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A higher than normal incidence of thromboembolic events has been observed in adult patients with beta-thalassaemia major (TM) and certain haemostatic anomalies found in these patients suggest the existence of a chronic hypercoagulable state. Thalassaemic red blood cells (RBC) were demonstrated to facilitate thrombin formation due to altered asymmetry of the membrane phospholipids with enhanced exposure of phosphatidylserine. Since RBC anomalies exist in thalassaemia from the first months of life, we studied markers of hypercoagulability and thrombophilia in 36 adult patients (range 19-38 years) and 26 children (range 2-18 years) with beta-TM. All the patients were in steady state and none had experienced clinical signs or symptoms of thrombosis. Highly elevated urinary levels of 11-dehydro-thromboxane B2 and significantly elevated plasma levels of thrombin-antithrombin III (TAT) complexes were observed to the same extent in TM children and adults. The levels of factor II were decreased while factors V, VII + X and plasminogen were within the normal range. The natural coagulation inhibitors, protein C (PC) and protein S (PS) were significantly decreased in all TM patients investigated, regardless of age, but the PS binding protein (C4bBP) and antithrombin III levels were normal. The frequency of other thrombophilic mutations was not increased. Thus, a chronic hypercoagulable state already exists in thalassaemia in childhood and may contribute to the cardiac and pulmonary anomalies and the thrombotic events which occur later.

Polycythemia vera (PV) is associated with a high incidence of thrombosis. The association of apparent and secondary polycythemia with thrombosis is not clear. It was suggested that activation of the coagulation system contributes to thrombus formation in PV. However, the mechanism of activation is unknown. Monocytes generate a potent tissue factor (TF) upon stimulation with various substances, which is involved in thrombus formation in various disorders. Therefore, we studied the possibility that the factor is involved in the activation of coagulation and thrombus formation also in PV. Unstimulated peripheral blood mononuclear cells (PBMC) from each of the different types of polycythemia expressed weak TF activity (2 U) and antigen (41.4 to 52.9 pg/ml), which were similar to normal controls. Following stimulation with endotoxin, PBMC from normal controls and from apparent and secondary polycythemia showed a 3.9- to 4.5-fold increase in TF, while cells from PV showed a 21-fold increase (P<0.001). Similar levels were generated by PBMC after treatment of PV and at the spent phase. TF was generated by monocytes but not by lymphocytes. Plasma prothrombin fragment1+2 (F1+2) levels, assayed at the same time, were significantly higher in PV (2.46 nm) compared to normals and apparent and secondary polycythemia (0.22 to 0.32 nm), and were in a significant correlation with monocyte TF activity and antigen levels (r = 0.77, 0.87). The high levels of F1+2 confirm that the coagulation system is activated in PV. The increased capacity of monocytes to generate TF may be responsible for the activation of the coagulation system and thrombus formation. The hypercoagulability state that is induced by this mechanism suggests that long-life oral anticoagulation should be considered once thrombosis has been developed in PV.

Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with beta-thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p-selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual-colour flow cytometry. A significantly (P < 0.01) higher fraction of FITC-annexin V-labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation (P < 0.001) was found in TM patients between the number of RBC-bound annexin V molecules and the fraction of CD62p (p-selectin) or CD63-positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelerate thrombin generation in vivo which, in turn, triggers platelet activation.

"Autonomous" development of erythroid colonies in erythropoietin (EPO)-free semi-solid culture has been used as an in vitro assay for diagnosis of polycythemia vera (PV). These colonies, however, are small and poorly hemoglobinized, rendering the assay in many cases unreliable. We report here on the use of a novel assay; it combines a modified culture procedure that maximizes the growth of EPO-independent erythroid cells, and immunofluorescence flow cytometry for their detection and quantitation. Peripheral blood mononuclear cells are cultured for 2-5 days in the presence of a combination of growth factors. During this phase, early erythroid committed progenitors, burst forming units (BFUe), proliferate and differentiate into colony forming units (CFUe)-like progenitors. In the second phase, the latter cells, in the presence of stem cell factor, hemin, and iron-saturated transferrin, continue to proliferate and mature into hemoglobin (Hb)-containing orthochromatic normoblasts. Neither phases contained EPO. The culture produced large, pure, and synchronized erythroid cell populations. The cells were then dually labeled with fluorescent probes, nuclear DNA with thiazole orange and intracellular hemoglobin (Hb) with phycoerythrin-conjugated monoclonal antibodies against human Hb. Cells positive for both labels were assigned as Hb-containing nucleated precursors. The presence of such cells in EPO-free cultures indicated "autonomous growth." None of the EPO-free cultures derived from normal donors or patients with secondary polycythemia contained such cells. Cultures derived from PV patients contained from 5 to 92% "autonomously grown" cells. These culture and analysis methods should minimize false negative results with PV patients and provide objective and quantitative data.

