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The lipid composition of fat cells in the bone marrow of normal individuals and patients with aplastic anemia was investigated, using thin-layer and gas-liquid chromatography. The results were compared to those obtained from fat cells grown in vitro. Thin-layer chromatography (t.l.c) revealed that triacylglycerols were the major component of normal and aplastic marrow fat cells and of fat cells in culture. Analysis by gas-liquid chromatography (g.l.c) of the triacylglycerols, diacylglycerols, and nonesterified fatty acids detected minor differences between fat cells from normal and aplastic marrow. Although there were no statistically significant differences in the proportions of saturated fatty acids and their unsaturated analogues, the relative amounts of gamma-linolenic acid were increased in aplastic anemia and those of linoleic and myristoleic acids were reduced. In contrast, fat cells obtained from in vitro cultures contained a much higher proportion of unsaturated fatty acids. These findings indicate that there is no marked abnormality in the composition of bone marrow fat cells in aplastic anemia, but that minor differences can be detected; the fatty acid composition of bone marrow fat cells is not reproduced in vitro.

Acquired aplastic anaemia remains a devastating and frustrating disease from which a proportion of patients still die as a result of failure of support measures. Its pathogenesis remains a mystery.

Sixty-four patients with aplastic anemia were treated with antilymphocyte globulin (ALG Merieux) between 1980 and 1985. The actuarial survival for all patients was 53% at 6 years, with 79% survival for nonsevere aplastic anemia (NSAA) and 36% for severe aplastic anemia (SAA). The neutrophil and platelet counts before treatment with ALG were highly predictive of survival, whereas sex, age, and etiology were not. Duration of disease prior to ALG treatment was inversely related to survival, although this was not statistically significant. Survival was closely associated with response to ALG, especially for patients with SAA. The response to one course of ALG was 33%. Eighteen patients who did not respond to an initial course of ALG received a second course; of these, four (22%) responded. The overall response to one or two courses of ALG was 40%. The mean RBC volume (MCV) measured after ALG treatment was a useful early indicator of response. Both the minimum lymphocyte count during ALG therapy and the mean lymphocyte count after therapy, however, were not significantly different between responders and nonresponders. The disappointing survival of patients with SAA in this study may reflect the poor clinical condition of severely affected patients referred to us and/or the presence of longstanding aplasia. The importance of studying a large series of patients with long-term follow-up to assess ALG in the treatment of aplastic anemia is shown by this investigation.

A 9-yr-old boy with a 2-yr history of Fanconi anaemia developed worsening pancytopenia that was unresponsive to oxymetholone therapy. Bone marrow was difficult to aspirate but showed the presence of megakaryoblasts. Bone marrow trephine was hypercellular with large clusters of abnormal megakaryocytes and a small population of megakaryoblasts, giving a diagnosis of megakaryoblastic leukaemia.

We present the case of a 60-year-old woman with drug-induced aplastic anaemia with a healthy monozygotic twin. Proof of monozygosity was confirmed by studies using the hypervariable minisatellite probe to obtain identical DNA fingerprints in donor and recipient. In vitro co-culture studies performed showed no evidence of a recipient-derived cellular or humoral inhibitor of donor haemopoiesis. Despite this, there was no engraftment following simple marrow infusion without preconditioning. A second syngeneic transplant following high dose cyclophosphamide therapy produced trilineage engraftment but severe thrombocytopenia developed at 3 months, followed later by pancytopenia with generalized marrow failure. Following a third syngeneic transplant with cyclophosphamide and total lymphoid irradiation there was good initial engraftment but graft failure occurred at 14 weeks. A fourth transplant using Campath 1G as preconditioning resulted in no engraftment and the patient died of septicaemia 8 weeks following her fourth transplant. We suggest that the cause of the recurrent aplastic anaemia in this case was a defect of marrow stroma as neither an inhibitor of donor haemopoiesis nor an intrinsic defect of donor stem cell growth could be demonstrated in vitro.

Graft versus host disease affecting the large bowel causes destruction of the crypt epithelium. There is a selective sparing of enterochromaffin cells in the majority of cases. As a consequence, single as well as small clumps of enterochromaffin cells are to be seen in the sites formerly occupied by the destroyed crypt epithelium. The reason for this phenomenon is unclear, but it may be related to the fact that the enterochromaffin cells are end-stage and non-proliferating cells. This is useful diagnostically. However, cytotoxic drugs or irradiation must be excluded as the cause of the mucosal damage to bowel as there are theoretical reasons to expect that a similar phenomenon will be seen after these forms of therapy.

