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Admission of patients with hematological malignancies to intensive care unit (ICU) raises recurrent ethical issues for both hematological and intensivist teams. The decision of transfer to ICU has major consequences for end of life care for patients and their relatives. It also impacts organizational human and economic aspects for the ICU and global health policy. In light of the recent advances in hematology and critical care medicine, a wide multidisciplinary debate has been conducted resulting in guidelines approved by consensus by both disciplines. The main aspects developed were (i) clarification of the clinical situations that could lead to a transfer to ICU taking into account the severity criteria of both hematological malignancy and clinical distress, (ii) understanding the process of decision-making in a context of regular interdisciplinary concertation involving the patient and his relatives, (iii) organization of a collegial concertation at the time of the initial decision of transfer to ICU and throughout and beyond the stay in ICU. The aim of this work is to propose suggestions to strengthen the collaboration between the different teams involved, to facilitate the daily decision-making process, and to allow improvement of clinical practice.

From the beginning of the century, autoimmune haemolytic anaemia (AIHA) was the first model of an auto-antibody mediated disease. Despite the variety of the clinical features, the diagnosis of AIHA provides few difficulties since the introduction of the popular Coombs' test. The clinical presentation of AIHA depends on the subclass type and on the thermal range activity of the causative auto-antibody, so that two main pictures occur usually: warm auto-antibody and cold auto-antibody types, the latter being less frequent than the former. In more than half the cases, AIHA is associated with another disease that must be considered more as the background of immune dysregulation than the cause of the disease. Systemic disorders, chronic lymphoid malignancy, primitive or acquired immunodeficiencies are the most common disorders associated with AIHA. Acute infections or drugs may give rise to acute transient AIHA. The clinical aspects and the links between AIHA and associated diseases are emphasised. No decisive advancement in therapy has arisen over the last decades. Some patients are still resistant to all therapeutic manoeuvres and may die. Much labour is to be done in order to discover more rational methods of therapy to restore a state of normal tolerance towards erythrocyte auto-antigens. Suppressing the production of pathogenic auto-antibodies by immunomodulation may be the first step of this task.

Intensive chemotherapy produces a lower complete remission (CR) rate in the myelodysplastic syndromes (MDS) than in de novo acute myeloid leukaemia (AML), possibly due in part to a higher incidence of P glycoprotein (PGP) expression in MDS blast cells. We designed a randomized trial of intensive chemotherapy with or without quinine, an agent capable of reverting the multidrug resistance (mdr) phenotype, in patients aged < or = 65 years with high-risk MDS. Patients were randomized to receive mitoxantrone 12 mg/m2/d days 2-5 + AraC 1 g/m2/12 h days 1-5, with (Q+) or without (Q-) quinine (30 mg/kg/d). 131 patients were included. PGP expression analysis was successful in 91 patients. In the 42 PGP-positive cases, 13/25 (52%) patients in the Q+ group achieved CR, compared to 3/17 (18%) patients in the Q- group (P = 0.02) and median Kaplan-Meier survival was 13 months in the Q+ group, and 8 months in the Q- group (P = 0.01). No life-threatening toxicity was observed with quinine. In conclusion, the results of this randomized study show that quinine increases the CR rate and survival in PGP-positive MDS cases treated with intensive chemotherapy.

Although the FAB classification of the myelodysplastic syndromes (MDS) allows to classify most patients, a few clinical patterns do not fit the FAB categories, including borderline forms with manifestations usually seen in other diseases and forms with atypical or unusual clinical or laboratory features that do not immediately suggest a MDS. Borderline forms are characterized by the presence of any of the following: high or very high platelet counts, myelofibrosis, bone marrow hypoplasia, eosinophilia, or systemic diseases such as relapsing polychondritis. Unusual or atypical forms include manifestations such as hemolysis, high reticulocyte counts, erythroblastopenia, or an abnormality of a single cell line such as isolated thrombocytopenia, isolated neutropenia, or isolated macrocytosis. The definitive diagnosis of these forms of MDS can require a number of investigations such as cytogenetic studies, bone marrow biopsy, and/or radionuclide evaluation, and may not be possible until the patient has been followed for some time.