Random and directed migration, O2- production, degranulation and adhesion were studied in neutrophils obtained from patients with homozygous beta-thalassaemia and iron overload, in the presence or absence of thalassaemic serum. The only significant defect found was an impairment in directed chemotaxis, further depressed after addition of thalassaemic serum. The chemotactic defect was encountered in all the patients that have suffered from pyogenic infections except one, and was not correlated with the severity of the iron overload. It is suggested that the described neutrophil migration impairment may contribute to the tendency towards infection in certain patients with homozygous beta-thalassaemia.

Investigation of red blood cells from beta-thalassemia patients by means of prothrombinase assay reveals phosphatidylserine in the outer leaflet of the plasma membrane. This might explain their elevated susceptibility to phagocytosis by macrophages and the chronic hypercoagulable state, frequent thrombotic events, and life-long platelet activation that are found in thalassemic patients.

The chronic severe anemia of patients with beta-thalassemia major stimulates extensive erythropoiesis, which results in circulating nucleated normoblasts. We devised a dual staining flow cytometric procedure in order to analyse the cell cycle and ploidy of these normoblasts. Peripheral blood cells of O blood-group type were first stained with Fluorescein Isothiocyanate (FITC)-conjugated anti-H lectin which labels erythroid cells (RBC and normoblasts) by green fluorescence, and then with propidium iodide (PI) which binds to DNA and thereby labels nucleated cells (leukocytes and normoblasts) by red fluorescence. The leukocytes and normoblasts present in the blood sample of thalassemic patients could be distinguished and "gated" based on their green fluorescence. The PI (red) fluorescence, i.e., the DNA histogram of each population, was thus obtained. The results indicated no statistically significant difference in the PI fluorescence of these two populations. Thus, in spite of the abnormal erythropoiesis in beta-thalassemia, the resultant orthochromatic normoblasts are normal with respect to their DNA content.

The levels, structure and composition of plasma lipoproteins were determined in 67 patients with homozygous beta-thalassaemia and compared to healthy or heterozygous members of the same families and to patients with either sickle cell or iron deficiency anaemia. Plasma total and LDL and HDL cholesterol levels were low in patients with homozygous beta-thalassaemia and with sickle cell anaemia. Plasma triglycerides did not differ between subjects. The low plasma and lipoprotein cholesterol was independent of age, transfusion requirements and splenectomy. Abnormal structure and composition of lipoproteins was found in homozygous beta-thalassaemia. The LDL was of higher density and was triglyceride-rich and cholesterol ester-poor. HDL separated to three populations. HDL2 was prominent (in spite of low plasma HDL cholesterol). HDL3 was of normal density and an intermediate HDL population, not found in normal subjects, was identified and designated HDL2-3. All three HDL populations were enriched with triglycerides and poor in cholesterol ester content. The modified LDL and HDL particles may then be possibly cleared rapidly from the plasma by activated monocytes and macrophages.

We have recently described a new two-phase liquid culture that supports the development of human erythroid progenitors (Fibach et al., Blood 73:100, 1989). The procedure separates the erythroid burst-forming units (BFUe) from the erythroid colony-forming units (CFUe) stage and enables quantitation of the proliferation and differentiation of BFUe into CFUe. In the present study we have utilized this system to study erythroid progenitors in polycythemia vera (PV). The abnormality of the erythroid series in PV has been shown to be associated with an increased responsiveness of the progenitors to the hormone erythropoietin (Epo). A basic question in this clonal stem cell disorder is at what developmental stage this abnormality of the PV clone is phenotypically expressed. We have studied this question by comparing the development of Epo-dependent and Epo-independent CFUe from peripheral blood BFUe of the PV patient during the BFUe to CFUe transition in the liquid culture. The results indicated that both types of CFUe are generated and that in all cases tested the ratio of Epo-independent progenitors at both the BFUe and CFUe stage was similar indicating no preferential development of Epo-independent CFUe. These results suggest that the abnormality of the PV erythroid progenitors is expressed only at the CFUe level. Moreover, since the liquid culture did not contain Epo, the results also support the conclusion that BFUe do not require Epo for proliferation or differentiation into CFUe.

