Showing papers in "British Journal of Haematology in 1988"

Bone marrow transplantation (BMT) versus immunosuppression for the treatment of severe aplastic anaemia (SAA): a report of the EBMT* SAA Working Party

Abstract: This is an analysis of 509 patients with severe aplastic anaemia (SAA) treated in Europe between 1981 and 1986; 218 patients were treated by allogeneic bone marrow transplantation (BMT) from HLA identical sibling donors and 291 with immunosuppressive therapy (IS) with antilymphocyte globulin (ALG). The overall actuarial survival was 63% after BMT and 61% after IS therapy at 6 years. All patients fulfilled the criteria of SAA; however, most patients with a neutrophil count of less than 0.2 x 10(9)/l also had infections and haemorrhages. Therefore a further subclassification was defined by pretreatment peripheral blood neutrophil count: very severe aplastic anaemia (vSAA) (less than 0.2 x 10(9)/l neutrophils) and moderately severe aplastic anaemia (mSAA) (0.2-0.5 x 10(9)/l neutrophils). A Cox regression analysis showed that the only significant pre-treatment variables were a low neutrophil count (P = 0.001) and increasing age (P = 0.05). Thus it seemed reasonable to analyse survival data after combined stratification for neutrophils (vSAA versus mSAA) and age (cut off at 20 years). BMT was superior to IS in patients with vSAA under 20 years of age (64% v. 38%; P = 0.01). IS was superior to BMT in patients with mSAA aged 20 or more (82% v. 62%; P = 0.002). The two treatments gave comparable results in young patients with mSAA (BMT = 58%, IS = 62%; P = 0.1), and in older patients with vSAA (BMT = 44%, IS = 43%; P = 0.06). Overall 75/218 and 87/291 patients, given BMT or IS respectively, died. The major cause of failure in BMT patients was graft rejection (n = 22) or problems associated with graft-versus-host disease. For ALG patients the major problem was persistence of the aplasia with haemorrhage (n = 32) or infections (n = 46). This study indicates that over 60% of patients with SAA can be successfully treated with either BMT or IS. Overall survival does not differ in the two groups, though significant differences emerge after stratification for severity of the aplasia and age.

Plasminogen activator inhibitor (PAI-1) in plasma and platelets

Abstract: The distribution of PAI-1 in the plasma and platelets of normal individuals and of patients with platelet abnormalities was studied. An ELISA, capable of measuring PAI-1 in plasma at 1.5 ng/ml, and a functional assay of t-PA inhibition were used to assay platelet-free plasma (PFP), platelet-rich plasma in which the platelets were lysed (PRP) and serum. The PAI-1 concentration of normal PFP was 21.0 +/- 7.2 ng/ml (mean +/- SD) and those of PRP and serum were 282.6 +/- 68.0 and 270.3 +/- 71.9 ng/ml. The concentration of PAI-1 in PRP was proportional to the platelet count with 0.67 +/- 0.18 ng/10(6) platelets. Patients with thrombocytopenia had approximately normal PAI-1 concentrations in PFP; the extremely low concentrations in serum or PRP reflected the platelet count. A patient with grey platelet syndrome showed a comparable pattern, confirming that PAI-1 occurs in the platelet alpha-granules and indicating that the plasma concentration of PAI-1 is independent of the platelet pool of PAI-1. The median inhibitory activities towards t-PA were 1.6, 8.7 and 8.3 units/ml in normal PFP, PRP and serum respectively. PAI-1 in PFP had a median specific activity (units/mg PAI-1) about 5-fold higher than platelet PAI-1. Plasma and platelets represent two distinct pools of PAI-1, both of which should be considered in studies on the relationship between circulating PAI-1 and thrombotic disease.

Selective growth response to IL‐3 of a human leukaemic cell line with megakaryoblastic features

Abstract: A new human leukaemic cell line (M-O7) with the phenotypic characteristics of CFU-mega is described. Its cells are positive for T200 leucocyte common antigen (LCA) and negative with MAbs recognizing T and B cells and mature myelomonocytic antigens. In contrast, they react with MAbs recognizing antigenic determinants common to multi-lineage (CD13, CD33, CD34) and to bipotent erythromegakaryoblastic (CD36, H25) haemopoietic precursors, and with MAbs specific for platelet glycoproteins (CD41w, CD42w). A small proportion (10%) of the cells were large and multinucleated, and on electron-microscopy examination showed peripheral splitting of platelet-like cytoplasm particles. When transferred to a serum-free Iscove modified Dulbecco's medium supplemented with human insulin and transferrin, M-O7 cells stop proliferating. Of the haemopoietic growth factors tested for their ability to restore the proliferative activity of this quiescent population, only rH IL-3 proved effective. Moreover, it also increased the cloning efficiency in methylcellulose more than any other CSFs. The M-O7 cell line may provide a valuable tool for the biological assay of IL-3, and a model for biochemical studies of the megakaryocytic lineage.

