Bone marrow transplantation in thalassemia: modifications of hepatic iron overload and associated lesions after long-term engrafting

Abstract: Among 450 thalassemic patients treated in the Hematologic Department, 50 patients who were disease-free 4–6 years after allogeneic bone marrow transplantation were sequentially studied by liver biopsy. The patients received marrow from siblings who were genotypically HLA identical at A, B, C and DR loci. For evaluation of siderosis and associated lesions, each patient underwent liver biopsy before, and again 6 months and yearly for 4 to 6 years after bone marrow transplant. Spontaneous reversibility of liver iron overload, once the need for transfusions ceased when a functioning graft had been established, was observed in the youngest patients, aged 1–8 years, whereas iron excess remained at the end of follow-up in many patients aged 9–15 years. Hypotheses about the mechanism of the iron decrease are discussed. Several cases also obtained improvement of associated pathologies such as hepatitis, probably through modifications in the mechanisms controlling their immunological status.     

Serum soluble transferrin receptor and the prediction of marrow aspirate iron results in a heterogeneous group of patients

Serum soluble transferrin receptor (sTfR) concentration has been evaluated in the diagnosis of iron deficiency in otherwise healthy individuals and in patients with rheumatoid arthritis, but has not been studied in a general population of patients with complicated clinical presentations. In this study, 145 anaemic patients with a variety of medical conditions undergoing diagnostic bone marrow aspiration for any reason were tested by a complete blood count, a panel of biochemical tests to evaluate iron status, bone-marrow aspirate iron stain, and serum sTfR concentration. Sixteen per cent lacked stainable iron in the marrow aspirate. All biochemical parameters differed significantly between patients with or without stainable marrow iron. The sTfR assay was significantly more sensitive but less specific than other iron status assays in identifying the absence of stainable iron. Logistic regression analysis demonstrated that only sTfR and ferritin contributed independently to the prediction of marrow iron status. Serum ferritin alone was highly specific but insensitive. A decision algorithm combining serum ferritin and sTfR was as sensitive as TfR and as specific as serum ferritin. The measurement of serum sTfR, especially in conjunction with serum ferritin, is a valuable addition to the existing methods for predicting the results of marrow aspirate iron stains.     

Alpha thalassaemia is associated with increased soluble transferrin receptor levels

Although α⁺ thalassaemia is the commonest haemoglobinopathy in the world, it is not known if it is associated with significant ineffective erythropoiesis, a fact of importance in interpreting its complex interaction with malaria. To study this problem, we have measured the concentrations of soluble transferrin receptor (sTfR) and ferritin in 181 children from Vanuatu with heterozygous (68) and homozygous (46) α⁺ thalassaemia, and normal controls (67). sTfR concentrations were significantly higher in both homozygotes (mean 3.1 mg/l, range 2.8–3.4) and heterozygotes (2.86 mg/l, 2.6–3.2) compared to the normal controls (2.48 mg/l, 2.3–2.7), suggesting that although globin chain imbalance is minimal, there is ineffective erythropoiesis in both these conditions. Age was also shown to significantly affect sTfR, with peak levels occurring in the 5–9 years age group. Ferritin concentrations showed a similar trend, being higher in the thalassaemic groups, although this did not reach statistical significance. No individuals had low ferritin concentrations, although two had significantly elevated sTfR levels. These observations suggest that the α⁺ thalassaemia phenotype includes an expansion of the erythron, and may suggest possible mechanisms for the increased susceptibility in babies with α thalassaemia to both P. falciparum and P. vivax malaria.     

