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.     

Relevance of Colony Forming Assays to Bone Marrow Transplantation with Particular Reference to Grafting for Aplastic Anaemia

The value of bone marrow colony-forming assays in monitoring transplanted patients has been assessed by comparing results from two pairs of bone marrow recipients. One pair received marrow from their identical twins for acute lymphoblastic leukaemia; the other pair were grafted with allogeneic marrow from their siblings for aplastic anaemia. One of each pair showed successful engraftment while in the others the grafts failed. The colony-forming assay was then used to investigate marrow function in five grafted aplastic patients. Of these, four rejected their first grafts and required further immunosuppression before engraftment could be accomplished. The remaining patient was immunosuppressed at the outset with antithymocyte globulin (ATG) and her first graft was successful. Sera from all five patients inhibited colony formation by normal human marrow and it is suggested that this activity was related to graft rejection as well as to the pathogenesis of the condition.     

Refractory Anaemia with Excess of Myeloblasts in the Bone Marrow: a Clinical Trial of Androgens in 90 Patients

In a cooperative trial, 90 patients with refractory anaemia with an excess of blast cells in the marrow were evaluated and treated with androgens. The clinical presentation was very similar to previously published observations: features of medullary insufficiency were less marked than in primary aplastic anaemia; bone marrow blastic infiltration varied from case to case, and remained stable until death or until an acute leukaemic change. All the patients were treated with high doses of androgens as for aplastic anaemia. The efficacy of this therapy was poor. The average life expectancy was 13 months, 64% of deaths being associated with a change to acute myeloid leukaemia. A severe bone marrow deficiency foreshadowed early death, but myeloblastic transformation was observed whatever the initial degree of blastic infiltration of the bone marrow. A comparison with the literature suggests that androgen therapy may accelerate the change to acute leukaemia.     

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.     

Selective Hypoplasia of Pelvic Bone Marrow

6 patients with thrombocytopenia and anaemia had fatty marrow replacement at the site of an iliac crest. ⁵²Fe scans showed marked abnormalities of bone marrow distribution. Particularly, the uptake of radioiron into the pelvis was almost absent. The sternal marrow was cellular. Studies of the bone marrow which included cytology, histology and electron microscopy failed to reveal why the pelvic marrow was aplastic. Occasional vascular lesions could be seen in the iliac crest bone marrow sections. The cellular morphology showed slight maturation abnormalities. Ferrokinetics were consistent with erythroid marrow hypoplasia. The pelvic and sternal marrow cellularity in patients with pancytopenia may not be representative of the cellularity of the whole marrow, and the pelvic marrow especially may be prone to aplasia.     

Peripheral Blood Cells from Patients with Aplastic Anaemia in Partial Remission Suppress Growth of their Own Bone Marrow Precursors in Culture

S ummary In 12 patients with severe aplastic anaemia who had achieved self-sustaining autologous bone marrow function after treatment with antilymphocyte globulin, or with cyclophosphamide given for attempted bone marrow transplantation, colony formation by all haemopoietic precursors remained far below normal. Precursors from peripheral blood, erythroid precursors in particular, failed to form a normal number of colonies. This paucity of colony formation does not reflect a true lack of precursor cells but is due to circulating cells which impair maturation. Addition of low density peripheral blood cells to autologous bone marrow cultures diminished colony formation by granulocyte-macrophage precursors (GM-CFC) and abolished 'burst' formation by BFU-E. Strong auto-inhibition preceded relapse in four of eight patients. The phenomenon was not observed in five normals, in five aplastic anaemia patients with stable haemopoietic grafts and in three polytransfused control patients.
The T-cell poor subpopulation of peripheral blood cells, containing mainly B-cells and macrophages, was especially inhibitory. Accordingly, removal of plastic adherent cells from bone marrow cell suspensions improved plating efficiency in aplastic anaemia patients, but not in normals. Isolated E-rosette positive cells had no negative effect.
Inhibition could only be demonstrated in the autologous situation. Colony formation by normal allogeneic peripheral blood precursors was not impaired by patient cells. The phenomenon is likely to reflect residual disease activity which is compensated in vivo but can be demonstrated in vitro. It may be of help in early recognition of patients who are at risk of relapse after autologous bone marrow reconstitution.     

