111Indium-Chloride Bone Marrow Scintigraphy in Aplastic Anaemia

Bone marrow scintigraphy, using ¹¹¹Indium-chloride, was performed in 24 patients with acquired aplastic anaemia to investigate: (1) a possible relationship between bone marrow scintigraphy and peripheral blood cell values, (2) a possible relationship between scintigraphy and histology of the bone marrow and (3) the ability to distinguish various aplastic anaemia subtypes with bone marrow scintigraphy. For this purpose a semi-quantitative scoring of scintigraphic results was used. Only a weak correlation was found between the radionuclide studies and blood counts. It appeared that an abnormal ¹¹¹In-scintigraphic activity in the pelvis was related to an abnormal quality and quantity of haematopoietic tissue. To study a correlation with histological subtype grading, the patients were grouped in 4 categories based on clinical-histological results. Thus it could be demonstrated that the presence of ¹¹¹In-activity in long bones (‘scintigraphic extension’) is an important parameter in distinguishing patients who are believed to suffer from a primary stem-cell defect, from patients who may suffer from an auto-aggressive disorder.     

Gelatinous Transformation of Bone Marrow in Prolonged Self-Induced Starvation

In bone marrow from 3 patients with prolonged, severe self-induced starvation, fat atrophy, hypoplasia of haemopoietic cells and characteristic gelatinous transformation of marrow were noted. The gelatinous substance appeared amorphous and stained pink with the Wright-Giemsa stains. Histochemical and ultrastructural studies indicated that it consisted of acid mucopolysaccharides and was extracellular in nature. Similar marrow abnormalities were produced in rabbits by limitating their food intake for 4 months. These marrow abnormalities in the experimental animals could be reverted to normal by restroing their nutritional status. It is proposed that the gelatinous transformation of marrow is caused by excessive production of mucopolysaccharides of the ground substance to compensate for the mobilization of marrow fat which occurs to meet the energy requirement. It is further postulated that excessive production of acid mucopolysaccharides may provide a microenvironment unsuitable for haemopoietic proliferation. The relevance of these findings to other conditions associated with marrow aplasia, is discussed.     

Erythrophagocytosis in the Human Bone Marrow as Disclosed by Iliacal Bone Biopsies

Erythrophagocytosis in human bone marrow has been studied in selected series of iliacal bone biopsies embedded in methacrylate or epoxy resin. Using established light microscopic criteria for recognition of the early digestive erythrophagic vacuole, intense erythrophagic activity was encountered in a series of 5 patients with haemolytic disease. A series of 32 biopsies, selected to represent ‘normal’ bone marrow, demonstrated low or modest erythrophagic activity in all cases. The findings are in accordance with previous demonstration of erythrophagocytosis in human bone marrow at autopsy and point to the bone marrow as the main site for destruction of aged red cells in the healthy human organism.     

Aplastic Anaemia with Marrow Defective in Formation of Fibroblastoid Cell Colonies in Vitro

The formation of fibroblastoid colonies by marrow cells in vitro has been used as a putative assay for a stem cell of haemopoietic stroma. Bone marrow from one patient with aplastic anaemia did not form any of these colonies, while its growth in diffusion chambers as an indirect measure of a haemopoietic stem cell was even better than normal. On the other hand, marrow from the other aplastic anaemia patient showed only quantitative decrease in the formation of fibroblastoid colonies and simultaneously grew very poorly in diffusion chambers. These patients were indistinguishable by the cytological examination of their marrow, however, the peripheral blood abnormalities were expressed less severely in the first patient. These results suggest the contribution of the defect in marrow cells, which form fibroblastoid colonies in vitro to the development of aplastic anaemia in these patients.     

Acquired Aplastic Anaemia in Adults

A follow-up study of 10 patients suffering from acquired aplastic anaemia, comprising methacrylate-embedded bone marrow biopsies and CFU cultures, is presented. The haematopoietic recovery patterns and changes in the inflammatory infiltration after permanent engraftment could be distinguished from those in non-transplanted patients. After anti-thymocyte globulin treatment followed by allogeneic bone marrow infusion, the recovery pattern resembled that in non-transplanted patients. The persistently low colony-forming capacity in some patients could be explained by the existence of lymphoid inhibitory cells, which suggests an immunologic auto-destructive mechanism.     