We designed a scoring system to rank acute care hospital projects and allocate resources between them. The evaluation tool assessed projects on an ordinal scale; the criteria scored were medical interest, feasibility, interest for teaching and research, and compatibility with the hospital's strategy. Clinical and technical projects were ranked separately. In 1994, 25 new projects, representing a total cost of $1.4 million, were reviewed by two independent reviewers. The scores ranged from 30 to 18 over 36. Projects presented by clinical departments scored higher than projects presented by medicotechnical departments.

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired disorder of haematopoietic stem cells. Although knowledge about the pathophysiology of the disease is increasing, no multivariate analysis of factors influencing survival has been undertaken, mainly because the disease is rare. We undertook such an investigation., Methods: Data were collected on 220 patients with PNH diagnosed over a 46-year period (1950-1995) from participating French centres. Diagnosis of the disease required, at least, an unequivocally positive Ham's test., Findings: The Kaplan-Meier survival estimate was 65% (SE 4) at 10 years and 48% (6) at 15 years after diagnosis. 8-year cumulative incidence rates of the main complications (pancytopenia, thrombosis, and myelodysplastic syndrome) were 15% (3), 28% (4), and 5% (2), respectively. Demographic data, presenting features, initial treatment, complications, and causes of death were similar to those previously reported. In multivariate analysis, seven factors were significantly associated with survival in patients with PNH. Poor survival was associated with the occurrence of thrombosis as a complication (relative risk 10.2 [95% CI 6-17], p < 0.0001), evolution to pancytopenia (5.5 [2.8-11], p < 0.0001), myelodysplastic syndrome or acute leukaemia (19.1 [7.3-50], p < 0.001), age over 55 years at diagnosis (4 [2.4-6.9], p < 0.0001), need for additional treatment (2.1 [1.3-3.6], p < 0.003), and thrombocytopenia at diagnosis (2.2 [1.3-3.8, p < 0.003). Better survival was shown for patients in whom aplastic anaemia antedated PNH (0.32 [0.14-0.72], p < 0.02). Factors associated in multivariate analysis with a high risk of thrombosis during the disease course were thrombosis at diagnosis (5.1 [2.5-10.6], p = 0.0002), age over 54 years (2.6 [1.5-4.6, p = 0.0014), and infection at diagnosis (2.6 [1.3-5.2], p = 0.0099). The risk factors for progression to pancytopenia were absence at diagnosis of anaemia (4.03 [1.3-12.2], p = 0.03) and neutropenia (2.45 [1.1-5.7], p = 0.03). The risk factors for development of myelodysplastic syndrome or acute leukaemia were abdominal pain crisis at presentation (10.5 [2.5-44.0], p = 0.004) and year of diagnosis after 1983 (8.45 [1.8-40.7], p = 0.004)., Interpretation: This large number of cases permitted a detailed analysis of prognostic factors for the first time, in this rare disease. Estimates of PNH prognostic factors may serve as baseline data in the assessment of current and future treatments for this disease.

Magnetic resonance (MR) imaging is a method of choice for assessing vascular patency and parenchymal iron overload. During the course of paroxysmal nocturnal hemoglobinuria (PNH), it is clinically relevant to differentiate abdominal vein thrombosis from hemolytic attacks. Furthermore, the study of the parenchymal MR signal intensity adds informations about the iron storage in kidneys, liver, and spleen. Twelve PNH patients had 14 MR examinations of the abdomen with spin-echo T1- and T2-weighted images and flow-sensitive gradient echo images. Vessels patency and parenchymal signal abnormalities--either focal or diffuse--were assessed. MR imaging showed acute complications including hepatic vein obstruction in five patients, portal vein thrombosis in two patients, splenic infarct in one patient. In one patient treated with androgens, hepatocellular adenomas were shown. Parenchymal iron overload was present in the renal cortex of eleven patients with previous hemolytic attacks. On the first MR study of the remaining patient with an acute abdominal pain showing PNH, no iron overload was present in the renal cortex. Follow-up MR imaging showed the onset of renal cortex iron overload related to multiple hemolytic attacks. Despite the fact that all our patients were transfused, normal signal intensity of both liver and spleen was observed in three of them. MR imaging is particularly helpful for the diagnosis of abdominal complications of PNH.