Sickle cell anemia, the most prevalent type of hemoglobinopathy, appears almost exclusively in backs. Since the first report of sickle cell anemia in a Caucasian by Cooley and Lee in 1929 [1], additional cases have been reported, mainly from Mediterranean countries, the Persian Gulf, and India [2]. Several cases were also found among Arabs in Israel with a relatively benign clinical course [3, 4]. The present report describes a unique case of sickle cell anemia associated with alpha-thalassemia in a non-black young adult. The diagnosis was made at the age of 23 years. Severe priapism was almost the first manifestation of the disease and subsided following transfusion of five units of packed red blood cells.

The human promyelocytic cell lines HL-60 can be induced to undergo differentiation to either granulocyte- or macrophage-like cells. We followed the changes in the synthesis and content of ferritin in this and other cell lines during differentiation. Ferritin content of HL-60 cells ranged from 11 to 81 fg/cell, depending on the clone tested. Following exposure to dimethylsulfoxide (DMSO) or retinoic acid (RA) an increase in ferritin and a decrease in total protein synthesis was observed, resulting in increased ferritin content, reaching a peak after 2 days. This increase occurred prior to the appearance of the typical morphological and functional characteristics of mature granulocytes. A correlation was found between concentrations of DMSO effective in inducing differentiation and the increase in ferritin content. Other inducers of granulocyte differentiation had a similar effect, while 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of macrophage differentiation, had not. Another human cell line (U-937), which was induced into monocyte-like cells by RA, showed a twofold increase in ferritin content following differentiation. Addition of iron to the culture medium increased ferritin content of both differentiating and non-differentiating cells, but the former responded to lower concentrations of iron. The increase in ferritin during differentiation, however, was not related to an accelerated iron uptake. The present results suggest that changes in the intracellular ferritin of the developing myeloid cells may play a regulating role in the process of maturation of these cells.

In order to clarify the indications for splenectomy in patients with homozygous beta thalassaemia we studied, retrospectively, the basal pre-transfusion haemoglobin levels and blood transfusion requirements before and after splenectomy, in a series of patients with this disorder. Thirty-six patients, of whom 20 underwent splenectomy, were included in this study. Three groups of patients with homozygous beta thalassaemia were identified on the basis of clinical and laboratory findings. Two of the three groups consisted of 24 patients with beta thalassaemia major, subdivided retrospectively according to their response to splenectomy. The third group consisted of six patients with beta thalassaemia intermedia who had a comparably mild clinical course. In the first group of 16 patients with beta thalassemia major, monthly transfusions were required from the age of 6 months to 24 months and splenectomy resulted in temporary improvement for a period of 1-2 years. Thereafter, the transfusion requirements returned to pre-operative levels with no sustained improvements in base-line pretransfusion haemoglobin levels. Despite the fact that haematological improvement was temporary, it is felt that splenectomy is indicated in this group since the development and the general well-being of the children was significantly improved. In the second group of eight patients with beta thalassaemia major, transfusion requirements were relatively infrequent before 1-2 years, but after several years the transfusion requirement increased simultaneously with the development of 'hypersplenism'.

Physiological or experimental decrease in sialic acid (SA) content on the red blood cell (RBC) membrane is believed to play an important role in the recognition of these cells by macrophages. Since there is a 20-30% decrease in the SA content on the membrane of thalassaemic RBC, the interaction between macrophages and these RBC was studied in vitro. Using mouse peritoneal macrophages, it was found that these macrophages 'recognize' and phagocytize thalassaemic RBC while RBC from normal donors are hardly phagocytized. The average level of phagocytosis of thalassaemic RBC from splenectomized patients was found to be 22-fold higher than that of RBC from normal donors. The phagocytized cells consisted of both mature and nucleated RBC. Mouse peritoneal macrophages seem to be a useful in vitro system for the study of the accelerated sequestration and shortened life span of thalassaemic RBC.

The ability of circulating progenitor cells from patients with polycythaemia vera (PV) and myelofibrosis with myeloid metaplasia (MMM) to develop erythroid colonies was studied in cultures with and without erythropoietin. In all normal controls, patients with secondary polycythaemia and MMM, erythroid colonies developed only after the addition of erythropoietin. Only in patients with PV, both in the active and spent phases of the disease, erythroid colonies developed in the absence of erythropoietin. The results indicate the perpetuation of erythropoietin-dependent, as well as erythropoietin-independent progenitors in both phases of this disease. Although spent PV often clinically resembles MMM, there is a basic difference in the behaviour of the circulating erythroid progenitors in these diseases which may serve as a useful tool in discriminating MMM from spent PV, when there is no history of active PV.