This study shows that hitherto unexplained thrombocytopenia and neutropenia occurring after bone marrow allografting and autografting may have an immune basis. In the case of autografts, such antibodies are, by definition, auto-antibodies, and it is suggested that similar antibodies occurring after allografting are also auto-immune.

Between December 1983 and November 1985 we treated 39 patients with chronic myeloid leukaemia by chemoradiotherapy and transplantation from HLA-identical sibling donors using bone marrow that had been depleted of T cells ex vivo with the rat monoclonal antibody Campath-1. Twenty-eight of the patients were in the chronic phase (good-risk group) and 11 patients were in more advanced phases of the disease (accelerated phase or blastic transformation; poor-risk group). Of the patients of good risk 23 (82%) survive; the median duration of follow-up is 461 (range 111-776) days; of the 11 patients of poor risk four survive; the median duration of follow-up is 280 (range 189-658) days. Acute graft-versus-host disease (GVHD) of grade II or greater occurred in three (11%) of the patients of good risk and in six (55%) of the patients of poor risk. In the patients of good risk haematological evidence of relapse was seen in four and cytogenetic evidence of persisting or relapsed leukaemia (based on the finding of Philadelphia-chromosome-positive marrow metaphases more than 6 months after transplant) was seen in three other patients. In comparison with the patients of good risk transplanted with untreated marrow between February 1981 and December 1983, the incidence of acute GVHD was reduced significantly (P less than 0.001) but the risk of leukaemic relapse (including patients with only cytogenetic evidence of relapse) was increased (P less than 0.005). We conclude that T-cell depletion used in this manner may be associated with an increased risk of leukaemic relapse.

Despite its considerable toxicity to haemopoietic colony-forming cells, 4-hydroperoxycyclophosphamide (4-HC) has successfully been used to purge marrow of leukaemic cells before it is used to rescue patients from high-dose chemoradiotherapy. These conflicting observations indicate that haemopoietic progenitor cells that are not detected by the established colony-forming assays survive exposure to 4-HC and repopulate the marrow. The recent finding that murine spleen colony-forming cells (CFU-S) are resistant to 4-HC [Porcellini A, et al. (1983) Expl Hemat. 11 (suppl 14) 331 (abstract)] [14] also indicates that sensitivity to 4-HC can be used to distinguish primitive progenitor cells from committed progenitor cells. As part of a study on the nature of a population of blast colony-forming cells in human bone marrow, we tested their sensitivity to 4-HC to see whether they also are spared by the drug. We found that 4-HC had much less effect on the blast colony-forming cells than on the granulocyte-macrophage colony-forming cells (GM-CFC). This result suggests that the blast-colony-forming cells may be early human haemopoietic progenitor cells.

Bone marrow transplantation is the first line of treatment for all patients with aplastic anaemia who have an identical twin. Age is not an important consideration in these circumstances. For patients with an HLA identical sibling donor bone marrow transplantation should be offered as a matter of urgency to those patients with SAA under the age of 50. The most controversial issue is the use of unrelated volunteers, or non-sibling family members who are phenotypically matched or who have a minor degree of mismatch of the HLA system. A strong case could be made for offering this form of transplant to all patients under the age of 20 who have VSAA and using it as the second line of treatment in patients who fail to respond to ALG after 4 months if they have VSAA.

Data were obtained from 46 patients with severe aplastic anemia (SAA) who received bone marrow transplants (BMT) from donors other than genotypically HLA-identical siblings. The data were collected in the SAA Registry of the European Bone Marrow Transplant Group. The donors were non-HLA-identical siblings in six cases, parents in 28 cases, a son in one case and unrelated individuals in 11 cases. Fifteen donor-recipient pairs were HLA-A, -B and -DR identical and mutually non-reactive in mixed lymphocyte culture; nine were mismatched at one locus, 17 were mismatched at two or more loci and in five cases data were not available for D/DR determinants. Actuarial survival was predicted by the degree of mismatch. It was 45% for phenotypically HLA-identical grafts, 25% for grafts mismatched at one locus and 11% for graft mismatched at more than one locus. Whether the graft was derived from a family member or an unrelated donor seemed to be less important and results were comparable. Age, patient sex and year of transplant had no significant influence on survival. The use of cyclosporine (CSA) for graft-versus-host disease (GVHD) prophylaxis (n = 21, survival 34%) appeared superior to both methotrexate (n = 9, survival 11%) and to CSA with T cell depletion of donor marrow (n = 13, survival 14%). The causes of death were rejection (n = 15), GVHD (n = 13), pneumonitis (n = 5) and infection (n = 1). Twelve patients are alive at 16-84 months post-BMT.(ABSTRACT TRUNCATED AT 250 WORDS)