Late haematological complications in severe aplastic anaemia

Abstract: 137 patients with severe aplastic anaemia (SAA) were treated in Basel from 1976 to 1986. 34 underwent bone marrow transplantation (BMT) and 103 received antilymphocyte globulin (ALG) therapy. We have analysed the incidence of late haematological complications in both groups of patients. 20 patients treated with ALG developed a late haematological complication. A myelodysplastic syndrome or frank leukaemia occurred in eight and paroxysmal nocturnal haemoglobinuria (PNH) in 13 patients. Nine of the 13 patients with PNH had clinical signs of haemolysis, four only had positive laboratory tests. One patient had PNH and acute leukaemia. The risk of developing a haematological complication increased continuously and reached 57% at 8 years. Neither PNH nor leukaemia occurred in patients treated with BMT. The increased survival rate and the long observation time after ALG therapy have revealed a new perspective of the prognosis of aplastic anaemia. Patients treated with BMT appear to be cured whereas those treated with ALG remain at risk for late complications.

Tumour necrosis factor may contribute to the anaemia of malaria by causing dyserythropoiesis and erythrophagocytosis.

Abstract: Among the unexplained changes caused by malaria in several host species, including man and mouse, are erythrophagocytosis and dyserythropoiesis. In order to see whether tumour necrosis factor (TNF) could contribute to these changes we injected recombinant human TNF intravenously into mice made very susceptible to this monokine by low density infection with a mouse malaria (Plasmodium vinckei) or prior injection of an extract of Coxiella burneti. Appreciable erythrophagocytosis, involving nucleated erythroblasts as well as mature red cells, was observed in bone marrow preparations from TNF-treated mice and those with severe illness due to P. vinckei, but in no other group. Dyserythropoiesis, involving irregularly-shaped nuclei and karyorrhexis, had the same distribution. TNF at concentrations up to 8 ng/ml was detected in the serum of all mice with severe malaria, but not uninfected animals or those with light infections. These findings are consistent with TNF making an important contribution to erythrophagocytosis and dyserythropoiesis, and thus to anaemia, in malaria and other conditions.

Changes in the plasma levels of vitamin K-dependent proteins C and S and of C4b-binding protein during pregnancy and oral contraception

Abstract: Summary The plasma concentrations of protein S, protein C and C4b-binding protein (C4BP) were analysed during pregnancy, in the postpartum period and in women using oral contraceptives. Free protein S. measured after precipitation of the C4BP-protein S complexes with 5% PEG 6000, was found to be 8.3 mg/l in the control group, which represents 36.3% of the total plasma protein S content (average 23.5 mg/l). The concentration of protein S was significantly decreased during pregnancy, the lowest levels occurring in the second trimester (14.8 mg/1). The values returned to normal within a few days after delivery. The concentration of free protein S was also decreased, down to an average of 3.7 mg/l at delivery, and did not return to normal within the first week postpartum. The mean concentration of protein S in women using oral contraceptives decreased to 17.7 mg/l and the free fraction went down to 6.6 mg/l. Unlike that of protein S, the plasma concentration of protein C increased during pregnancy, reaching a maximum of 135% in the second trimester. Also, it was significantly higher in the postpartum period and in women using oral contraceptives, than in controls. The level of C4BP was increased throughout pregnancy, with a maximum of 143.4% at delivery. These changes in the plasma levels of proteins C and S during pregnancy indicate that the two proteins differ in the regulation of their synthesis. The major decrease in the level of free protein S may predispose to thrombotic episodes during pregnancy, whereas the increased level of protein C may have the reverse effect. These results indicate the importance of taking into account the normal changes in the plasma levels of protein C and S during pregnancy and the use of oral contraceptives, when evaluating patients with increased risk of thromboembolic disease.

Refined chromosome study helps define prognostic subgroups in most patients with primary myelodysplastic syndrome and acute myelogenous leukaemia.