A highly sensitive antiglobulin test based on rosette formation due to the interaction between IgG bearing red blood cells (RBC) and Fc receptors on K562 cells, was used to study the immunoglobulin molecules present on human senescent RBC. Normal human RBC were separated into young and senescent subpopulations on the basis of age-dependent differences in density by centrifugation on a discontinuous density Percoll gradient, and by flotation on phthalate ester mixtures. The senescent but not the young RBC were found to bear membrane bound IgG. Most of the bound IgG molecules could be specifically eluted by galactose in its alpha-anomeric form. Antigalactosyl (anti-Gal) IgG antibodies with similar reactivity were found to be present in high titres in every one of the 400 normal human sera tested. The natural anti-Gal antibodies isolated from normal sera by affinity chromatography could bind to IgG depleted senescent RBC but not to young RBC. Erythrophagocytosis experiments indicated that the anti-Gal bound to the senescent RBC induced their destruction by macrophages. It is suggested that the natural anti-Gal antibodies interact with cryptic alpha-galactosyl residues which are exposed in the course of the RBC senescence and mediate the removal of these RBC from circulation by cells of the reticuloendothelial system.

Iron binding in the sera of 35 patients with beta thalassaemia major and intermedia was studied. In patients receiving regular blood transfusions since infancy transferrin was completely saturated and about 2.7--7.1 mumol/l of the serum iron could be removed by dialysis or ultrafiltration in the presence of a chelating agent or by filtration on DEAE-Sephadex-catecholdisulphonic acid columns. In contrast, less than 1.0 mumol/l of transferrin bound iron was removed when subjected to the same procedures. The non-specific iron of thalassaemic sera could no longer be demonstrated after incubation with normal serum. These findings indicate that non-specific iron is a chelatable with normal serum. These findings indicate that non-specific iron is a chelatable compound which is readily available for transferrin binding. In view of the known toxicity of unbound iron, its identification in thalassaemic sera might be of relevance to the pathogenesis of tissue damage and the protective effect of iron chelating therapy in this disease.

Thoracic masses resulting from extramedullary hematopoiesis developed in two sisters of Moroccan origin with congenital dyserythropoietic anemia type II (HEMPAS). In one patient, the diagnosis of extramedullary hematopoiesis was confirmed histologically. The appearance of extramedullary foci of hematopoiesis mimicking mediastinal tumors has not been previously described in HEMPAS. These masses result from persistent erythropoietic stimulation associated with chronic hemolytic anemia. In both patients, detection of the asymptomatic masses was preceded by normalization of hemoglobin levels. Thus unexpected correction of a chronic refractory anemia associated with the appearance of mediastinal masses might be the heralding manifestation of an effective extramedullary hemopoiesis.

Disseminated intravascular coagulation (DIC) is a frequent occurrence in acute promyelocytic leukemia (APL), especially after onset of chemotherapy. We have used a human promyelocytic leukemic established cell line (HL-60) and various other human leukemic cells to investigate the effect of cytotoxic drugs on generation of procoagulant activity (PCA). The results indicate that, unlike normal human peripheral blood monocytes and certain other cell types where PCA induction requires active mRNA and protein synthesis, in HL-60 cells, compounds such as actinomycin D, puromycin, and cytosine arabinoside and a variety of other cytotoxic agents, induced generation of a potent PCA. Although different in its mechanism of induction, this HL-60 cell PCA was similar, and may be identical, to mononuclear cell tissue factor. The PCA induction was rapid and preceded the lytic effect of the drugs. It was first detected on the outer cell surface but, following prolonged exposure to the drugs, upon lysis of the cells, it was also found in the extracellular medium. This in vitro effect mimics the development of DIC in patients with APL. The system may, therefore, serve as a model for the study of the cellular and molecular events associated with PCA generation by malignant promyelocytes and DIC occurrence in patients with APL and other malignancies.

We report the presence of sickle cell trait in several members of a white Jewish family. The trait was discovered when the propositus developed massive splenic infarction at high altitude. No erythrocyte markers characteristic of African ancestry were detected in any of the family members. This is the first bona fide documentation of sickle trait among white Jews.