A knowledge of the bacterial ecology of a haematology unit should help in the management of the febrile patient with or without neutropenia. We studied the prevalence and the susceptibility profiles of bacteria isolated during a six-year period among patients hospitalized in a 44-bed haematology unit. Antibiotic use over this period was also studied. The most prevalent bacteria were coagulase-negative staphylococci (CNS) (35.1%), Escherichia coli (11.4%), Staphylococcus aureus (9.9%), Enterococcus spp. (8.2%), and Pseudomonas aeruginosa (7.5%). The susceptibility of CNS to oxacillin decreased from 67-44% over six years, while that of enterobacteriaceae to amoxycillin and piperacillin was reduced by about 50%. P. aeruginosa susceptibility to ceftazidime remained remarkably stable at around 90%, despite extensive empirical use. Imipenem and ciprofloxacin were used restrictively and ceftazidime-resistant P. aeruginosa remained susceptible to these two agents in most cases. Our antibiotic policy was found to be compatible with the frequency of the bacterial strains isolated in our department and with their susceptibility profiles.

Managing new innovations in medicine is a particularly timely subject. There is an abundant history concerning over expectations resulting from the development of new treatments or diagnostic procedures, some shown to be less effective than promised, others even found to be dangerous. A new aspect to the question is the importance of economic pressures which require rational investment decisions when diffusing innovating technologies. In 1991, the Commission for the evaluation and diffusion of innovating technologies (CEDIT) at the University Hospitals of Paris (Assistance Publique-Hôpitaux de Paris) developed a programme aimed at better managing the distribution and use of polyvalent intravenous immunoglobulins (IgIV), a new promising therapeutic tool with both a high cost and a certain number of risks. The programme was designed to assist prescribers in elaborating better therapeutic strategies and to help hospital managers rationalize expenditures for IgIV. The results of this experience are presented here together with certain conclusions concerning the way management decisions can be applied to the diffusion of an innovation in health care.

We report the set-up of a denaturant gradient gel electrophoresis (DGGE) assay to screen for mutations in the whole coding sequence of the p53 gene. These DGGE experimental conditions were applied to the analysis of the p53 gene in acute leukemias. Forty adults with acute myelogenous leukemia (AML) and 21 with acute lymphoid leukemia (ALL) were investigated. Eleven of the AML patients were investigated at the time of the initial diagnosis and at relapse. In contrast with most reports based on amplified fragments analyzed by single-strand conformation electrophoresis and focusing on exons 5 to 8, we analyzed the whole coding sequence of the gene. Two of the 40 AML patients displayed a point mutation in exon 7; it was either an A to G substitution that converted Tyr-234 to Cys, or a G to A change that converted Arg-248 to Gln. The screening procedure led to the discovery of several intronic and exonic polymorphisms. These results confirm the low incidence of p53 mutations in acute leukemias and suggest a limited role of the p53 protein in leukemogenesis. The computerized modeling and electrophoresis parameters presented here provide a powerful tool for the exhaustive characterization of p53 mutants in all kinds of malignancies.

Anaemia is frequently encountered in daily practice. With full knowledge of its pathophysiology a rational classification is possible allowing a suitable approach for diagnostic investigations. In a first stage, the data provided by blood counts, erythrocyte constants and reticulocyte counts guide the diagnostic rationale. In cases with microcytic and hypochromic anaemia, measurement of ferritin level separates iron deficiency anaemia from the so-called "inflammatory" anaemias. A high number of reticulocytes points to haemolytic anaemia. Among the many causes of haemolysis, one must first look for autoimmune haemolysis. Elsewhere, the clinical context and morphological red cell abnormalities point to a hereditary disease affecting the erythrocyte membrane, enzymes or globin content. Although rare, microangiopathic anemia with schizocytosis and paroxysmal nocturnal haemoglobinuria must not be misdiagnosed. Bone marrow examination remains the clue in non-regenerative normochromic, normo- or macrocytic anaemias. In difficult cases, other investigations, such as cytogenetics, isotopic examination and progenitor culture, may help in characterizing the anaemia. Serum erythroproietin essays and plasma transferrin receptor counts will soon figure among the methods used to explore anaemias.

Ten adult patients with warm antibody haemolytic anaemia at initial presentation and seven other patients with either refractory AIHA (two patients) or who relapsed after an initial response to prednisone (five patients) were treated with both Danazol and prednisone. 80% of the first group, but also 60% of the second group displayed long-lasting responses (mean follow up 21 months). Minimal side-effects occurred. Finally, addition of Danazol at presentation in warm AIHA may decrease the duration of prednisone therapy and markedly reduce the necessity of second-line splenectomy which is usually required in many patients.