Abstract: Summary Based on a 6·1/2-year study of 284 consecutive adult patients with primary myelodysplastic syndrome (MDS) and and acute myelogenous leukaemia (AML), we have found that refined chromosome analysis can be used as an independent prognostic indicator in the great majority of patients with MDS and AML. In MDS, the FAB subtype was also found to have prognostic value and this was enhanced when the chromosomal findings were taken into consideration. In AML, the age of the patient correlated more closely with the chromosomal changes in predicting prognosis in most patients than did the FAB classification. Previously we reported that refined chromosome analysis of bone marrow specimens from 161 adult patients with primary or non-therapy related MDS and AML identified three prognostic chromosomal categories in each disease, representing 40% of all patients (Yunis et al, 1984, 1986). By extending our study to 284 patients, as well as a longer follow-up, it was possible to determine the prognostic implications of two additional chromosomal categories in MDS and five in AML. Since 73% of all patients are now represented in well-defined chromosomal subgroups with prognostic significance, refined chromosome analysis emerges as a tool that could have considerable impact in clinical decision making and in the development of treatment protocols.

Inheritance and bleeding in factor XI deficiency.

Abstract: A study of 20 Jewish and four non-Jewish kindreds transmitting factor XI deficiency (164 individuals) confirmed inheritance to be autosomal with severe deficiency in homozygotes (mean factor XI level 3.8 u/dl, SD 2.91) and partial deficiency in heterozygotes (mean factor XI level 57 u/dl, SD 10.42; normal mean factor XI level 96 u/dl, SD 11.6). The probability of an individual being heterozygous can be predicted from the factor XI level using a graph derived from this data. The accuracy is increased by including the prior probability derived from the pedigree. A high frequency of heterozygote to heterozygote mating was observed in the Jewish families consistent with an estimated gene frequency of 13.4% in this racial group. The relationship between factor XI level and bleeding tendency is poor; a third of heterozygotes had bled excessively after surgery, including six with factor XI levels above 50 u/dl, showing this condition to have clear signs of expression in heterozygotes. The lower limit of the normal range (2 SDs from the mean) was found to be 72 u/dl.

Bone marrow transplantation for chronic myeloid leukaemia in first chronic phase: importance of a graft-versus-leukaemia effect.

Abstract: We analysed the incidence of graft failure, graft-versus-host disease (GVHD) and relapse of leukaemia in 208 patients undergoing allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia in chronic phase in eight transplant centres in Europe and the United States. 106 patients received unmanipulated donor bone marrow (Group 1) and 102 patients received marrow depleted of T-cells by incubation with the monoclonal antibodies Campath-1 or CT-2 and complement (Group 2). The incidence of graft failure was higher and of GVHD was lower in Group 2 than in Group 1. Relapse of leukaemia occurred more frequently in patients in Group 2 than in Group 1 (17 v. 2, P less than 0.001). Multivariate analysis showed that the following factors were associated with an increased risk of relapse: the use of T-cell depletion, the absence of GVHD and a high platelet count at the time of admission for transplant. The findings support the concept that a graft-versus-leukaemia effect mediated by T-lymphocytes is important for cure of leukaemia after BMT.

Prognostic features of chronic myelomonocytic leukaemia: a modified Bournemouth score gives the best prediction of survival.

Abstract: We have studied the clinical and laboratory features of 53 cases of chronic myelomonocytic leukaemia (CMML) defined according to the FAB criteria. A granulocyte count of greater than 16 X 10(9)/l or a monocyte count of greater than 2.6 X 10(9)/l correlated with a poorer survival, but the best predictor of poor survival was the modified 'Bournemouth' score in which one point each was allocated for Hb less than 10 g/dl, neutrophils less than 2.5 X 10(9)/l or greater than 16 X 10(9)/l, platelets less than 100 X 10(9)/l and bone marrow blasts greater than 5%. A score of 2 or more was predictive of poor survival (Chi2 = 10.25; P less than 0.001). Those patients with low monocyte and neutrophil counts and a low modified Bournemouth score tended to have a clinical course similar to refractory anaemia (RA) with an indolent course not requiring treatment and long survival whereas those with high monocyte and neutrophil counts and a high modified 'Bournemouth' score have a clinical course resembling that of refractory anaemia with excess of blasts (RAEB).

Enhancement of erythropoiesis in vitro by human growth hormone is mediated by insulin-like growth factor I.

Abstract: Insulin-like growth factor I (IGF-I) is the presumed paracrine or autocrine growth-promoting mediator of growth hormone in peripheral tissues. In order to evaluate the role of IGF-I as mediator of human growth hormone (hGH) in erythropoiesis, we compared the effects of both peptides upon in vitro colony formation by primitive (BFU-E) and relatively mature (CFU-E) human erythroid precursors. Biosynthetic IGF-I (2 ng/ml) and hGH (25 ng/ml) induced a significant increase in the growth of both BFU-E and CFU-E. BFU-E growth was maximally enhanced by 6 ng/ml IGF-I and by 50 ng/ml hGH, resulting in an increase in burst numbers of 62 +/- 12% and 52 +/- 12%, respectively. Maximal enhancement of CFU-E growth was detected at higher concentrations of IGF-I (20 ng/ml) and hGH (150 ng/ml), with respective increases of 121 +/- 35% and 137 +/- 18% in colony numbers. Enhancement of bone marrow and peripheral blood erythroid progenitor cell growth by hGH required the presence of monocytes and was abrogated by specific monoclonal antibodies directed against IGF-I membrane receptors. The in vitro growth-promoting effect of hGH upon human erythroid precursors thus appears to be mediated by paracrine IGF-I.

Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults.

Abstract: Myelodysplasia (MDS) and leukaemia following acquired aplastic anaemia has been reported as a rare event occurring in about 5% of patients. Improved results in survival of patients with severe aplastic anaemia (SAA) and subsequent prolonged follow-up created the possibility of evaluating the occurrence of MDS and leukaemia in 38 adult patients with acquired SAA surviving two or more years without bone marrow transplantation. Five patients, age 22, 35, 47, 56, 72 years, two females, three males, all with idiopathic SAA and normal cytogenetic analysis developed a refractory anaemia (RA) 7, 30, 48, 56, 142 months after diagnosis of SAA. In 3/5 RA evolved into an acute myeloid leukaemia (AML) either via a chronic myelomonocytic leukaemia (CMML) (2/3) or via RA with excess of blasts (RAEB) (1/3). Three patients revealed a monosomy 7 during MDS and/or leukaemic phase. One patient died during RA phase without cytogenetic abnormalities. A pattern of evolution could be identified in these patients revealing well-documented SAA - improvement of bone marrow haematopoiesis - dyshaematopoietic features of one or more cell lines with predominance of dyserythropoiesis - RA - RAEB or CMML - AML. These five patients represent more than 10% of all patients surviving at least 2 years. This implies that the risk of developing MDS and leukaemia in SAA patients surviving with autologous marrow, might increase with longer follow-up.

The molecular basis of thalassaemia major and thalassaemia intermedia in Asian Indians: application to prenatal diagnosis

Abstract: A study of the molecular pathology of beta thalassaemia in the Asian Indian immigrant population in the U.K. included 37 patients with thalassaemia major and 14 with thalassaemia intermedia. Using a combination of oligonucleotide probe hybridization and restriction endonuclease analysis the mutations in 100/102 (98%) of the beta thalassaemia genes were characterized. Nine different types were found, of which six are associated with beta zero, one with severe beta+ and two with mild beta+ thalassaemia. Comparison of the beta-globin gene cluster haplotypes, alpha globin genotypes and beta gene mutations of the thalassaemia major group with the thalassaemia intermedia group suggests that the co-inheritance of a high Hb F determinant associated with the - + - + + 5' beta haplotype and the inheritance of a mild beta-thalassaemia mutation are the major ameliorating factors of disease severity in Asian Indians. In comparison with other population groups. beta thalassaemia in Asian Indians is not associated with one or two predominant mutations. Despite this, prenatal diagnosis by direct detection is possible in the majority of families by restriction analysis and a limited number of oligonucleotide probes since the majority of severely affected individuals are homozygous for a single mutation. The characterization of these mutations should be useful for the planning of prenatal diagnosis programmes for beta thalassaemia in other Asian Indian communities.

Relapse after allogeneic bone marrow transplantation for acute leukaemia: a survey by the E.B.M.T. of 117 cases.

Abstract: A multi-centre retrospective analysis on 117 patients relapsing after bone marrow transplantation (BMT) for acute leukaemia was carried out by the Leukaemia Working Party of the European Group for Bone Marrow Transplantation (E.B.M.T.). Forty-one patients had acute myeloid leukaemia (AML) and 76 had acute lymphoblastic leukaemia (ALL). Relapse occurred between 3 and 30 months after BMT and where investigated the leukaemia was found to have relapsed in recipient cells. In 10 cases the relapse was associated with new cytogenetic abnormalities. 74 patients received further treatment for leukaemia. Of these 21 out of 50 with ALL and 11 out of 24 with AML achieved a complete remission and had a median survival of 12 months compared with a median survival of 4 months for untreated patients or patients not achieving complete remission (P less than 0.001). Factors predictive for successful remission induction were a long interval between bone marrow transplant and relapse in ALL patients; and isolated extramedullary relapse. Presenting blast count, karyotype and remission status and number at the time of BMT were not predictive. Donor bone marrow was shown to be responsible for haemopoietic recovery occurring in the 21 out of 31 patients tested who achieved remission using donor karyotype or red blood cell antigens as markers. Nine patients received a second bone marrow transplant but only one became a long-term survivor. The results show that chemotherapy can usually prolong survival in selected patients with acute leukaemia relapsing after BMT but further BMT has a poor outlook.