Bone Marrow Transplantation for Aplastic Anaemia

Indications for treatment of aplastic anaemia (AA) by marrow transplantation are discussed. Selection of donor-recipient pairs by histocompatibility testing, conditioning of patients for transplantation, technique of grafting, post-grafting care and immunosuppressive drug regimens are reviewed. Data are presented on 28 patients with advanced AA treated by marrow grafts from HL-A matched siblings following conditioning with cyclophosphamide (50 mg/kg) on each of 4 successive days. Survival and haematological reconstitution in more than half the patients indicates that marrow grafting in patients with advanced AA should be undertaken early, before major infections and refractoriness to blood transfusions occur provided that an HL-A matched sibling can be identified as a donor. The results have shown that survival in marrow grafted patients with severe AA compares favourably to that of patients treated with conventional therapy.     

Aplastic anaemia with 'hot pockets'.

4 patients with chronic severe aplastic anaemia and with persistent foci of intense haematopoietic activity, so-called 'Hot Pockets', in their bone marrow were studied. In all patients the remainder of the bone marrow was morphologically and erythrokinetically hypoplastic. The cellular morphology in these 'Hot Pockets' displayed megaloblastic features and definite maturation abnormalities in erythroblasts, but no consistent changes in the myeloid or megakaryocytic series. The presence of persistent 'Hot Pockets' presents a conceptual challenge since these pockets contain multipotential stem cells capable of differentiation and self-renewal, but obviously incapable of repopulation of the bone marrow. In view of additional evidence for dyserythropoiesis in aplastic anaemia such as changes in erythroblast morphology and the production of macrocytes, fetal haemoglobin and complement sensitive cells, it seems likely that the bone marrow of aplastic anaemia patients is totally dyserythropoietic rather than hypoactive and that bone marrow transplantation in many cases may be both justified and necessary.     

Bone Marrow Transplantation in Severe Aplastic Anaemia: a Survey of the European Group for Bone Marrow Transplantation (E.G.B.M.T.) *

Summary . A survey of the results of bone marrow transplantation for severe aplastic anaemia in 13 European teams is reported. 159 questionnaires were analysed by univariate and multivariate analysis. The overall 1 year survival was 41.2%. The chance of survival was decreased by the presence of infection before grafting and by the use of female donors. The conditioning regimen did not influence survival. Graft rejection was influenced by the conditioning regimen and the sex of the donor. Graft-versus-host disease was influenced by the conditioning regimen, and the age and the sex of the recipient. This study defines prognostic indices which may be helpful in determining the likelihood of success after marrow transplantation for severe aplastic anaemia.     

Neutrophil Function in Bone Marrow Transplant Recipients

The neutrophil function of seven patients receiving allogeneic bone marrow transplantion was studied. Five of the patients had been transplanted for aplastic anaemia and two for acute leukaemia. Determinations were made of neutrophil phagocytosis, chemotaxis, random migration, and microbicidal activity for Candida albicans and Staphylococcus aureus. One patient showed a decreased ability to kill C. albicans at a time when she had active pneumonia due to Pneumocystis carinii. The remainder of the studies showed normal neutrophil functions. No differences were observed in the patients who had graft versus host disease [GvH] from those without GvH. These studies suggest that defects in phagocytic neutrophil function do not contribute significantly to the impaired host defenses in recipients of bone marrow transplantation.     

Bone marrow transplantation from identical twins in the treatment of aplastic anaemia: implication for the pathogenesis of the disease

S ummary . Treatment of aplastic anaemia by bone marrow transplantation from a syngeneic (identical twin) donor has provided insights into the pathophysiology of the disease.
We report from patients with severe anaemia who were treated by syngeneic bone marrow transplantation. None of the patients had sustained recovery of peripheral blood counts. All four received second transplants from the same twin donor after immunosuppressive conditioning treatment. Each had prompt recovery of haematopoiesis. A review of the literature indicates that failure of syngeneic bone marrow transplantation in patients with aplastic anaemia is not uncommon. These data indicate that aplastic anaemia may be caused by a mechanism other than an absence or intrinsic abnormality of haematopoietic stem cells in many patients.     

Pathophysiology of aplastic anaemia

It is the conventional opinion that acquired aplastic anaemia is a heterogenous disease including basically different conditions, such as idiopathic or virus induced pancytopenia, toxic-allergic marrow damage or autoimmunity. Here, an alternative concept is proposed, according to which aplastic anaemia is one disease, but multifactorial in all patients, apparent differences being due to the relative prevalence of one or the other pathophysiological component in individual patients. Bone marrow from patients in the severe phase of aplastic anaemia does not grow in culture and is therefore not suitable for experimentation. Alternatively, bone marrow from patients who have resumed some degree of autologous bone marrow function, but still have residual signs of the disease after non-invasive therapy, offers the possibility to study pathophysiological mechanisms in vitro. The majority of experiments presented in this chapter have been done in such patients, assuming that their status of disease in some way reflects the original, more serious pretreatment condition. Three major pathophysiological components will be discussed, and it will be proposed how these factors act in concert to cause or aggravate aplasia.     

