Tatsuya Chuhjo

Relative increase of granulocytes with a paroxysmal nocturnal haemoglobinuria phenotype in aplastic anaemia patients : the high prevalence at diagnosis

Abstract: To clarify the pathologic significance of granulocytes exhibiting the paroxysmal nocturnal haemoglobinuria (PNH) phenotype in patients with aplastic anaemia (AA), we examined peripheral blood from 100 patients with AA for the presence of granulocytes deficient in glycosylphosphatidylinositol (GPI)‐anchored proteins using a sensitive flow cytometric assay. A significant increase in the frequency of CD55−CD59−CD11b+ granulocytes (>0.003%) compared to normal individuals was observed in 31 of 35 (88.6%) patients with untreated AA at diagnosis. The proportions of patients showing increased PNH granulocytes in treated AA patients with a short (<5 yr) and long (>5 yr) disease duration were 68.6% (11/16) and 20.4% (10/49), respectively. When 19 patients showing increased frequency of PNH granulocytes before therapy were studied 6–12 months after antithymocyte globulin plus cyclosporin A therapy, the frequency decreased to 0.01–90% of pretreatment values in 15 recovering patients. These findings suggest that a relative increase in the number of PNH granulocytes is a common feature of AA at diagnosis, and that it may represent the presence of immunologic pressure to normal haematopoietic stem cells as a cause of AA.

Graft rejection and hyperacute graft-versus-host disease in stem cell transplantation from non-inherited maternal antigen complementary HLA-mismatched siblings

Human leukocyte antigen (HLA)‐mismatched stem cell transplantation from non‐inherited maternal antigen (NIMA)‐complementary donors is known to produce stable engraftment without inducing severe graft‐versus‐host disease (GVHD). We treated two patients with acute myeloid leukemia (AML) and one patient with severe aplastic anemia (SAA) with HLA‐mismatched stem cell transplantation (SCT) from NIMA‐complementary donors (NIMA‐mismatched SCT). The presence of donor and recipient‐derived blood cells in the peripheral blood of recipient (donor microchimerism) and donor was documented respectively by amplifying NIMA‐derived DNA in two of the three patients. Graft rejection occurred in the SAA patient who was conditioned with a fludarabine‐based regimen. Grade III and grade IV acute GVHD developed in patients with AML on day 8 and day 11 respectively, and became a direct cause of death in one patient. The findings suggest that intensive conditioning and immunosuppression after stem cell transplantation are needed in NIMA‐mismatched SCT even if donor and recipient microchimerisms is detectable in the donor and recipient before SCT.

Successful treatment of persistent erythroid aplasia caused by parvovirus B19 infection in a patient with common variable immunodeficiency with low‐dose immunoglobulin

Parvovirus B19 causes persistent erythroid aplasia in immunocompromised hosts. From April through July 1996, we encountered five adult patients presenting with reticulocytopenia and fever caused by parvovirus B19 infection. The reticulocyte count of four patients with normal immunity recovered within two weeks after the onset of fever. However, in the one remaining patient with common variable immunodeficiency (CVI), reticulocytopenia, and other symptoms including fever and the elevation of lactate dehydrogenase (LDH) levels persisted beyond 16 days of onset. Although the DNA of parvovirus B19 was detected in the peripheral blood of the CVI patient, neither immunoglobulin Ig‐G nor Ig‐M antibodies specific to the virus were detectable. We administered 50 mg/kg of Ig to the CVI patient for six days. The reticulocyte count recovered promptly on the sixth day of the treatment and parvovirus B19 DNA was not detectable 30 days after therapy. This indicates that although patients with CVI may be susceptible to persistent erythroid aplasia during an endemic of parvovirus B19, the complication can be treated successfully with relatively low‐dose Ig. Am. J. Hematol. 60:222–224, 1999.

Lymphocyte collection for donor leucocyte infusion from normal donors: estimation of the minimum processed blood volume and safety of the procedure

To estimate the minimum volume of processed blood necessary for the purpose of donor leucocyte infusion (DLI), we determined the number of CD3+ cells harvested by apheresis from normal donors and examined adverse events during the procedure.

Cyclosporine therapy for acquired aplastic anemia: predictive factors for the response and long-term prognosis

Although cyclosporine (CsA) is a key drug in the treatment of acquired aplastic anemia (AA), the role of single-agent therapy with CsA remains unclear. To determine the efficacy of CsA in the treatment of AA, we treated 38 AA patients with CsA alone and followed up the patients for 6 months to 16 years. Twenty patients (53%) achieved either a partial or complete remission within 1 year of starting CsA therapy. Thirteen (81%) of 16 patients who showed an increase in the reticulocyte count of >20 x 109/L within 2 months achieved remission, whereas the response rate was only 32% in patients who failed to show such an increase in the reticulocyte count. The actuarial overall survival and failure-free survival rates at 5 years were 91% and 37%, respectively. These data indicate that CsA alone can achieve a sustained remission in approximately 40% of AA patients, with a low probability of inducing secondary clonal diseases. Given its low toxicity and because the effectiveness of CsA can be judged within 2 months of therapy, CsA may be the first drug of choice at outpatient clinics for AA patients not requiring transfusions.