Changes in serum erythropoietin levels during allogeneic bone marrow transplantation

Serial serum erythropoietin levels were measured in 10 consecutive patients undergoing allogeneic bone marrow transplantation. Observed erythropoietin levels are compared with those predicted from a large control population of anaemic patients not receiving chemotherapy. There was an initial acute rise in serum erythropoietin, peaking between days 1 and 4 after marrow transfusion, which was unrelated to changes in haemoglobin concentration. Patients maintained serum erythropoietin concentrations at around twice the predicted level for the first 2 weeks following transplantation, with a gradual fall into the expected range by wk 3. Erythropoietin levels did not change with episodes of bacterial infection or acute graft-versus-host disease. A patient with severe aplastic anaemia had initial successful engraftment with normalisation of erythropoietin levels, but showed a marked and amplified rise in erythropoietin 2 wk before falling peripheral blood counts indicated failure of the bone marrow graft.     

Correlation between serum level of soluble L-selectin and leukocyte count in chronic myeloid and lymphocytic leukemia and during bone marrow transplantation

Abstract: L-selectin is a glycoprotein which is one of three members in a family of cell adhesion molecules called selectins. L-selectin is present in distinct forms on both neutrophil granulocytes and lymphocytes, and it appears to play an important role in the early stages of leukocyte-endothelial cell interaction. Activation of leukocytes leads to shedding of the extracellular part of L-selectin which thus forms a soluble adhesion molecule, sL-selectin, which retains functional capacity and can be detected in serum. In the present study we have developed a specific, sensitive sandwich ELISA to measure the serum level of sL-selectin in patients with hematological and infectious disorders. Three patients with acute myeloid leukemia in remission and 1 patient with chronic myeloid leukemia in chronic phase were followed during bone marrow transplantation and the level of sL-selectin was found to correlate closely to the leukocyte counts with no detectable sL-selectin during periods of severe leukopenia. In 11 patients with chronic phase chronic myeloid leukemia and 13 patients with chronic lymphocytic leukemia the sL-selectin level was also found to correlate closely to the leukocyte count (R = 0.98; p = 0.001 and R = 0.83; p = 0.004 respectively). One CML patient with a leukocytosis of 385 times 10⁹/1 was found to have an sL-selectin concentration 625 times above normal. Ten patients with acute pneumonia were evaluated at diagnosis and at the time of follow-up 4–8 weeks later. In all patients the initial sL-selectin level was higher than at follow-up. However, no close correlation between sL-selectin and leukocyte count or CRP (C-reactive protein) at the time of diagnosis was found. In summary, we have found that the sL-selectin level in human serum closely correlates to the leukocyte count in both CML and CLL and during bone marrow transplantation. –     

Reticulocyte haemoglobin content vs. soluble transferrin receptor and ferritin index in iron deficiency anaemia accompanied with inflammation

Ferritin concentration, as a parameter of iron status that is commonly used in the diagnosis of iron deficiency anaemia (IDA), often has limited values if the iron deficiency is accompanied by inflammatory disease. This study evaluated the value of reticulocyte haemoglobin content (CHr) and soluble transferrin receptor-ferritin index (sTfR/F) in the diagnosis of IDA and differential diagnosis of IDA and anaemia of chronic disease. The study included 66 nonanaemic individuals as controls, 86 patients with IDA divided into noninflammatory and inflammatory subgroups, and 32 patients with anaemia of chronic disease. Blood count, iron, transferrin saturation, total iron binding capacity, ferritin, C-reactive protein, sTfR and CHr were determined. Receiver operator characteristic curve analysis showed very high discriminating power for CHr, soluble transferrin receptor (sTfR) and sTfR/F in the diagnosis of IDA. In patients with anaemia of chronic disease these parameters showed no significant difference from the control. CHr and sTfR enabled recognition of iron deficiency and were not affected by acute phase reaction. They are sensitive markers of body iron status with additional value to conventional tests for the detection of iron deficiency.     

Soluble c-kit levels in acute GVHD after allogeneic bone marrow transplantation

Serum soluble c-kit concentrations were measured in 11 patients with or without acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. The serum soluble c-kit levels in the patients with moderate to severe acute GVHD (grade II-IV) were significantly lower than those in the patients with no or mild acute GVHD (grade 0-I) following the onset. The data from this study indicate that measurement of serum soluble c-kit concentration is a useful indicator of severe acute GVHD.     