3'-Azido-3'-deoxythymidine (AZT) inhibits proliferation in vitro of human haematopoietic progenitor cells.

Abstract: Peripheral blood cytopenias are a serious, dose-limiting toxicity of AZT therapy in patients infected by HIV. To evaluate the mechanism by which cytopenias develop, AZT effects of haematopoietic differentiation and growth were measured in serum-free, nucleoside-depleted cultures of normal human bone marrow. In contrast to native thymidine, AZT suppressed the proliferation of erythroid, granulocyte/macrophage and primitive haematopoietic stem cells in a dose-related and time-dependent fashion. Relative progenitor sensitivity varied, with half-maximal concentrations of 1-5 microM and 20-40 microM AZT for inhibition of erythroid and nonerythroid progenitor cell growth, respectively. Inhibition was observed over full ranges of concentrations of haematopoietic tissue-specific regulators (human erythropoietin, colony-stimulating activity, interleukin-3 and lymphocyte conditioned medium) and of platelet-derived growth factor (PDGF), an agent that enhances erythropoiesis in vitro via accessory marrow stromal elements. Furthermore, suppression was similar in cultures of marrow cells that were depleted of accessory populations, suggesting that its action is directed at progenitors. Finally, when deoxythymidine was added in increasing amounts to cultures with a half-maximal concentration of AZT, inhibition was abrogated. We conclude that AZT is a potent inhibitor of haematopoiesis in vitro, and that erythroid progenitors are particularly sensitive to its action. These results may explain the marrow hypoplasia that occurs during AZT administration in vivo.

A prognostic classification of myelofibrosis with myeloid metaplasia.

Abstract: The dependence of survival time on a set of prognostic factors was explored by means of Cox's regression model in 137 cases of myelofibrosis with myeloid metaplasia (MMM). The following parameters recorded at diagnosis proved to be important independent indicators of a poor prognosis: a higher value for age, a lower value for Hb concentration, a higher value for immature myeloid cells in peripheral blood (IMC), a lower value for total erythroid iron turnover (TEIT), and a bone marrow red cell aplasia (RCA). A prognostic classification tree was constructed whose terminal nodes (risk groups), described by simple logical conditions upon important indicators, were characterized by significantly different expected survival. The two extreme risk groups lend themselves to a simple, but complete description. The low-risk group (19.7% of the sample) comprises cases who had the diagnosis of MMM before age 45 and a number of IMC constantly lower than 24%. The actuarial proportion of patients surviving at 15 years was 100%. The high-risk group (29.9% of cases) comprises patients with age greater than 45 and Hb lower than 13 g/dl, associated with RCA, or with a relatively decreased erythropoiesis (TEIT lower than 2 times the normal) or with IMC greater than 24%. Seven out of the 11 who died within this group developed blastic crisis. Median survival time of the group was 69 months.

Alkaline phosphatase positive precursors of adipocytes in the human bone marrow.

Abstract: Developing fat cells in the bone marrow of leukaemic patients treated with chemotherapy were found to be endowed with membrane-bound alkaline phosphatase. Since alkaline phosphatase is a cytochemical marker of 'reticular' cells, this observation provides cytochemical evidence that reticular cells may convert to adipocytes when marrow cellularity abruptly decreases.

Acquired alpha-2-antiplasmin deficiency in acute promyelocytic leukaemia.

Abstract: We report a prospective study in nine consecutive adult patients with acute promyelocytic leukaemia (APL). The study objective was to assess the prevalence of activation of blood coagulation and/or activation of fibrinolysis in APL. Coagulation and fibrinolytic parameters relevant to the objective included antithrombin III, plasminogen, fibrin/fibrinogen degradation products and alpha-2 antiplasmin activity and antigen levels. The results of this study revealed consistently normal antithrombin III levels, both before and in the course of antileukaemic treatment. Plasminogen levels were slightly decreased or normal. However, a distinct alpha-2 antiplasmin activity deficiency in all patients was observed with levels even reaching zero in three patients, during chemotherapy. Alpha-2 antiplasmin activity levels were consistently lower than the alpha-2 antiplasmin antigen levels. The in vitro binding of alpha-2 antiplasmin activity to fibrin clots was severely reduced which appeared to be due to the reduced alpha-2 antiplasmin plasma levels. Upon crossed-immunoelectrophoresis against alpha-2 antiplasmin antiserum two alpha-2 antiplasmin antigen peaks were observed in the plasma of all nine patients. All abnormalities were reversible 4 d after completion of chemotherapy. In a second series of 12 consecutive APL patients we confirmed the consistency of the alpha-2 antiplasmin activity deficiency and normal antithrombin III plasma levels. In addition Protein C activity and antigen levels were normal or near normal in 10 and reduced in two patients. Thrombin-antithrombin III complexes were increased in 10 and normal in two patients. We conclude that some activation of blood coagulation is present in APL (increased thrombin-antithrombin III complex levels) but its contribution to the coagulopathy seems to be minor (normal antithrombin III and only slightly reduced protein C levels). The observed reduced alpha-2 antiplasmin content of the fibrin clot in vitro may result in vivo in a fibrin clot that is highly susceptible to fibrin degradation, thus aggravating the coagulopathy in APL.