Bone marrow transplantation for severe aplastic anaemia a review of the Westminster experience of 24 cases

Summary Twenty-four patients have received bone marrow transplantation for severe aplastic anaemia at the Westminster Hospitals since 1974. Twelve patients are long term survivors. Infectious complications in association with graft rejection, graft versus host disease or prolonged neutropenia were the major cause of death. In the last 18 months the introduction of more effective conditioning regimes and Cyclosporin A as graft versus host disease prophylaxis has improved the survival rate to 85%. One patient has required regrafting for late graft failure without evidence of graft rejection.     

Bone Marrow Studies in Myelomatosis

The percentage of fat-cell areas in bone marrow particles from 22 patients with untreated myelomatosis was estimated. In only 1 patient was the mean fat cell area below 25 % of the bone marrow area measured. A negative correlation was found between the area of fat cells and plasma cells, indicating a displacement of the fat cell area by the plasma cells. 28 % of the patients had empty bone marrow deposits of iron. However, based on a normal iron saturation of S-transferrin and a normal sideroblast count in the bone marrow, the supply of iron to the erythropoiesis was considered sufficient. All patients but one had normoblastic bone marrows. Using a deoxyuridine suppression test in 10 patients, no biochemical defect could be demonstrated. To judge from the correlation coefficient a minor degree (9–14 %) of the variation in Hb values could be predicted from the cellularity in the bone marrow while a major degree (70 %) could be predicted from the renal glomerular filtration rate. The results do not support a displacement of blood-forming elements, iron deficiency, vitamin B₁₂ or folic acid deficiency to be of general significance in the pathogenesis of anaemia, but agrees with a causal relationship between anaemia and renal failure.     

Assessment of Bone Marrow and Splenic Erythropoiesis in Myelofibrosis

The iron uptake in bone marrow and spleen was measured in 29 patients with myelofibrosis using ⁵²Fe and quantitative scanning. In 10 patients, no iron uptake in the marrow could be observed and active erythropoiesis was extramedullary only. In the bone marrow of patients with myelofibrosis, the iron uptake per nucleated red cell was less than that observed in conditions without myelofibrosis or extramedullary erythropoiesis. Increasing splenic iron uptake was likely to be associated with a decreasing bone marrow iron uptake and was related to the size of the spleen. The data suggest that in myelofibrosis, the spleen dominates iron uptake through intense erythropoiesis and a high splenic blood flow, thus restraining iron supply to the bone marrow.     

Oral cyclosporin-A is effective treatment for untreated and also for previously immunosuppressed patients with severe bone marrow failure

16 patients with transfusion-dependent, life-threatening bone marrow failure (14 with severe aplastic anaemia, 1 with systemic lupus erythematosus and 1 with pure red cell aplasia) were treated with cyclosporin-A (Cy-A) after either lack of response to conventional immunosuppression with antithymocyte-globulin/high-dose methylprednisolone for 95 to 1190 days (median 186.5) (group I, 8 patients) or as a primary treatment due to ineligibility for conventional immunosuppression (group II, 8 pat.). Cyclosporin-A was given orally to maintain trough levels of 200 to 300 ng/ml (RIA). In group I, 6 out of 8 patients responded 30 to 480 d (median 53) and are currently alive 627 to 1482 d (median 731) after initiation of Cy-A, respectively. In 3 of the responders Cy-A has been withdrawn, without relapse. In group II, 5 of 8 patients responded 26 to 170 d (median 63) and are currently alive 142 to 697 (median 420) d following initiation of Cy-A, respectively. These data indicate a place for cyclosporin-A in the management of patients with life-threatening bone marrow failure in whom a) immunosuppressive therapy with antithymocyte-globulin and high-dose methylprednisolone had failed and b) who are not candidates for vigorous immunosuppression or bone marrow transplantation, for medical or other reasons.     