435名女性体检者血清铁蛋白与骨密度的相关性

目的了解正常女性随年龄增长体内铁含量与骨密度变化的关系,分析铁含量与骨密度的相关性。方法收集2011年1月至2012年12月在苏州大学附属第二医院435名22~80岁体检女性血清铁蛋白和骨密度数据。采用电化学发光法检测血清铁蛋白,采用双能X线吸收仪测定骨密度。按每5岁为1个年龄段分组,分别统计各组受试者血清铁蛋白和不同部位骨密度的平均值,观察血清铁蛋白、骨密度随年龄变化的情况。将血清铁蛋白按5等分法分类,运用非条件Logistic回归法,分析随血清铁蛋白升高,发生骨量减少风险的变化。采用多元逐步回归分析、偏相关分析法,了解正常女性血清铁蛋白变化与骨密度的相关性。结果女性血清铁蛋白随年龄增加而增加,骨密度随年龄增加而下降,两者变化趋势在围绝经期、绝经期变化显著。骨量减少组(-2.5T值-1.0)血清铁蛋白平均值显著高于骨量正常组(P0.001),骨质疏松组(T值≤-2.5)血清铁蛋白平均值显著高于骨量减少组(P0.01)和骨量正常组(P0.001)。校正混杂因素后,血清铁蛋白按5等分法分组,非条件Logistic回归显示血清铁蛋白最高组与最低组相比,股骨颈和腰椎发生骨量减少的OR值(95%CI)分别为2.82(1.25~6.38)和2.04(0.92~4.51),提示随血清铁蛋白增加,骨量减少发生的风险增加。多元逐步回归分析显示,与骨密度变化相关指标为年龄、体重、血清铁蛋白和体重指数(BMI),校正年龄、体重、BMI、C反应蛋白(CRP)等混杂因素后,血清铁蛋白与不同部位骨密度均呈显著负相关(P0.05)。结论随着年龄增长,女性血清铁蛋白逐渐升高、骨密度逐渐下降,二者呈显著负相关。血清铁蛋白增高时,骨量减少、骨质疏松发生率升高。     

Delineation of erythropoiesis in normal and malignant bone marrow using monoclonal antibody AS-E1 directed against transferrin receptors (CD71)

Abstract: We have delineated the erythropoietic compartment in normal and malignant bone marrow (BM) by using the monoclonal antibody (mAb) AS-E1 directed against the transferrin receptor by flow cytometric (FCM) analysis. In normal BM we found a bimodal expression in antigen density with a minor subset (?3%) expressing AS-E1^high and a larger subset (?15%) expressing AS-E1^low. By fluorescence activated cell sorting, morphological examination of smears stained by immunocytochemistry and by BFU–E assays the AS-E1^high fraction was shown to contain cells of erythroid origin (proerythroblasts, basophilic erythroblasts and polychromatic erythroblasts), whereas the AS-E1^low fraction consisted mainly of promyelocytes and myelocytes. In patients with malignant hematological disorders we found a more pronounced heterogeneity in the density and the degree of AS-E1^low expression compared to normal BM, and to further characterize the AS-E1^low cells in patients and to exclude that this broad reactivity interfered with the identification of the AS-E1^high cells, we employed triple-color FCM assays with mAbs directed against the myeloid surface markers CD13 and CD66 in addition to AS-E1. In all patients we found that 80–90% of the AS-E1^low cells co-expressed CD13 and/or CD66 and thus were of myeloid origin. Finally, we evaluated 2 methods for determination of the AS-E1^high subset and found an assay involving forward light scatter and logAS-E1 density to be sufficient. We conclude that AS-E1^high is a valid FCM marker for the normal erythropoiesis.     