The Colony Forming Cell in the Myeloproliferative Disorders and Aplastic Anaemia

Bone marrow colony forming cell (CFC) concentration and the proportion of CFC in DNA synthesis were studied in myeloproliferative disorders and aplastic anaemia. Growth patterns of bone marrow cells in agar cultures were able to supplement traditional morphological and clinical criteria in the diagnosis of these haematolog-ical conditions. Bone marrow CFC concentration tended to be increased in chronic myeloid leukaemia (CML) and polycythaemia vera (PV), but decreased in myelo-fibrosis, erythroleukaemia, paroxysmal nocturnal haemoglobinuria (PNH) and the aplastic phase of aplastic anaemia. The proportion of CFC in DNA synthesis was decreased in CML, myelofibrosis and aplastic anaemia, but increased in blastic transformation, PV, PNH and during regeneration from aplastic anaemia. The proportion of CFC in DNA synthesis in bone marrow from patients with CML in blastic transformation was directly related to the percentage of myeloblasts in the bone marrow. CFC kinetics in blastic transformation have been demonstrated to be different from those in acute leukaemia.     

The colony forming cell in the myeloproliferative disorders and aplastic anaemia.

Bone marrow colony forming cell (CFC) concentration and the proportion of CFC in DNA synthesis were studied in myeloproliferative disorders and aplastic anaemia. Growth patterns of bone marrow cells in agar cultures were able to supplement traditional morphological and clinical criteria in the diagnosis of these haematological conditions. Bone marrow CFC concentration tended to be increased in chronic myeloid leukaemia (CML) and polycythaemia vera (PV), but decreased in myelofibrosis, erythroleukaemia, paroxysmal nocturnal haemoglobinuria (PNH) and the aplastic phase of aplastic anaemia. The proportion of CFC in DNA synthesis was decreased in CML, myelofibrosis and aplastic anaemia, but increased in blastic transformation, PV, PNH and during regeneration from aplastic anaemia. The proportion of CFC in DNA synthesis in bone marrow from patients with CML in blastic transformation was directly related to the percentage of myeloblasts in the bone marrow. CFC kinetics in blastic transformation have been demonstrated to be different from those in acute leukaemia.     

Origin of Human Bone Marrow Fibroblasts

We investigated the origin of bone marrow fibroblasts in three bone marrow transplant recipients with aplastic anaemia and leukaemia who received grafts from HLA-identical siblings of opposite sex. The patients were conditioned for transplantation with high doses of cytotoxic drugs and 300–1000 rads total body irradiation. After transplantation, bone marrow cells wrere cultured in T flasks for 3 weeks and the adherent cells were then trypsinized and passaged weekly. After several passages the cells had the typical morphology and growth pattern of fibroblasts. Metaphases from these cells were all of recipient sex type. In contrast, haematopoietic cells and lymphocytes obtained at the same time were of donor sex type. Our findings indicate that the human bone marrow fibroblast is not derived from a precursor common to haematopoietic cells or lymphocytes. The bone marrow fibroblast appears to be a mesenchymal cell, unrelated to haematopoietic stem cells, which is capable of in vitro proliferation after as much as 1000 rads of total body irradiation.     

Increased apoptotic cells in bone marrow biopsies from patients with aplastic anaemia

In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anaemia (AA) we determined the proportion of apoptotic cells in paraffin-embedded bone marrow biopsies from patients with aplastic anaemia using an in situ TdT-catalysed DNA nick end labelling (TUNEL) staining method. A significant increase in the proportion of mononuclear apoptotic cells was demonstrated in biopsies from patients with aplastic anaemia (8.19 ± 1.45%) when compared with controls (2.07 ± 0.86%). These data support the view that apoptosis may play a role in the pathophysiology of bone marrow failure.     

The Aplastic Anaemia–Paroxysmal Nocturnal Haemoglobinuria Syndrome

Seven patients are described who suffered from the aplastic anaemia-paroxysmal nocturnal haemoglobinuria syndrome. All had been first diagnosed as having aplastic anaemia. In one case the bone marrow became repopulated but the patient developed severe intravascular haemolysis and died of liver failure due to intrahepatic venous thrombosis. Two patients died of marrow aplasia; haemolysis was minimal and was only a laboratory phenomenon. Another patient also died of marrow aplasia but in his case haemolysis had contributed significantly to his illness. In three patients the bone marrow became repopulated completely or partially and the patients have recovered clinically despite the persistence of a small proportion of PNH red cells.
The relationship between aplastic anaemia and PNH is discussed. It seems likely that the link between the diseases is the development of an abnormal clone of haemopoietic cells in a regenerating, previously aplastic, marrow.
The effect of the superimposition of the PNH defect on the prognosis in aplastic anaemia is discussed. In five cases haemolysis had little or no influence on the course of the disease; but in two patients intravascular haemolysis was severe and was undoubtedly harmful.
The influence of splenectomy on the course of the disease was studied. It was performed in four of the patients, but in only one did it seem to have a beneficial effect.     

