-HBs antibodies. Seven patients had anti-HBs antibodies; two had anti-HBs antibodies without anti-HBc IgG.
HCV: All 15 serum samples collected at the time of hepatitis diagnosis were negative for anti-HCV antibodies by both EIA and RIBA tests.
HDV, HEV and HGV RNA viral sequences were negative for all 15 patients from whom serum samples were collected at the time of hepatitis.
Following the above mentioned investigation, 12 serum samples remained. Parvovirus B19 DNA sequences were not detectable in 10 of these 12 tested cases; the positive results in cases Nos 11 and 12 were obtained from serum samples taken following blood transfusion, making the interpretation of these positive results difficult.
TTV DNA sequences were not detectable in 12 cases tested.
In all instances where serum was available, other common viral agents (such as EBV, CMV, HSV, VZV, and HIV), as well as drugs, metabolic disorders, and autoimmune hepatitis were excluded as causes of disease.
Conditioning protocols prior to BMT
In this study group summarizing the data of AA patients between 1981 and 1997, five conditioning protocols were used during this time period: the first protocol involved nine hypertransfused patients who were transplanted from fully HLA-matched siblings using unmanipulated inoculum. The conditioning regimen included fractionated total lymphoid irradiation (TLI; 200 cGy X6), followed by cyclophosphamide (CY; 50 mg/kg/day × 4 days). Five out of nine patients received post-transplant GVHD prophylaxis consisting of methotrexate (10 mg/m2 i.v. once a week for 3 months). In the era of cyclosporin A, GVHD prophylaxis was based exclusively on this drug in 5/9 patients for 3–6 months post BMT. In the second protocol 31 patients received T cell-depleted allografts from HLA-matched sibling donors using CAMPATH 1M in vitro. Conditioning included fractionated TLI (150 cGy × 12 during 6 days), followed by CY (50 mg/kg/day × 4 days). In the third protocol 18 patients received T cell-depleted allografts from HLA-matched sibling donors using CAMPATH 1G ‘in the bag’. In the fourth protocol six patients received T cell-depleted allografts from HLA-matched sibling donors using CAMPATH 1G in vivo (i.v., 10 mg/kg/day for 4 days). In the last protocol, four hypertransfused patients lacking a family matched donor underwent BMT from mismatch siblings in two cases and unrelated matched donors in another two. The first two patients were treated according to the second protocol, while the latter two patients received the first protocol.
All 17 HAAA patients underwent allogeneic stem cell transplantation from HLA-matched donors (Table 2). Sixteen of 17 were transplanted from a sibling donor and one from an unrelated donor. The donor's sex was identical in 10 patients, and different in five male patients and two females. Major ABO incompatibility was present in 4/17 cases. In 16 of 17 patients, the conditioning regimen prior to BMT was based on cyclophosphamide 200 mg/kg, and addition of either total lymphoid irradiation (1800 rad in 12 fractions) or monoclonal anti-T cell antibody (CAMPATH 1G). All patients received T cell-depleted allografts with a total number of mononuclear cells between 1.57 and 8.74 × 108/kg. Only case No. 17 received anti-T cell globulin, fludarabine and T cell-depleted peripheral blood stem cells.
The mean post-BMT follow-up period of AAAH was 38 months (range 1 day–123 months). The Kaplan–Meier survival curve of 17 patients with AAAH was similar to that of 51 AA patients who did not have an underlying history of hepatitis (see figure). The differences in survival rates were non-significant in these patients groups (P = 0.51). Of 17 AAAH patients, 15 achieved bone marrow engraftment and normal blood counts. Five patients died due to sepsis following BMT, between 1 day and 5.3 months after the procedure (mean 1.9 months). Two of five cases did not achieve bone marrow engraftment (cases 4 and 6) and died immediately following BMT, while the other three patients survived longer (0.03–1.5 and 2–5.3 months, respectively). Twelve AAAH patients were alive at the end of the follow-up period (mean 52.8 months; range 31–123 months). All these 12 patients achieved bone marrow engraftment. Only three patients (cases 2, 9 and 15) had chronic graft-versus-host disease (GVHD) treated with steroids and cyclosporin A. All 12 normalized ALT levels post BMT. However, three cases developed a new rise in ALT during the post-BMT period: case No. 10 – serum ALT levels were 207 IU 2 months after BMT, coinciding with Candida sepsis. Case No. 15 – serum ALT levels were 313 IU 7 months after BMT associated with severe chronic GVHD. Case No. 17 – ALT level elevated to 237 IU shortly before the patient died from Diphtheroid and Candida sepsis 2 months post BMT. Hepatitis virus serology remained unchanged in all three cases at the time of ALT elevation. There was no evidence of relapsing hepatitis or relapsing AA in any of the 17 patients post BMT.
HAAA accounts for 25% of AA patients who underwent allogeneic BMT in our institute during the last 16 years. Presentation of hepatitis prior to AA occurrence, manifested by ALT elevation in 16 patients and sub-fulminant hepatitis in one case. The development of AA was unrelated to age, sex or severity of hepatitis. More than one third of reported pediatric patients who presented with non-A, non-B fulminant hepatic failure also developed AA either before or shortly after liver transplantation.23 In our patients the interval between onset of hepatitis and that of AA was 14–225 days (mean 62.3). Length of the interval was unrelated to age, sex or severity of hepatitis. In about 10% of reported cases, AA occurred more than 1 year after diagnosis of hepatitis. HAAA cases reported in previous studies were generally younger (age of 18 to 20 years) than in our study group. Most were male, similar to our observation, and their survival was shorter (10 weeks)1718 than that of patients in this study. In previous reports, hepatitis resolved partially or completely 4 to 12 weeks before AA was noted.