Reversal of drug-resistance in multiple myeloma with verapamil.

Abstract: Summary In vitro testing with a new doxorubicin resistant myeloma cell line revealed the reversal of drug resistance with as little as 100 ng/ml of verapamil (a calcium channel blocker), a dose easily achievable in humans. A first patient with IgGk Myeloma is presented in whom resistance to VAD (vincristine/doxorubicin infusion plus dexamethasone) chemotherapy was reversed with the administration of verapamil. A subsequent clinical study has been initiated. The potential for further evaluation of calcium channel blockers in multiple myeloma is discussed.

Extracellular matrix of the marrow microenvironment.

Abstract: Haemopoiesis in vivo and in vitro is closely associated with specific niicroenvironmental conditions provided by stromal cells and their products and by certain non-stromal cells such as macrophages. The long-term bone marrow culture (LTBMC) system (Dexter et al, 1977) has permitted detailed investigation of relationships between haemopoietic cells and the microenvironment in vitro and it is clear that the extracellular component of the haemopoietic microenvironment plays an important role in the regulation of cell proliferation and differentiation.

Effects of platelet derived growth factor, epidermal growth factor and transforming growth factor-β on the growth of human marrow fibroblasts

Abstract: Summary. The effects of three growth factors contained in platelets on human bone marrow fibroblasts as well as fibroblast colony-forming cells were investigated to clarify the pathogenesis of marrow fibrosis frequently associated with myeloproliferative disorders. Two growth factors, platelet derived growth factor (PDGF) and epidermal growth factor (EGF), cooperatively stimulated the growth of passaged fibroblasts as well as fibroblast colony-forming cells. Transforming growth factor-β augmented the growth of fibroblasts at low concentrations in the presence of EGF and/or PDGF, but inhibited that of colony-forming cells in the presence of PDGF or EGF. These results suggest that the growth of fibroblasts causing marrow fibrosis might be finely regulated, depending on the concentrations of growth factors released or leaked from platelets or megakaryocytes into marrow environment.

Effects of preparative procedures and of cell activation on flow of white cells through micropore filters

Abstract: Using newly developed filtration techniques to analyse the flow resistance of white blood cells (WBC), the effects of preparative procedures and of various treatments (including activation) have been tested. Flow rates of WBC were measured using both 5 and 8 micron pore filters. It was found that EDTA was the anticoagulant of choice over heparin and sodium citrate, and that calcium-containing media should be avoided because they promoted cell-cell aggregation. Exposure to density separation media did not significantly alter filtration rates, but storage of prepared WBC for even 1 h caused deterioration in flow. Storage of whole blood before preparation of the WBC had a much lesser effect. Storage appeared to be linked to cell activation, and use of the activating agents phorbol ester (PE, 4.5 x 10(6) M/l) and f-methyl-leucyl-phenylalanine (FMPL, 10(-7) and 10(-9) M/l) also increased WBC resistance to pore transit. The effect of PE was greater, causing rapid pore blockage, but was non-specific, while the lesser effect of FMLP was restricted to granulocytes and was concentration dependent. Cell volume was increased by these agents, but measurement of the filterability of hypotonically swollen granulocytes showed that volume changes alone only partly explained the activation effects. These results suggest that activation in vivo would have a significant rheological effect, detectable by filtration methods.