The usefulness of consensus conferences has been questioned. We have assessed the impact of a 1989 consensus conference about the treatment of hypovolaemia on prescribing practice at our hospital. Data available from the blood bank enabled us to follow albumin use and costs by department between 1987 and 1991. Medical journals, direct mail, and meetings were used to disseminate the recommendations of the consensus conference, which were that the only indications for albumin are massive haemorrhage, plasmapheresis, massive hypoalbuminaemia, and volume expansion in pregnancy; for hypovolaemia caused by septic shock or vasoplegia, fluid gelatins and crystalloids should be used. In the year after the conference, and in subsequent years, albumin use and costs were 40% lower than before the conference, even though total medical expenditure continued to rise and numbers of patients admitted did not change. We believe that consensus conferences can be a useful means of improving medical practice. The features that ensured success in this programme were the small number of prescribers, the homogeneous setting, and the frequent restatement of the recommendations.

CD8+/Leu-7+ T cells which circulate in increased proportions in the blood of long-term surviving BMT patients are for the most part high-density resting lymphocytes lacking IL2R-alpha (p55) expression. We show that they can be induced by IL2 to manifest cytolytic function after 24-48 hr stimulation by using rather high concentrations of IL2 (at least 50 U/ml). This function was much more readily induced in high-density CD8+/Leu-7+ T cells than in high-density CD8+/Leu-7+ T cells and occurred in the presence of minimal cell proliferation. Other cytokines involved in primary CTL differentiation (IFN-gamma, IL4 and IL6) were without effect suggesting that CD8+/Leu-7+ T cells are, in the BMT model, in vivo preactivated CTL ready to differentiate into cytolytic effectors under the sole IL2 stimulus. TU27 Mab directed at IL2R-beta (p75) subunit almost completely prevented IL2-induced cytolytic function of CD8+ T cells while 33B3.1 Mab directed at IL2R-alpha (p55) subunit was ineffective, suggesting that the signal for this function has its origin in IL2R-beta chains constitutively expressed by these cells.

We analyzed the functional status of the small CD8+/Leu-7+ T-lymphocytes that circulate in increased proportions in the blood of many allogeneic bone marrow transplant (BMT) patients. Purified CD8+/Leu-7+ T cells were tested for their effect on T-cell proliferative responses. In contrast to CD8+/Leu-7-T-lymphocytes, such cells behaved as suppressor cells for lectin-induced mitogenic responses of the donor's peripheral blood lymphocytes. However, they did not interfere with the in vitro responsiveness to specific stimuli such as protein purified derivative (PPD) or alloantigens. We demonstrate that CD8+/Leu-7+ T cells are resting pre-cytotoxic T-lymphocytes (CTL) that can be induced by mitogenic lectins to express their cytolytic program in a non-specific, non-major histocompatibility complex-restricted manner against phytohemagglutinin-treated lymphoblasts or K562 target cells. The lectin-triggered cytotoxicity was achieved within a few days, together with limited cell division. Our results suggest that circulating CD8+/Leu-7+ T cells from BMT recipients are in vivo primed CTL awaiting cellular activation.

The polymerase chain reaction (PCR) allows the detection of minimal amounts of nucleic sequences and has been successfully used to test for the chronic myeloid leukemia-specific bcr/abl transcripts. We studied blood samples from 17 patients who had undergone allogeneic bone marrow transplantation for CML, using a modified polymerase chain reaction-based assay for the detection of leukemic mRNA. This nested PCR technique was found to be highly sensitive, detecting the chimeric bcr/abl transcript in 16 of 17 patients including several long-term survivors. Cytogenetic techniques failed to detect Ph mitoses. The clinical significance of the persisting bcr/abl transcript for long periods following BMT is poorly understood and remains to be elucidated by further studies.

An 18 year old boy presented with microcytic hypochromic anemia. Erythrocytic abnormalities and family studies suggested congenital sideroblastic anemia (CSA), but atypical features included absence of clinical iron overload, scanty iron deposits in mitochondria of late erythroblasts and reticulocytes, and a high platelet count. An unusual adhesion between bone marrow macrophages and reticulocytes was observed by electron microscopy. Haematological response was seen following pyridoxine administration, thus fending support to the diagnosis of CSA.