Pregnancy associated aplastic anaemia: a report of five cases and review of current management

The occurrence of aplastic anaemia in pregnancy has been long recognized but its rarity has made it difficult to establish the relationship between the two conditions and the optimal management. We now report five cases of aplastic anaemia in pregnancy and offer some recommendations for treatment. In two patients the pregnancy was allowed to continue to term and the disease persisted post-partum leading to death in one case. The other three patients had their pregnancies terminated; one subsequently deteriorated and died, two had spontaneous remissions of their aplasia. We suggest that patients presenting with severe aplastic anaemia in early pregnancy should be offered termination because this may be followed by haematological improvement. If haematological improvement does not occur allogeneic bone marrow transplantation (BMT) may be considered. Aplastic anaemia presenting in late pregnancy should be treated with supportive care until delivery. On the basis of our experience, antilymphocyte globulin may safely be given during pregnancy.     

Bone Marrow Fibroblastoid Colony-Forming Cells (F-CFC) in Aplastic Anaemia: Colony Growth and Stimulation of Granulocyte-Macrophage Colony-Forming Cells (GM-CFC)

S ummary . Colonies of fibroblast-like cells have been grown from the mononuclear cell fractions of bone marrow aspirated from normal individuals and patients with aplastic anaemia. Some of the characteristics of the fibroblastoid cells have been determined and their granulocyte-macrophage colony-stimulating activity (CSA) in semi-solid agar culture has been used as a functional test of their influence on granulopoiesis.
The incidence and growth rates of fibroblastoid colony-forming cells (F-CFC) from aplastic patients'bone marrows were not markedly different from normal either before or after treatment by allogeneic bone marrow transplantation or with antilymphocyte globulin (ALG).
Confluent monolayers of fibroblastoid cells grown from normal marrow were, on the whole, poor stimulators of granulocyte-macrophage colony-forming cells (GM-CFC) and CSA was not detected in the supernatant medium. Fibroblastoid monolayers derived from many of the aplastic bone marrows studied were efficient stimulators of GM-CFC but, like the monolayers grown from normal cells, did not release CSA into the culture medium.
Addition of methylprednisolone (MP) to the cultures had little effect on fibroblastoid cell growth, induced fat-accumulation by some of the fibroblastoid colonies comprising the monolayer and reduced the abilities of the monolayers to stimulate GM-CFC.     

A phase I/II trial of recombinant human interleukin-6 in patients with aplastic anaemia

Summary. In a phase I/II study, 11 patients with marrow failure (10 with acquired aplastic anaemia and one with pancytopenic Fanconi anaemia) were treated with recombinant human interleukin-6 (rhIL-6) to assess the safety and tolerability of rhIL-6 and its effects on peripheral blood counts, bleeding complications and transfusion requirements. All patients with acquired aplastic anaemia were refractory to immunosuppressive treatment or had relapsed after immunosuppressive therapy and were not bone marrow transplantation candidates. Recombinant hIL-6 was to be given as a once-daily subcutaneous injection for 28 d at doses ranging from 0-5 to 5-0βg/kg. After an observation period of 2 weeks, five patients received a second treatment course of 28 d.
Only one patient had a sustained increase in platelet count from 18 000 to 72 000/ fA. Bleeding occurred in four patients and caused premature discontinuation of rhIL-6 therapy in three patients. A deterioration of pre-existing anaemia was observed in nine patients. No significant changes of leucocyte counts were observed during the first cycle. During the second cycle the peripheral blood monocyte counts decreased significantly. No significant changes in bone marrow cellularity were observed. Recombinant hIL-6 induced a dose-dependent increase in acute-phase reactants in all patients. Other adverse events included fever, headache, arthralgia, tachycardia and hypertension. In conclusion, rhIL-6 given alone at low doses does not increase platelet counts in the majority of patients with aplastic anaemia and can precipitate a sudden worsening of pre-existing anaemia and thrombocytopenia. This study was discontinued prematurely on account of the toxicity of rhIL-6 seen in patients with aplastic anaemia.