Fibrinolysis during normal human pregnancy: complex inter-relationships between plasma levels of tissue plasminogen activator and inhibitors and the euglobulin clot lysis time

Abstract: Summary. Although it has been previously considered that blood fibrinolytic capacity is reduced during pregnancy, this has been disputed. Also the mechanisms underlying any change in fibrinolysis in pregnancy require clarification. We have therefore measured the plasma activity of tissue plasminogen activator (t-PA) and inhibitors (t-PAi) and the concentration of the pregnancy specific inhibitor (PA12) antigen, as well as the euglobulin clot lysis time (ECLT) during normal pregnancy. Plasma concentrations of fibrinogen, plasminogen, fibrin(ogen) degradation products (FDP) and cross-linked products (D-dimer) were also monitored. We confirm a marked reduction of the fibrinolytic activity of the plasma euglobulin fraction from the second trimester, and a parallel reduction in t-PA and increase in t-PAi activities, with rapid return to non-pregnant levels post-partum. In contrast, PAI2, whilst undetectable in non-pregnant control plasma, was already measurable in the first trimester, increased through pregnancy, and remained at a high concentration up to at least 48 h post-partum. Fibrinogen and plasminogen concentrations rose progressively through pregnancy and FDP and D-dimer were frequently detectable in late pregnancy plasma. Changes in the ECLT and plasma t-PA and t-PAi activities in pregnancy cannot therefore be directly related to the concentration of PAI2 antigen. Also, despite the apparent marked reduction in fibrinolytic capacity fibrin(ogen) breakdown products are frequently present in increased plasma concentrations in late pregnancy.

Characterization of two deletions that remove the entire human zeta-alpha globin gene complex (- -THAI and - -FIL).

Abstract: We have fully characterized two alpha thalassaemia mutants that occur in Southeast Asia, - -THAI and - -FIL. Each mutant is due to a deletion that removes the entire zeta-alpha-globin gene complex. Localization of the 5' breakpoints described here, allows the identification of unique fragments that are specific for each of the two mutations. This information can be used to assess the frequency of these mutants in Southeast Asia and will be of value in prenatal testing for alpha thalassaemia in this area.

Polyclonal integration of HTLV-I proviral DNA in lymphocytes from HTLV-I seropositive individuals : An intermediate state between the healthy carrier state and smouldering ATL

Abstract: Summary We have studied 15 individuals (aged 14-74 years) with antibodies to HTLV-I in their serum and random integration of HTLV-I proviral DNA in their peripheral blood lymphocytes. All but one of these patients suffered from a variety of non-specific complaints which did not correspond to those of adult T-cell leukaemia (ATL). All of them were born in Kyushu and Okinawa which are endemic areas for HTLV-I infection; 25% of their family members were also seropositive for HTLV-I. The only haematological abnormality in these patients was the presence of few atypical lymphoid cells in the peripheral blood. The CD4/CD8 ratios were normal but the proportion of Tac positive cells was slightly higher than normal. These individuals with polyclonal integration of HTLV-I proviral DNA seem to represent an intermediate state between smouldering ATL (monoclonal integration) and healthy HTLV-I carriers (with antibodies but no detectable HTLV-I proviral DNA). Patients with this intermediate state of HTLV-I infection may be at risk to progress to ATL. The natural history of HTLV-I infection in humans leading to the development of ATL is reviewed in the light of these new findings.

Elevated red cell adenosine deaminase activity: a marker of disordered erythropoiesis in Diamond-Blackfan anaemia and other haematologic diseases.

Abstract: Red-cell adenosine deaminase (ADA) activity in children with Diamond-Blackfan anaemia is significantly increased (1.91 +/- 0.90 U/g Hb) compared to that seen in transient erythroblastopenia of childhood (0.80 +/- 0.16 U/g Hb) or normal individuals (0.61 +/- 0.13 U/g). These data thus further support that measurement of this purine metabolic enzyme is useful in diagnosing the cause of pure RBC aplasia in children. Of interest, however, elevated RBC-ADA activity also is seen in some children with acute leukaemia and other haematologic disorders. In children with acute lymphoblastic leukaemia (ALL), the increase in RBC-ADA activity is proportional to the degree of anaemia. However, the elevated RBC-ADA activity in this leukaemic population is not related to the fetal haemoglobin concentration. These data suggest increased RBC-ADA activity may be a non-specific manifestation of abnormal erythroid stem cell function, an alteration distinct from that seen with reactivation of fetal erythropoiesis. However, since almost all patients with Diamond-Blackfan anaemia manifest elevated RBC-ADA activity, this chemical alteration yet may reflect the specific erythroid differentiation lesion in this disorder.