Between 1980 and 1986 we transplanted 49 patients from genotypically identical sibling donors for severe aplastic anaemia (SAA). Patients were predominantly adults, median age 22 years (3-47). Forty-six were multiply transfused prior to referral. The median pre-transplant disease duration was 4 months (1-72). Pre-transplant conditioning was with cyclophosphamide (CY) 200 mg/kg. Cyclosporin A (CSA) was given from 1 day before the transplant and continued for 9-12 months. Eight of 48 evaluable patients did not achieve initial engraftment (early graft failure). Six of these episodes occurred in the eight cases transplanted more than 1 year after diagnosis, four of whom died. Thirteen of 44 (30%) evaluable patients (with stable engraftment after one or two transplants) had grade III-IV acute graft-versus-host disease (GVHD). Only three of 35 patients surviving more than 100 days with sustained engraftment developed generalized chronic GVHD; two died. An additional 10 patients developed localized chronic GVHD, which was very mild, transient and related to CSA withdrawal. Four long-term survivors are known to have autologous marrow function. All survivors have Karnofsky scores of greater than 90%. We conclude that the use of CSA after bone marrow transplantation for SAA is associated with good long-term survival and minimal on-going chronic GVHD. Early graft failure was frequent when transplantation was delayed beyond 1 year from diagnosis.

Under most circumstances 52Fe, 111In, and colloid show a similar distribution of marrow. The lesser uptake of 111In by liver and spleen may occasionally be of value in permitting visualization of that portion of the spinal marrow obscured by these organs in the colloid scan. However, in red cell aplasia, when there is dissociation between phagocytic and erythropoietic functions, scanning with 111In gives no information about erythropoietic tissue distribution. Therefore, indium cannot be used as an analog for iron in the study of the hematopoietic system.

Primitive myeloid progenitor cells will adhere to stromal feeder layers of human bone-marrow-derived adherent cells grown in the presence of methylprednisolone (MP+ layers). These progenitors form colonies of blast cells on the MP+ stromal layers, but not on stromal layers grown in the absence of MP (MP- layers). The present study was designed to determine whether this failure of colony formation was caused by inability of the progenitors to adhere to the MP- layers or inability to proliferate in their presence. We also compared the capacities of the blast progenitors to adhere to MP+ and MP- stromal cells with those of mixed (GEMM-CFC), erythroid (BFU-E), megakaryocytic (Mk-CFC), and granulocyte-macrophage (GM-CFC) colony-forming cells. Incubation of bone marrow mononuclear cells with MP+ stromal layers removed 90% of the blast progenitors, but did not remove the majority of the GEMM-CFC, BFU-E, Mk-CFC, and GM-CFC; incubation of bone marrow mononuclear cells with MP- stromal layers did not remove the blast progenitors or the GEMM-CFC, BFU-E, Mk-CFC, and GM-CFC. Thus, the blast progenitors adhere to MP+ stromal feeder layers, but not to MP- stromal layers. In this respect they differ from the other more mature colony-forming cells that do not show any marked tendency to adhere to either type of stromal layer.

The technique of scanning with indium-111 autologous leukocytes has been used to assess gastrointestinal graft-versus-host disease (GVHD) following allogenic marrow transplantation. In patients with active disease, abdominal scans showed extensive abnormal localization in the bowel, while in those whose disease was quiescent after responding to treatment, scans showed localized ileocecal involvement. Rectal histology showed excellent agreement with scanning in the diagnosis of GVHD, but in three of six cases with active disease underestimated disease severity. Indium-111 leukocyte scanning is a useful noninvasive technique for the diagnosis and assessment of gut GVHD.

We describe 4 patients with a haemophagocytic syndrome resembling histiocytic medullary reticulosis (HMR) complicating chronic lymphocytic leukaemia (CLL) of 9 months to 8 years duration. Surface marker studies in 2 cases showed that the CLL lymphocytes were of B-cell type in one and of T-cell type in the other. 2 of the patients had histiological evidence of co-existing immunoblastic sarcoma at the time of diagnosis of the HMR-like syndrome and all 4 patients died within 3 weeks of this diagnosis. The pathogenesis of the HMR-like syndrome in these patients is discussed and it is concluded that it is probably reactive to an underlying opportunistic viral function related to their immunodepressed state secondary to CLL and/or the cytotoxic therapy.