Increased levels of soluble flt-3 ligand in serum and long-term bone marrow culture supernatants in patients with chronic idiopathic neutropenia

The levels of serum and long-term bone marrow culture supernatant soluble flt-3 ligand (sFL) were determined in 54 patients with chronic idiopathic neutropenia (CIN) and 16 normal controls. Both serum and supernatant sFL levels were significantly increased in the patients compared with controls. Individual sFL values inversely correlated with the number of circulating neutrophils and the proportion of bone marrow CD34+ cells. Supernatant sFL values positively correlated with the levels of supernatant G-CSF. These findings suggest that the impaired myelopoiesis in CIN patients is accompanied by a compensatory mechanism attempting to increase the neutrophil production at the myeloid progenitor cell level.     

A function of CD10 on bone marrow stroma

Summary. Bone marrow (BM) stromal cells express CD10 (cALLA), a surface antigen now known to be a neutral endopeptidase (NEP-24.11). The function of CD10 in BM stroma is unknown, although purified NEP-24.11 is known to degrade different substrates including interleukin 1β (IL-1β). We have therefore employed a CD10-positive BM stromal cell line (L2AK) which proliferates in response to IL-1β to test the hypothesis that degradation of this cytokine is one of the functions of stromal CD10. We first showed that [³H]thymidine incorporation by L2AK cells is enhanced by IL-1β in a clear dose-dependent manner. Addition of the CD10 inhibitor, phosphoramidon, together with IL-1β resulted in a left shift in the dose-response curve which corresponded to a 10-fold potentiation of the IL-1β effect. These results indicate that CD10 on bone marrow stromal cells can degrade IL-1β and therefore provide a local control of the effects of this, and possibly other, growth factor(s).     

Expression of cell-surface transferrin receptor following in vitro stimulation of peripheral blood lymphocytes in patients with beta-thalassaemia and iron-deficiency anaemia

The ferritin concentration in peripheral blood lymphocyte extracts was measured in 10 normal subjects, 7 patients with homozygous beta-thalassaemia, and 5 patients with iron-deficiency anaemia. The mean intracellular ferritin content was found increased in beta-thalassaemia and reduced in iron-deficient patients. Incubation of mononuclear cells in phytohaemagglutinin medium led to an increase of DNA synthesis concomitant with an increased number of lymphocytes bearing transferrin receptor and interleukin-2 receptor as measured by immunofluorescent technique. Although there was an immunological impairment of lymphocytes in patients with either iron depletion or iron loading compared to normal subjects, their ability to express transferrin receptor and interleukin-2 receptor on their cell surface was normal.     

Relationship between mixed chimerism and rejection after bone marrow transplantation in thalassaemia

Background

Thalassaemia is a genetic disease that requires a hypertransfusion regimen to treat the anaemia caused by enhanced red blood cell destruction. The only radical cure for thalassaemia is to correct the genetic defect by bone marrow transplantation from an HLA-identical donor capable of producing and maintaining a normal haemoglobin level in the recipient. Complete donor haematopoiesis is not essential for sustained engraftment and the simultaneous presence of haematopoietic cells of both donor and recipient origin is not a rare event after a transplant.

Patients and methods

The evolution of marrow engraftment of 93 transplanted thalassaemic patients, all from Middle East or Asian countries, was monitored by analysis of short tandem repeats.

Results

Forty-three of 93 (46%) patients experienced a status of mixed chimerism early after bone marrow transplantation. Results of further engraftment analysis in these patients showed in 27 complete donor engraftment; rejection occurred in seven, while eight maintained the presence of both host and donor-derived cells. Interestingly, five out of the seven patients who rejected their transplant showed more than 25% residual host cells early after transplantation.

Discussion and conclusion

Our study confirmed that the presence of large amounts of residual host cells within the first 2 months after a transplant is a risk factor for graft rejection also in a group of patients with wide ethnic heterogeneity, irregular transfusion regimens and/or poor chelation treatment. Ten percent of the transplanted thalassaemic patients maintained coexistence of donor and recipient cells, showing a stable functional graft, characterized by normal production of beta globin chains and high levels of haemoglobin. A mechanism responsible for peripheral tolerance induction, such as the production of specific regulatory T-cell clones, seems to play a key role in the induction of long-term tolerance after the transplant.     