All HAAA patients in this study were non-A, non-B, non-C, non-D, non-E and non-G. Eight patients had anti-HBc IgG and/or anti-HBs antibodies in serum, an indication of past exposure, or a result of immunization. Thus, there was no evidence of recent or acute HBV infection in our patient group. In this group of patients HGV infection was not a causative agent for HAAA, as previously reported.2425 Similar to the reported data, our study supports the lack of an association between HAAA and HCV,2627 or HEV.14 Brown,24 Zaidi,25 Byrnes28 and their colleagues report five patients with HAAA who had HGV RNA detected in their serum at presentation (before any blood transfusion was administered). A newly described novel non-A-G DNA virus, named transfusion-transmitted virus (TTV), was recently detected with high prevalence in Japanese patients with fulminant hepatitis and chronic liver disease of unknown aetiology. TTV viraemia is frequent in the blood-donor population, and transmission of TTV through transfusion of blood components may have occurred extensively. The high prevalence of active TTV infection in the general population, both in the UK and in Japan, and the lack of significant liver damage, suggest that TTV, similar to hepatitis G virus (HGV), may be an example of a human virus with no clear disease association.293031 However, there are no available data regarding TTV in the HAAA group. In the group of HAAA patients reported here, TTV was not detected, suggesting that TTV is not a major etiological agent for HAAA.
A number of other viruses, which are not strictly hepatotropic, may induce bone marrow failure. Epstein–Barr virus (EBV) which might cause hepatitis has been detected in marrow cells. The mechanisms of marrow aplasia following EBV infection may be related to a direct cytotoxic effect, or an immunologic reaction of the host to the virus. Parvovirus B-19 infection leads to transient erythroid aplasia, induces severe AA, and only rarely causes hepatitis.233233 HIV infection is frequently associated with varying degrees of cytopenia and in rare cases hepatitis. The marrow is often cellular, but occasional cases of AA have been reported.834 In these patients, marrow hypoplasia may result from direct viral suppression on the bone marrow cells, induction of an immune-mediated effect against bone marrow cells, and from the many drugs used to control viral replication in this disorder.
AA is more common in the far east, an area with a high prevalence of HAV, HBV, HCV, and HIV infection. In addition, HAAA is associated with low socioeconomic status,35 rice farming (with attendant exposure to water and insects),36 and a past exposure to HAV.37
One of the possible mechanisms of both AA and hepatitis is T cell-mediated suppression of bone marrow38 and liver infiltration with activated CD8 cells, respectively. The current medical treatment of AA and HAAA for patients without an available HLA-matched donor is immunosuppression. Immunosuppressive therapy with anti-lymphocyte serum (ALS) and anti-lymphocyte globulin (ATG) appears to act by reducing the circulating cytotoxic T cells, and possibly by mediating the release of hematopoietic growth factors from certain T cells to enhance the growth of hematopoietic stem cells.3940 Response to ALS or ATG therapy in AA patients is about 50%.4142434445 Additional therapeutic modalities have also been suggested: (1) marrow recovery can also occur following high doses of glucocorticoids,4647 or cyclosporin A.484950 (2) Androgens were not effective as a primary treatment for severe or moderate AA.5152 Combined immunosuppressive treatment where androgens have been employed in an attempt to improve response and survival, have resulted in both negative52 and positive53 responses. (3) Other therapeutic modalities including the use of hematopoietic growth factors,545556 intravenous gamma globulin (IVIG),57585960 and splenectomy had a marginal or no effect.
BMT has been highly successful for patients with AA, curing 75–85% of untransfused patients and 55–60% of those with multiple previous transfusions.6162 Relapse of AA occurs in about 15% of patients following BMT, although underlying rejection can not be excluded.63
The prognosis of patients with HAAA has been reported to be extremely poor.151664 Previous publications have suggested that HAAA of the same degree of severity responds as well as does that with no prior history of hepatitis to treatment with both immunosuppression and with BMT.24656667686970 Children appear to respond better to BMT than do adults with HAAA. Successful allogeneic BMT in patients with HAAA has also been reported in a number of case reports.656667686970 In other reports however, BMT was associated with poor survival in HAAA patients compared to AA of other etiologies.7172 Survival, graft rejection, and incidence of acute and chronic GVHD after marrow transplantation for HAAA are similar to those in patients transplanted for AA of other etiologies. Previous hepatic damage from viral hepatitis and liver function abnormalities existing at the time of grafting do not appear to increase the risk of post-transplant morbidity and mortality from hepatocellular damage or veno-occlusive disease in cyclophosphamide-conditioned patients.73
In summary, our study suggests that HAAA was probably not related to known viral hepatotropic agents. No cases of recurrent hepatitis occurred after BMT during the follow-up period, with patients having reasonable survival rates.
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We would like to thank all staff members of the Department of Bone Marrow Transplantation, and the Liver Unit, for the support in this study, in particular Nili Daudi, Ruth Adler, Rima Barsak, Ludmela Rivkin and Simcha Samuel for their valuable assistance.
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Safadi, R., Or, R., Ilan, Y. et al. Lack of known hepatitis virus in hepatitis-associated aplastic anemia and outcome after bone marrow transplantation. Bone Marrow Transplant 27, 183–190 (2001) doi:10.1038/sj.bmt.1702749
- aplastic anemia
- bone marrow transplantation
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