The kinetics of homologous platelets, labelled in plasma with 111In-tropolonate, have been studied in five recipients with chronic marrow hypoplasia and severe thrombocytopenia, who were refractory to platelet transfusions as a result of alloimmunization. Mean platelet life span (MPLS), recovery, plasma 111In level and splenic and hepatic uptake kinetics were studied on two occasions, one using HLA-matched platelets and the other unmatched platelets. In each case, recovery of labelled platelets at 1 h post-injection and MPLS improved with HLA matching, although this improvement was highly variable. Only two of the five subjects would have derived any significant benefit from HLA-matched as compared with unmatched platelet transfusions. It was concluded that the need exists for additional cross-matching procedures, possibly related to platelet specific antigens, in patients who remain refractory to platelet transfusion.

A raised reticulocyte count is common in patients with immune or autoimmune haemolytic anaemia, and the result of the direct antiglobulin test (DAGT) is usually positive because of IgG or IgG and complement components on the red cells. We report on three patients who had low reticulocyte counts when they were most anaemic, and in whom no red cell autoantibodies could be detected by the DAGT. We postulate that reticulocytes may be selectively destroyed if antibodies are directed against antigenic sites on these young red cells, thus giving rise to a population of cells whose antigenic sites are poorly expressed. This theory might explain the low reticulocyte counts and the "absence" of antibodies (as tested by the DAGT) in such patients. Radioisotopic studies with 51Cr and 59Fe may provide useful information on the rate and sites of red cell destruction.

In 1979 two patients with Philadelphia (Ph1)-chromosome-positive chronic granulocytic leukaemia (CGL) were treated with chemoradiotherapy and transplantation of bone marrow from their respective identical twins. Subsequently twelve patients with Ph1-positive CGL in chronic phase were treated with chemoradiotherapy followed by transplantation of bone marrow from their HLA-identical sibs. Two of the fourteen patients have died of complications of the transplant procedure; twelve patients are alive and well. All the survivors have normal or nearly normal blood counts; there is no evidence of recurrent leukaemia or Ph1-positive cells in any patient after follow-up periods ranging from 97 to 1112 days. Bone-marrow transplantation should be considered in the management of any young patient with CGL who has a suitable marrow donor.

Hereditary haemolytic anaemia with basophilic stippling caused by pyrimidine 5'-nucleotidase deficiency is described in three members of two unrelated Irish families. In one family, the disease was moderately severe and the patient's condition was improved by splenectomy. In the other family the haemolytic anaemia was well compensated. In neither family was there a marked elevation of reduced glutathione. The implications are that pyrimidine 5'-nucleotidase deficiency is a heterogeneous disorder, both clinically and biochemically. In more severe forms splenectomy may be beneficial.

Although changes in the mechanical properties of infected red cells may contribute to the pathophysiology of malaria, such changes have not previously been described in detail. In this study, the physical properties of individual cells from both clinical and cultured samples infected with Plasmodium falciparum were tested using micropipette aspiration techniques. Cells containing ring forms took about 50% longer to enter 3 microns pipettes compared with nonparasitised cells, and there was a similar increase in the critical pressure required to induce cell entry. These abnormalities were similar in clinical and cultured samples. More mature cultured parasites (ie, trophozoites and schizonts containing pigment) caused much greater loss of deformability, with entry time and pressure increased four to sixfold. The decrease in deformability of the ring forms was attributable to a deficit in cell surface area/volume ratio (based on micropipette measurement of the surface area and volume of individual cells) and slight stiffening of the cell membrane (shear elastic modulus increased 13%, as measured by pipette aspiration of small membrane tongues). Measurement of the rate of cell shape recovery indicated that the membrane of parasitised cells was not more viscous. The main factor in the drastic loss of deformability of the trophozoites and schizonts was the presence of the large very resistant parasite itself. Otherwise, the cell surface area/volume deficit was slightly less and membrane rigidification slightly greater compared with ring forms. The above abnormalities should cause the trophozoites and schizonts to have great difficulty in traversing splenic or marrow sinuses and could contribute to microvascular occlusion and sequestration. On the other hand, the ring forms may be expected to circulate relatively unhindered.

1. The incorporation of (14)C into the brain glycogen of conscious rabbits with labelled glucose, bicarbonate and glutamate as precursors has been studied. 2. Substantial incorporation from all these precursors was demonstrated after an interval of 5hr. from their injection. 3. With [(14)C]glucose maximal incorporation occurred at about 8hr. from the time of injection. 4. Hydrocortisone led to increased incorporation of (14)C from labelled glucose. 5. Some comparisons between the turnover of brain glycogen and that of skeletal and cardiac muscle are reported.