Capillary leak syndrome associated with elevated IL-2 serum levels after allogeneic bone marrow transplantation

Summary The pathophysiological mechanisms involved in the development of a spontaneous systemic capillary leak syndrome (CLS) are unknown. In contrast, CLS is a well-known side effect of high-dose interleukin-2 (IL-2) therapy in solid tumors. We report on a patient who developed CLS with high serum levels of endogenous IL-2 under immunosuppressive therapy for chronic graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation (BMT). Generalized edema persisted for 10 weeks. The condition resolved after antibiotic therapy of a septic shock with hemolyzing streptococci group A. Thus, a latent infection may alter cytokine homeostasis and may cause CLS in BMT patients.     

Y-body study in bone marrow precursors, peripheral blood cells and alveolar macrophages for demonstration of haemopoietic engraftment in allogeneic bone marrow transplantation

The value of Y-body study for assessment of haemopoietic engraftment was analyzed in 50 consecutive patients submitted to allogeneic bone marrow transplantation (BMT) (sex-matched in 28 cases, sex-mismatched in 22). The study was performed weekly on bone marrow and peripheral blood smears in all cases, and alveolar macrophages were also studied in 15 patients in whom bronchoalveolar lavage was carried out because of concurrent respiratory disturbances. The analysis was performed blindly by 2 independent observers. In both sex-matched and sex-mismatched cases there was an absolute concordance between recipient and donor Y-body results, as well as with the simultaneous cytogenetic study. The engraftment of erythroid and granulopoietic lines was documented at day +14 in all cases of sex-mismatched BMT, whereas megakaryocyte and lymphocyte take was demonstrated at d +21. On the other hand, the results from alveolar macrophages were in accordance with those obtained in the simultaneous study of bone marrow precursors after BMT. The above results indicate that Y-body analysis is a simple and useful tool for the demonstration of bone marrow take in sex-mismatched BMT.     

Correlation between low CSA plasma concentration and severity of acute GvHD in bone marrow transplantation

Summary Between 1982 and 1986 51 patients were treated with ciclosporin a (CSA) to prevent graft versus host disease (GvHD) after bone marrow transplantation (BMT). Major side effects of the drug were tremor, hypertension, hepatotoxicity and nephrotoxicity. Acute GvHD 0° to II° occurred in 80% of our patients, and GvHD III° and IV° in 20% despite the use of CSA. Two to four days before the onset of GvHD, CSA serum levels were significantly lower on the average in patients who developed GvHD III° and IV° compared to the others. Our data indicate that plasma CSA concentrations higher than 250 ng/ml should be achieved to reduce the severity of GvHD after BMT.     

Little evidence for clonal evolution of malignant haematopoietic cells following relapse after autologous bone marrow transplantation

Abstract: By screening for immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements in bone marrow samples aspirated at different time points during the course of disease from 43 patients with acute leukaemia we have analysed the extent of clonal evolution after autologous bone marrow transplantation (ABMT) and addressed the issue of whether the Southern Blot method has the power to reveal clonal proliferations representing minimal residual disease (MRD) in the autologous bone marrow grafts. Our results show that no clonal proliferations were detectable in any of the 43 bone marrow grafts analysed, even after we analysed DNA preparations in 5 cases from cells highly enriched for cells of the original malignant immunophenotype. Moreover, as judged by the Ig- and TCR gene configurations in 11 patients, relapse arose from the original clone even though minor clonal variations did occur in about half of the relapsing patients. We conclude that while the Southern Blot method can detect gene receptor rearrangements in the majority of patients with acute leukaemias and high-grade non-Hodgkins lymphomas, it is not useful for predicting relapse after ABMT. On the other hand, it is possible – by employing it – to evaluate whether or not relapse after ABMT arises from the original malignant clone and to what extent clonal evolution has taken place.