Allogeneic bone marrow transplantation for severe aplastic anaemia-the London experience

Using the Seattle protocol with minor modifications, 23 patients with severe aplastic anaemia received allogeneic bone marrow transplants from HLA/ mixed leucocyte culture matched sibs in three London centres between 1973 and 1977. Ten patients (43.5%) are alive 6 months to 5 years after transplantation, and are well with full haemopoietic reconstitution, two with autologous bone marrow recovery following the graft procedure. A failure of the marrow graft to take, or take followed by rejection occurred in 12 patients (52%). Failure of marrow recovery was associated with a high early mortality from bacterial or fungal infection. The only survivors amongst those who rejected the first graft were four patients in whom a subsequent graft from the same donor was successful, and two in whom autologous recovery occurred. Graft versus host disease (GVHD) occurred in seven patients, and was fatal in one case. The most frequent complication after successful engraftment was varicella-zoster infection which occurred in five patients and was fatal in one patient. The overall results compare favourably with those from other transplant centres, but the high rate of graft rejection and low incidence of GVHD differ from other series. The results should encourage further referral of patients with severe AA for bone marrow transplantation.     

Morphologic Studies of the Erythropoietic Part of the Bone Marrow in Myeloid Leukaemias

The erythropoietic part of the bone marrow has been morphologically analyzed in 147 patients with various forms of myeloid leukaemias and in 20 healthy controls. In all the patients the percentage of basophilic erythroblasts was abnormally high and correlated to elevated mitotic indices. Megaloblastic changes were found to be relatively common in the patients. All these findings are compatible with an ineffective erythropoiesis where the normal differentiation towards more mature erythroblasts becomes increasingly blocked during the course of the disease regardless of the type of myeloid leukaemia.     

Correction of stromal cell defect after bone marrow transplantation in aplastic anaemia

Defects in stromal cell function have been demonstrated in a number of aplastic anaemia (AA) patients. Here we have studied a patient with severe AA and abnormal stromal cell function who underwent bone marrow transplantation (BMT). The objective of this study was to investigate the timing and the mechanism of correction of the stromal defect after transplantation. The patient, a 25-year-old woman with severe AA, underwent BMT from her brother. BM was obtained from the patient on five occasions: 2 weeks pre BMT, and 3, 8, 16 and 21 months post BMT. Stromal cells were grown to confluence and recharged with purified CD34+ cells from normal donors. The support of such cells, as assessed by weekly colony-forming assay (CFU) of non-adherent cells, was compared with that of stromal layers grown from normal BM. A novel technique of combined fluorescence in situ hybridization (FISH) and immunocytochemistry was used to determine the origin of specific stromal cell types on cytospins of stroma post BMT. Stromal function was defective at 2 weeks pre BMT and at 3 months post BMT, but returned to normal at 8 and 16 months post BMT. At 21 months post BMT, stromal fibroblasts and endothelial cells were shown to be of recipient origin, and macrophages and T cells were of donor origin. We present here evidence in a case of severe AA for defective stromal function before BMT and delayed normalization of function after BMT. This correlated with engraftment of donor macrophages and T cells, but not fibroblasts and endothelial cells.     

Bone Marrow Necrosis

ABSTRACT Bone marrow necrosis (BMN) is a rare finding in specimens from living patients. It is most commonly found in patients with neoplastic disorders, severe infections and sickle cell disease. We present a patient with Hodgkin's disease who developed extensive BMN 11 months before death. A concise review of the literature is also presented.     

Predicting response to immunosuppressive therapy and survival in severe aplastic anaemia

Horse anti-thymocyte globulin (h-ATG) and ciclosporin are the initial therapy for most patients with severe aplastic anaemia (SAA), but there is no practical and reliable method to predict response to this treatment. To determine whether pretreatment blood counts discriminate patients with SAA who have a higher likelihood of haematological response at 6 months to immunosuppressive therapy (IST), we conducted a single institution retrospective analysis on 316 SAA patients treated with h-ATG-based IST from 1989 to 2005. In multivariate analysis, younger age, higher baseline absolute reticulocyte count (ARC), and absolute lymphocyte count (ALC) were highly predictive of response at 6 months. Patients with baseline ARC ≥ 25 × 10⁹/l and ALC ≥ 1 × 10⁹/l had a much greater probability of response at 6 months following IST compared to those with lower ARC and ALC (83% vs. 41%, respectively; P  < 0·001). This higher likelihood of response translated to greater rate of 5-year survival in patients in the high ARC/ALC group (92%) compared to those with a low ARC/ALC (53%). In the era of IST, the baseline ARC and ALC together serve as a simple predictor of response following IST, which should guide in risk stratification among patients with SAA.     