Blood CFU-GM and BFU-E, grown from 17 patients who had undergone allogeneic bone marrow transplantation (BMT), were studied by the plasma-clot technique before day 45, and at a time when their blood count was approximately normal. The number of colonies varied from one patient to another, but was always lower than in normal subjects. Removal of cells forming rosettes with sheep erythrocytes (E+C) increased colony growth in four out of eight cases, whereas removal of adherent cells (AC) had the same effect in five out of six cases. Addition of E+C or AC after their initial removal restored the inhibition of colony growth. This suppression was noted at a 1:1 to 8:1 cellular ratio and ranged from 25% to 75%. The phenotype of the suppressive cells was further characterized by complement-mediated lysis with monoclonal antibodies (MoAbs) and fluorescent labeling. Two types of cells associated with inhibition of colony growth were identified: the first were E+C positive, characterized by the T3, HNK1, DR, and T8 determinants; the second were identified by the MO2 MoAb, indicating their monocytic origin, together with their properties of adherence. Similar suppressor cells of CFU-GM were found in the marrow of two other allogeneic BMT patients. A direct suppressive effect of the two types of cells was demonstrated in one experiment when MO2+ and/or HNK1+ cells collected by cell sorting were added back to cultures depleted in MO2+ and HNK1+ cells by complement-mediated lysis and were both found to decrease colony growth. Purified HNK1+ cells led to moderate inhibition of colony growth, which was not enhanced by increasing their concentration. This suppressive effect of hematopoiesis could be the consequence of an allogeneic reaction, since no inhibition was affected by T cells or monocytes in seven autologous BMTs and one syngeneic BMT.

In a series of 40 patients with Budd-Chiari syndrome, 5 (12%) were found to be afflicted with paroxysmal nocturnal hemoglobinuria. The clinico-pathological features in these 5 patients and in 26 well-documented previously reported cases could be ascribed to three groups of increasing severity: thrombosis limited to small-sized hepatic veins with no or transient ascites, partial thrombosis of large-sized hepatic veins with chronic ascites, and complete thrombosis of large-sized hepatic veins with a life-threatening course. These three groups did not differ with regard to sex, age, and duration and characteristics of paroxysmal nocturnal hemoglobinuria. In view of the relationship between prognosis and the extent of hepatic vein obstruction, we suggest that early therapy directed toward limiting the extension of thrombosis, or toward dissolving formed thrombi, should improve the prognosis of this severe complication of paroxysmal nocturnal hemoglobinuria.

Among nine cases of early erythroblastic leukemia previously diagnosed using a panel of antibodies, two patients have erythroid blasts expressing glycophorin A, seven patients have blasts with a more immature phenotype. These immature blasts were labeled by the FA6-152 monoclonal antibody when studied with the immunogold technique. The blasts exhibited large nucleoli, and their cytoplasm contained numerous ribosomes and large mitochondria. In the Golgi apparatus several granules resembled the theta granules as previously described and contained ferritin molecules in the absence of rhopheocytosis. A large proportion of these blasts exhibited a platelet peroxidase (PPO)-like activity. As the blasts from the two other patients with a more mature phenotype and glycophorin A reactivity lacked this PPO, this enzyme seems to be restricted to the more immature cells. Since in these leukemic samples immature erythroid blasts were admixed to promegakaryoblasts, immunogold labeling was also performed with antiplatelet antibodies. This latter population which was labeled with C17, a monoclonal antibody to platelet glycoprotein IIIa, showed strong PPO activity but lacked theta granules and ferritin. In the normal bone marrow enriched by panning for CFU-E (8%) and depleted in progenitors of other lineages, blast cells showing characteristics similar to leukemic erythroid blasts were seen. They exhibited theta granules and ferritin and a proportion of them also had a PPO-like activity. Thus, a PPO reaction is not restricted to the platelet-megakaryocyte line. In conclusion, a PPO-like activity and ferritin molecules were present in immature leukemic erythroid blasts. Similar cells could be identified from normal bone marrow.