Improvement in rejection, engraftment rate and survival without increase in graft‐versus‐host disease by high marrow cell dose in patients transplanted for aplastic anaemia

Abstract: Summary. Two hundred and fifty-two consecutive patients with severe aplastic anaemia were conditioned by cyclophosphamide and given marrow transplants from HLA-identical siblings or parents. The results were analysed to examine the influence of marrow cell dose on graft rejection, speed of engraftment, survival and acute and chronic graft-versushost disease (GVHD). Sixty patients received a corrected marrow cell dose below 2.2 × 108 nucleated cells per kg body weight, 73 patients between 2.2 and 3.2 × 108 cells/kg, 60 patients between 3.3 and 4.2 × 108 cells/kg, and 59 patients more than 4.2 × 108 cells/kg. The significant association between high marrow cell dose and low risk of graft rejection previously reported was confirmed. In addition, high marrow cell dose resulted in small but statistically significant reductions in time to granulocyte and platelet recoveries in patients with sustained engraftment. High marrow cell dose was also associated with a significant improvement in patient survival, a result solely due to the reduction in the incidence of graft rejection. Neither acute nor chronic GVHD were influenced by marrow cell dose. We conclude that the maximum marrow cell dose should be transplanted in patients with severe aplastic anaemia. Maximum marrow cell doses are likely to decrease the incidence of graft rejection, shorten the time to engraftment thereby decreasing the need for blood products and antibiotic support, and improve overall patient survival.

Use of leucocyte-poor blood components to prevent primary cytomegalovirus (CMV) infection in patients with acute leukaemia.

Abstract: de Graan-Hentzen et al(1987) reported the iuse of leucocytepoor blood components to prevent primary CMV infection in patients with acute leukaemia; 38 CMV-seronegative patients remained seronegative 3 months after the start of remission induction therapy, although there was no control group in their study. We report the incidence of CMV ~erocoiiver~ion i 180 acute leukaemia patients undergoing initial1 chemotherapy and receiving one of three different transfusion programmes depending on the level of leucocyte-depletion, as shown below and see Table I: Group A: plasma-reduced red cell transfusions and standard platelet concentrates. Group B: plasma-reduced red cell transfusions and leucocyte-poor platelet concentrates. Group C: leucocyte-poor red cell transfusions and leucocyte-poor platelet concentrates. A detailed description of the methods used for preparation of the leucocyte-poor blood components has been reported (Murphy et al. 1986). Serum samples were tested for CMV IgG antibodies at initial presentation and at the end of remission induction therapy using an indirect radioimmunoassay technique. Samples from platelet donors were routinely tested for CMV IgG antibodies and 50% were found to be CMV-seropositive; samples from red cell donors were not routinely tested, but it is likely that the incidence of CMV-seropositivity was similar. The three groups of patients were similar in terms of age, number of transfusions received and numbers of patients CMV-seronegative at presentation with acute leukaemia; there was a preponderance of males in group B. In group A, nine of the 39 patients (23%) were CMV-seronegative at initial presentation, and two patients (22%) became seropositive during remission induction therapy (see Table I). In group B. 34 of the 107 patients (32%) were seronegative before treatment, and four (12%) became seropositive during treatment. In group C. 11 of the 32 patients (32%) were seronegative before treatment and none became seropositive during treatment. The six patients who seroconverted to CMV-seropositive during treatment received a mean number of 30 (range 844) red cell concentrates and 22 (range 11-45) platelet concentrates; the mean number of CMV-seropositive platelet concentrates received by these patients was 7 (range 2-12). The results in this study, although not statistically significant, suggest that the incidence of primary CMV infection in patients being treated for acute leukaemia is lower in patients receiving leucocyte-poor blood components than in those receiving standard blood components, and support the observations of de Graan-Hentzen et al(1987).

A combination of factor Xa and phosphatidylcholine-phosphatidylserine vesicles bypasses factor VIII in vivo.

Abstract: A combination of phosphatidylcholine-phosphatidylserine lipid vesicles (PCPS), as a source of coagulant active phospholipid, when infused with factor Xa bypasses factor VIII in vivo. To demonstrate this, a reproducible model of bleeding in haemophilic dogs was used. Control studies were performed in normal dogs. In initial studies, factor Xa/PCPS at a dose of 6.5 x 10(-12) and 4.0 x 10(-7) moles/kg respectively failed to correct the abnormal bleeding in the haemophilic animals and initiated a bleeding diathesis in the normal controls. Coagulation studies and immunoblotting demonstrated activation of protein C and an anticoagulant effect resulting from significant falls in the levels of factors V and VIII. Adjustment of the dose of factor Xa/PCPS to 2.6 x 10(-11) and 4.0 x 10(-8) moles/kg respectively produced an immediate haemostatic effect in both haemophilic and normal animals with bleeding stopping within 15-30 s. Despite this observation, protein C activation was again noted. It is concluded that the presence of coagulant active phospholipid and factor Xa in prothrombin complex concentrates may explain the observed factor VIII bypassing activity of these preparations and that the use of a controlled formulation of these two components may provide a more effective approach to the management of patients with factor VIII inhibitors.