Bone marrow transplantation for acquired aplastic anemia: What's new

Bone marrow transplantation is a major therapeutic option for patients with acquired severe aplastic anaemia: improved survival has been achieved in younger patients, thanks to better donor selection, conditioning regimens and graft versus host disease prophylaxis, together with improved supportive care, including diagnosis and treatment of opportunistic infections. This has not been the case for older patients over the age of 40 years.We will discuss transplantation platforms as used for different donor types and we will analyse major breakthroughs of the last years: the combination of Fludarabine and cyclophosphamide as a conditioning regimen, the use of alternative donors including HLA haploidentical related donors and new strategies to prevent acute and chronic graft versus host disease, including post transplantation Cyclophosphamide. These changes extend the option of a bone marrow transplantation for patients who lack an HLA matched donor and appear to improve engraftment and reduce graft versus host disease: whether this will be true for all age groups is currently being investigated.     

Coexisting Suppressor Cells for Myeloid Colony Formation in the Bone Marrow of Patients with Aplastic Anaemia

Suppressor cells for colony forming cells (CFUc) were found in the bone marrow of 1 of 6 patients with aplastic anaemia. The assay applied was based on the fact that coexisting suppressor cells are more sensitive to dilution than CFU_C, as long as adequate stimulation is provided by a source of colony stimulating activity. In the patient with suppressor cells, a 4 d trial of 1000 mg methylprednisolone per d failed to improve the blood cell production and had to be discontinued due to gastric irritation.     

Efficacy and safety of romiplostim in refractory aplastic anaemia: a Phase II/III,multicentre, open‐label study

A previous dose‐finding study has suggested that romiplostim is effective in patients with refractory aplastic anaemia (AA) and 10 µg/kg once weekly was recommended as a starting dose. In this Phase II/III, multicentre, open‐label study, romiplostim was administered subcutaneously at a fixed dose of 10 µg/kg once weekly for 4 weeks (weeks 1–4) followed by weekly doses (5, 10, 15 and 20 µg/kg) titrated by platelet response for up to 52 weeks (weeks 5–52). A total of 31 patients with AA who were refractory to immunosuppressive therapy (IST) and thrombocytopenia (platelet count of ≤30 × 10⁹/l) were enrolled. The primary efficacy endpoint of the proportion of patients achieving any haematological (platelet, neutrophil and erythrocyte) response at week 27 was 84% [95% confidence interval (CI) 66–95%]. Trilineage response was 39% (95% CI 22–58%) at week 53. The most common treatment‐related adverse events (AEs) were headache and muscle spasms (each 13%). All AEs were mild or moderate except for three patients with Grade 3 hepatic AEs; no AEs necessitated romiplostim discontinuation. Two patients developed cytogenetic abnormalities, of whom one returned to normal karyotype at last follow‐up. High‐dose romiplostim is effective and well tolerated in the treatment of patients with AA refractory to IST.     

Bone Marrow Cell Culturing in Double Diffusion Chambers

A double diffusion chamber technique (DDC) has been established. The bone marrow cells (BMC) were cultured in the peritoneal cavity of mice in DDC consisting of 2 compartments separated from one another by a Millipore membrane. One chamber half contained the mouse bone marrow target cells, and the other half (regulator compartment) either medium (control), spleen cells or BMC. In the controls the BMC proliferated rapidly from day 2, and the cell yield on day 7 was reduced by only 20% when compared with single diffusion chambers. Diffusible factors from spleen cells stimulated the growth of CFU-S and CFU-C in the bone marrow, and increased the number of granulocytes and macrophages harvested in 7 day cultures. Conversely, BMC in the regulator compartment depressed granulopoiesis in the other chamber half.