Thrombocytopenic thrombotic purpura is a rare condition of controverted pathophysiology. The authors have observed and treated 10 cases between 1974 and 1979. The clinical features were uniform: haemolytic anaemia with schistocytosis (10/10), thrombopenia (10/10), fever (9/10), varying neurological disorders (9/10) which required assisted ventilation in 7 cases. Renal insufficiency was found in 7 patients, but associated with renal lesions only one and never interfered with the course of the disease. All patients received corticosteroids in high dosage and anti-platelet aggregation drugs. That treatment alone cured 3 patients. In addition, 5 patients underwent plasmapheresis, which seemed to be very effective in 2 cases. Five patients died, 3 of whom despite corticosteroids, anti-aggregants and plasmapheresis. Thus, none of these treatments seems to be effective in all cases, and prospective multicentre therapeutic trials are probably required.

4 months after a bone marrow transplantation performed for acute lymphocytic leukaemia, a 28-year-old man had encephalitis. A brain CT scan revealed bilateral and symmetrical temporal hypodense areas. Serological studies revealed a recent CMV conversion in serum and a higher conversion in cerebral spinal fluid. All other viral antibodies remained at low levels, especially for herpes simplex virus. Because of a high incidence of CMV infection after bone marrow transplantation, the responsibility of CMV as the cause of the encephalitis is discussed.

We reviewed 213 consecutive adult pancytopenic patients to determine the frequency of underlying pathology, to analyze our diagnostic procedure, and to determine the value of peripheral blood data for diagnosis. Pancytopenia was defined as the association of hemoglobin level below 12 g/dl in males and 11.5 g/dl in females, leukopenia below 4 x 10(9)/L, and thrombocytopenia below 150 x 10(9)/L. The bone marrow aspirates were normo- or hypercellular in 140 cases (66%). Bone marrow biopsies, performed in 93 cases, documented the presence of myelofibrosis in 67 cases. Aplastic anemia was diagnosed in 10% of the cases. Malignant myeloid disorders (acute myeloid leukemias, myelodysplastic syndromes, acute myeloid disorders with myelofibrosis) represented 42% of the cases and various malignant lymphoid disorders 18%. Vitamin deficiencies accounted for 7.5% and nonhematological pathology 10% of the cases. The bone marrow aspirate was sufficient for the diagnosis in 55% of the cases, and the trephine biopsy was necessary in 30%. In the remaining cases, other complementary tests were necessary to achieve final diagnosis. A discriminant analysis, focused on the hemogram data, showed that parameters obtained by analysis of blood smears were helpful for the diagnosis, especially the presence or absence of blast cells and/or of abnormal lymphoid cells.

A new case of cold agglutinin disease in a patient who had a long lasting Raynaud's phenomenon without hemolysis and a persistent HB antigen cirrhosis, is reported. The cold agglutinin is a monoclonal IgA kappa antibody which reacts at 4degreesC to a titer of 256. As the three other cases described in the literature, it demonstrates Pr1 specificity. The eluate from human cells reacts with rat and dog cells whose receptor is destroyed by both papain and neuraminidase, thus eliciting the characteristic Pr1d specificity.

The growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum-free medium, the cells require insulin, transferrin, and albumin. Two highly water-soluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen-stimulated lymphocytes were grown in serum-free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free-iron salts are taken up by the cells by a mechanism that is completely independent from transferrin-receptors.

Peripheral blood lymphocytes (PBL) and epidermal cells (EC) of 44 patients to be grafted with bone marrow from an HLA-identical sibling have been used as stimulator cells in primary cultures with effector PBL of one or several potential donors. Proliferative responses against PBL did not differ from those obtained with effector cells cultured in medium alone, whereas EC induced clearly positive proliferation in 21/53 (40%) of the pairs tested. Evaluation of 30 patients followed for more than 3 months after the graft shows that a high level of response in the mixed epidermal cell lymphocyte reaction is directly correlated with the incidence of acute graft-versus-host disease.

The expression of i-antigen during the in vivo aging of erythrocytes from normal subjects and beta-thalassemic patients was investigated by immunofluorescence labeling and by immunoagglutination. The results showed that the expression of i-antigenic determinants inversely related to the aging of RBCs. This phenomenon could be either the result of a progressive loss of i-antigenic structures or the consequence of i to I transformation occurring during RBCs aging in vivo.

An authentic case of Waldenstrom's macroglobulinaemia without a tumoural syndrome is reported, with initial signs of peripheral pancytopenia. Complete and persistent remission appears to have followed multiple sequential chemotherapy. This very satisfactory and, according to data in the published literature, very rare result suggests that this type of treatment should be prescribed for all patients with macroglobulinaemia with poor prognostic clinical and histological characteristics.