Clinical Significance of Increased PNH-Type Cells in the Peripheral Blood of Patients with Aplastic Anemia and Refractory Anemia

A small number of blood cells with the paroxysmal nocturnal hemoglobinuria (PNH) phenotype are occasionally detected in patients with aplastic anemia (AA) and those with myelodysplastic syndrome (MDS). In an attempt to determine the clinical significance of PNH cells in bone marrow failure syndrome, we examined peripheral blood red blood cells (RBCs) and granulocytes of AA patients and MDS/refractory anemia (RA) patients, for the presence of CD55CD59 (PNH) cells using a sensitive flow cytometry. A total of 100 AA patients and 105 RA patients with varying disease duration were studied. A significant increase (≥0.003%) in the percentage of PNH cells compared to normal individuals was detected in 52% of AA patients. The frequency of patients with increased PNH cells was particularly high as 88.6% in newly diagnosed AA patients. On the other hand, the increased PNH cells were detected only in 15 of 105 (14.3%)) RA patients. None of the 15 patients with increased PNH cells showed apparent trilineage dysplasia; in these patients, RA was diagnosed mainly based on the presence of erythroid dysplasia and/or mild trilineage dysplasia as well as hypercellularity in the bone marrow aspirates from the sternum or the iliac bone. None of them exhibited karyotypic abnormalities. Pancytopenia of these patients improved with cyclosporine or anabolic steroids, or remained stable without treatments. Thus, the presence of PNH cells in peripheral blood probably serves as a marker for good prognosis in RA patients.

Relative erythroid hyperplasia in the bone marrow at diagnosis of aplastic anaemia: a predictive marker for a favourable response to cyclosporine therapy

Predicting the treatment response of aplastic anaemia (AA) is essential when considering cyclosporine (CyA) therapy among several treatment options, because it requires at least 2 months to determine whether the therapy is beneficial to a patient with AA. To identify the characteristics of patients with AA who are likely to respond to CyA therapy we retrospectively reviewed the clinical records and bone marrow smears of patients treated with CyA. Among 30 patients who received the therapy for at least 3 months within 1 year after diagnosis of AA, and who had not been exposed to antilymphocyte or antithymocyte globulin, 16 (53%) responded with disease remission. CyA‐responsive patients had a significantly higher ratio of erythroblasts to granulocytes (E/G ratio) in the bone marrow at the time of diagnosis as compared with patients refractory to therapy (P = 0.004). Multivariate analysis revealed that a high E/G ratio (>0.6) was significantly associated with a good response to CyA (P = 0.03): 15 (83%) of the 18 patients with an E/G ratio >0.6 responded, but only one (8%) of the 12 with an E/G ratio ≥0.6 did. Although the presence of subclinical paroxysmal nocturnal haemoglobinuria was suspected from the relative erythroblastosis observed in the bone marrow of these patients, flow cytometric analysis of neutrophils in the peripheral blood failed to reveal neutrophils deficient for glycosyl‐phosphatidylinositol (GPI) anchored membrane proteins in all but one case. Identification of the presence of relative erythroid hyperplasia in the bone marrow when AA is diagnosed may help to predict a favourable response to CyA therapy, and therefore facilitate the selection of optimal therapy for AA.

Demonstration of Ph1-negative host haemopoietic progenitor cells in an allogeneic marrow transdant recbient with chronic myelocytic leikaernia;sing polymerase chain reaction

Abstract: In an attempt to characterize host‐derived haemopoietic cells in mixed haemopoietic chimeras, we studied bone marrow cells from male patients with chronic myelocytic leukaemia (CML) transplanted with marrow grafts from female donors. Amplification of a Y chromosome‐specific sequence (YDNA) in DNA from marrow mononuclear cells using polymerase chain reaction (PCR) revealed persistence of host cells in 2 of 3 patients studied. On the other hand, persistence of Phl‐positive cells was unable to be demonstrated in the marrow cells from these patients using reverse transcription PCR (RT‐PCR) for detecting bcr‐abl chimeric messenger RNA. RT‐PCR sensitivity for detecting minimal Phl‐positive cells in a background of Ph1‐negative cells proved better than that of the PCR for detecting male cells among female cells. When bone marrow progenitor cells from one of the documented mixed chimeras were analyzed after an in vitro colony assay using PCR, 2 of 12 erythroid burst‐forming units (BFU‐E) proved to be YDNA‐positive, i.e., of host origin, whereas none of them was shown to be Ph1‐positive, although 12 BFU‐E analyzed in the marrow cells obtained pretransplant from the same patient were all YDNA‐ and Phl‐positive. These findings indicate that, in some marrow transplant recipients with CML, a small number of host‐derived normal haemopoietic progenitor cells may persist following lethal chemoradiotherapy and allogeneic bone marrow transplantation despite the fact that Phl‐positive clones can be eradicated.

Non‐myeloablative stem cell transplantation for accelerated‐phase chronic myeloid leukaemia: circumvention of graft rejection with donor leucocyte infusion early after transplantation

Keywords: accelerated phase; chronic myeloid leukaemia; non-myeloablative stem cell transplantation; donor leucocyte infusion; fluorescence in situ hybridization