63 patients with Hodgkin disease of limited extent (I, II, III) are treated with two protocols: extended field irradiation versus chemotherapy (M.O.P.P.) + extended field irradiation. Three points are suggested by analysis of the results: 1) Advantage from the combination of chemotherapy-radiotherapy. 2) Resistance to chemotherapy frequent innodular sclerosis. 3) In patients treated with chemotherapy the reaction has a prognostic value: failure of treatment being seen only in patients who did not obtain a complete remission.

BFU-E from the blood of 14 normal adults have been grown by the plasma clot technique. The hemoglobins synthesized in burst colonies were purified from other proteins by affinity chromatography on Sepharose-haptoglobin. The radioactivity incorporated in the globin chains was estimated by CM-cellulose chromatography in urea. The number of bursts scored at the 14th day of culture fluctuated between 50-130 (average 86, s: 29) for 10(6) mononuclear plated cells. A constant reactivation of fetal hemoglobin was found (from 1.4% to 11%, mean value 5.8%, s:3.07), but was lower than previously described, mainly because of the highly selective purification of Hb. This reactivation of fetal hemoglobin was not dependent upon the concentration of erythropoietin (from 1 U/ml to 6 U/ml) nor on the purity of the erythropoietin preparations (from 6 U/mg of protein to 70 000U/mg of protein). In addition, the same subject exhibited a constant proportion of Hb F synthesized in culture over a period of time up to 6 months. A positive correlation exists between the proportion of Hb F in culture and that of F cells present in the blood, with the exception of two subjects. Such findings suggest that Hb F in culture is a characteristic of each individual and that this reactivation often represents an amplification of the Hb F synthesis in vivo.

Congenital dyserythropoietic anaemia type II (HEMPAS) is a hereditary disease believed to be caused by a membrane abnormality of erythroid cells. Since the molecular basis of this membrane abnormality has not yet been defined, membrane glycoproteins of HEMPAS erythrocytes were analysed by cell surface labelling and endo-beta-galactosidase digestion in this study. HEMPAS erythrocytes showed an abnormal glycoprotein profile when cells were labelled by the galactose oxidase/NaB[3H]4 method; Band 3 and Band 4.5 glycoproteins in HEMPAS are labelled but with less intensity although normally these proteins are the major components revealed by the same method. Instead, in HEMPAS, labelled lactosaminoglycans were found as a lower molecular weight glycoconjugate (HEMPAS glycan). HEMPAS glycan was characterized by micelle formation, a monomer molecular weight of 4000, susceptibility to endo-beta-galactosidase and resistance to protease. These characteristics suggest that HEMPAS glycan has the nature of macroglycolipid. Proteins of Band 3 and the glucose transport protein (a component of Band 4.5), which were detected by antibodies showed a slightly decreased molecular weight in HEMPAS erythrocytes compared to those from normal erythrocytes, which was consistent with the decreased glycosylation of these proteins. The results indicate that anomalies in glycosylation occurred specifically in lactosaminoglycan glycoproteins of HEMPAS erythrocytes.

A successfull bone marrow transplant was achieved in a case of post hepatitic aplastic anemia after cyclophosphamide immunosuppression. Caryotype analysis, erythrocytic phenotype and IgG Gm allotype demonstrated evidence of complete chimerism. Anti-thymocyte serum undoubtly was able to suppress a life threatening episode of graft versus host reaction. Severe long lasting skin lesions are now persisting 2 years after the graft.

The expression of myeloid and megakaryocytic markers of differentiation has been studied in one K-562 cell subline, in its clones, and in the original cell line. Cytotoxicity, electron microscopy, immunofluorescence studies with a panel of polyclonal and monoclonal antibodies, and radioimmunoassays were performed on K-562 cells before and after induction with hemin, sodium butyrate, and 12-O-tetradecanoylphorbol-13-acetate. Myeloid membrane markers were present in all K-562 cell lines. Only the early granulopoietic cell surface markers were expressed in 75 to 95% of the cells, while none of the late membrane markers was detected. In contrast, neither the early (myeloperoxidase) nor late (lactoferrin) cytoplasmic markers were present. Thus, K-562 cells showed a membrane phenotype similar to that of a normal or leukemic promyelocyte but lacking myeloperoxidase. Membrane megakaryocytic markers, such as platelet glycoprotein IIIa and platelet peroxidase, were also detected in K-562 cells. However, some other early megakaryocytic markers, such as platelet glycoprotein lb, Factor VIII-R-Ag, and platelet Factor 4, could not be detected by fluorescent labeling. Cloning of the cell line did not result in the selection of a unipotential cell line. These results could be explained by the expression of multilineage markers in a single cell. In all of the cell lines and clones, hemin slightly increased the expression of the myeloid membrane markers without any modification of the megakaryocytic markers. Sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate diminished most of the myeloid markers and very significantly increased the expression of the megakaryocytic markers.

The expression of carbonic anhydrase (CA) as a marker of erythroid differentiation was investigated by immunologic and enzymatic procedures. A polyclonal anti-CA antibody was obtained by immunizing rabbits with purified CA I isozyme. This antibody is reactive with CA I but not with CA II. Within blood cells, CA I was only present in erythrocytes, whereas CA II was also detected in platelet lysates by enzymatic assay. Concerning marrow cells, identifiable erythroblasts and some blast cells expressed CA I. Most of the glycophorin A-positive marrow cells were clearly labeled by the anti-CA I antibody. However, rare CA I-positive cells were not reactive with anti-glycophorin A antibodies. We therefore investigated whether these cells were erythroid precursors or progenitors. In cell sorting experiments of marrow cells with the FA6 152 monoclonal antibody, which among hematopoietic progenitors is reactive only with CFU-E and a part of BFU-E, was performed, CA I+ cells were found mainly in the positive fraction. The percentage of CA I+ cells nonreactive with anti-glycophorin A antibodies contained in the two fractions was in the same range as the percentage of erythroid progenitors identified by their capacity to form colonies. In addition, the anti-CA I antibody labeled blood BFU-E-derived colonies as early as day 6 of culture, whereas in similar experiments with the anti-glycophorin A antibodies, they were stained three or four days later. No labeling was observed in CFU-GM- or CFU-MK-derived colonies. The phenotype of the day 6 cells expressing CA I was similar to that of erythroid progenitors (CFU-E or BFU-E): negative for glycophorin A and hemoglobin, and positive for HLA-DR antigen, the antigen identified by FA6 152, and blood group A antigen. Among the cell lines tested, only HEL cells expressed CA I, while K562 was unlabeled by the anti-CA I antibody. In contrast, HEL and K562 cells expressed CA II as detected by a biochemical technique. Synthesis of CA I, as with other erythroid markers such as glycophorin A and hemoglobin, was almost abolished after 12-O-tetradecanoyl-phorbol-13 acetate treatment of HEL cells. In conclusion, CA I appears to be an early specific marker of the erythroid differentiation, expressed by a cell with a similar phenotype as an erythroid progenitor.

The Tn syndrome is an acquired clonal disorder characterized by the exposure of a normally hidden determinant, the Tn antigen, on the surface of human erythrocytes, platelets, granulocytes, and lymphocytes. Two distinct populations, Tn positive (Tn+) and Tn negative (Tn-), of mature hemopoietic cells are present in Tn patients. To determine whether the Tn antigen is already expressed on erythroid, myeloid, and pluripotent progenitors, light-density mononuclear blood cells from two patients with this syndrome were separated by fluorescent-activated cell sorting and by affinity chromatography into Tn+ and Tn- fractions, using their binding properties to Helix pomatia agglutinin (HPA). Burst-forming-unit erythroid (BFU-E), colony-forming-unit granulocyte/macrophage (CFU-GM), cells were assayed in plasma clot cultures. After 12-14 d of culture, colonies were studied by a double fluorescent labeling procedure. First, a fluorescein-conjugated HPA permitted evaluation of the presence or absence of the Tn antigen at the surface of the cells composing each colony, and second, the binding of a murine monoclonal antibody against either glycophorin A (LICR-LON-R10) or against a myeloid antigen (80H5), revealed by an indirect fluorescent procedure, was used to establish the erythroid or myeloid origin of each cell. The Tn+ fraction obtained by cell sorting gave rise to nearly 100% Tn+ colonies composed exclusively of cells bearing this antigen. The reverse was observed for the Tn- cell fraction. These results demonstrate that in the Tn syndrome, BFU-E, CFU-GM, and CFU-GEMM of the Tn+ clone express the Tn antigen at this early stage of differentiation.