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Les recommandations de la Societe de Reanimation en Langue Francaise (SRLF) [1], publiees en 2002, concernant les limitations et arrets de therapeutiques actives en reanimation adulte, sont a ce jour une initiative unique qui a modifie les pratiques professionnelles des services de reanimation. Dans un contexte different, de nombreuses situations posent au quotidien, dans les services d’hematologie, le probleme de la proportionnalite des soins et d’une eventuelle obstination deraisonnable, encore appelee communement acharnement therapeutique. En hematologie comme dans d’autres disciplines, cette conduite therapeutique peut etre cause de souffrance chez les malades, leurs familles et les soignants eux-memes, et souleve de graves problemes ethiques. C’est pourquoi le premier objectif du groupe Ethique de la Societe Francaise d’Hematologie (SFH), cree par son conseil d’administration en fevrier 2004, a ete de proposer a la discipline une reflexion et des recommandations. Ce texte a ete redige en s’inspirant du texte de la SRLF [1], des reflexions et de l’experience des membres du groupe et des donnees de la litterature. Il sera presente aux membres du conseil d’administration de la SFH et diffuse au sein de la discipline « hematologie ». Ce projet est apparu particulierement necessaire avec l’apparition de nouveaux textes legislatifs (en particulier la loi du 4 mars 2002), l’avis n° 60 du Comite Consultatif National d’Ethique du 27 janvier 2000 intitule « Fin de vie, arret de vie, euthanasie », la publication des recommandations de la Conference de consensus de l’ANAES des 14 et 15 janvier 2004 sur l’Accompagnement des patients en fin de vie et de leurs proches et la proposition de loi n° 1882 du 30 novembre 2004 relative aux droits des malades et a la fin de vie. Ce texte est propose comme une incitation a la reflexion plutot que comme une serie de recommandations qui s’ajouteraient aux textes officiels actuels ou en cours d’elaboration.

hma.2011.0608 Auteur(s) : Chantal Bauchetet, Dominique Bordessoule, Philippe Casassus, Joel Ceccaldi, Morgane Cheminant, Christian Bastard, Philippe Colombat, Diane Damotte, Eric Fiat, Dominique Jaulmes, Jean-Pierre Jouet, Sandra Malak, Genevieve Margueritte, Sarah Morin, Alice Polomeni, Henri Rochant, Jean-Jacques Sotto JJSotto@chu-grenoble.fr, Marc Zandecki, Robert Zittoun Commission d’ethique de la Societe francaise d’hematologie Tires a part : J. Sotto Notre pratique quotidienne [...]

Resume Propos. – Decrire les principales caracteristiques et les modalites evolutives des anemies hemolytiques auto-immunes (AHAI) de l’adulte et evaluer la pertinence et la rentabilite des examens complementaires realises a la recherche d’une maladie sous-jacente. Methodes. – Etude retrospective (1980–2000) monocentrique. Criteres d’inclusion : âge superieur a 16 ans, AHAI definie par une anemie (hemoglobine ≤ 12 g/dl chez l’homme, a 11 g/dl chez la femme) de type hemolytique, associee a un test de Coombs direct positif et/ou la presence d’agglutinines froides a un taux significatif (≥ 1/500) et/ou apres avoir elimine toute autre cause d’hemolyse constitutionnelle ou acquise. Resultats. – Quatre-vingt-trois patients inclus (56 femmes, 27 hommes), d’âge moyen 56 ans (± 21) au diagnostic, repartis en 72 (87 %) AHAI a anticorps « chauds » et 11 (13 %) maladies des agglutinines froides. Duree moyenne de suivi egale a 48 mois (mediane : 22 mois). Parmi les 72 AHAI « chaudes », la specificite du test de Coombs direct etait : IgG + complement (43 %), IgG seul (32 %) ou complement seul (25 %), des agglutinines froides etaient associees (taux :1/60 a 1/512) dans 15 cas (20 %). Les anticorps antinoyaux (AAN) etaient positifs (taux ≥ 1/80) chez 33 % des patients testes. La presence d’une hypogammaglobulinemie sur l’electrophorese des proteines plasmatiques (EPP) etait significativement correlee a la presence d'un lymphome malin non hodgkinien (LMNH). La tomodensitometrie (TDM) thoracoabdominale, realisee chez la moitie des patients, retrouvait une anomalie autre qu’une splenomegalie dans 25 % des cas. La biopsie medullaire (BOM) etait anormale dans 7/26 cas (27 %) mais permettait de porter a elle seule le diagnostic de LMNH dans 1 seul cas. Sur les 72 AHAI « chaudes », 37 (51 %) etaient finalement considerees comme « secondaires »: LMNH (n = 14), Hogdkin (n = 1), connectivite (n = 14), prise medicamenteuse (n = 3), causes diverses (n = 5). Dans 6 cas sur 14 (43 %), le diagnostic d’AHAI precedait le diagnostic de LNH, avec des delais variant de 24 a 66 mois. Les reponses aux differents traitements etaient comparables dans les AHAI « primitives » et « secondaires ». Treize patients (15,6 %) sont decedes au total, de complications infectieuses (n = 5), du LMNH sous-jacent (n = 5), ou d’autres causes (n = 3). Conclusions. – Les AHAI s’associent a une maladie sous-jacente dans plus de la moitie des cas et elles peuvent preceder de plusieurs annees l’apparition d’un lymphome. En dehors d’une cause evidente, la realisation systematique de la recherche d’AAN, d’une EPP et d’une TDM thoracoabdominale se justifie. En cas de normalite de ces examens, la realisation systematique d’une BOM parait tres peu rentable.

A prospective study of autologous bone marrow or peripheral blood stem cell transplantation after intensive chemotherapy in myelodysplastic syndromes

The CML 88 study was designed to evaluate the efficacy of maintenance therapy in a multicentric randomised protocol using IFN combined with low-dose Ara-C versus IFN alone, following an induction with IFN + HU. Between April 1988 and February 1991, 237 patients from 36 French Hematology Centres were entered in the study. Preliminary cytogenetic results show a slightly higher, although not statistically significant, proportion of major chromosomal responses, including complete cytogenetic remissions, in the IFN + Ara C arm.

We designed a randomized trial of IC with or without quinine, an agent capable of reverting the multidrug resistance (mdr) phenotype, in patients agedor = 65 years with high risk MDS. Patients were randomized to receive Mitoxantrone 12 mg/m2/d d2-5 + AraC 1 g/m2/12 h d1-5, with (Q+) or without (Q-) quinine (30 mg/kg/day). 131 patients were included. PGP expression analysis was successfully made in 91 patients and 42 patients (46%) had positive PGP expression. In PGP positive cases, 13 of the 25 (52%) patients who received quinine achieved CR, as compared to 3 of the 17 (18%) patients treated with chemotherapy alone (p = 0.02). In PGP negative cases, the CR rate was 35% and 49%, respectively in patients who received quinine or chemotherapy alone (difference not significant). In the 42 PGP positive patients, median Kaplan-Meier (KM) survival was 13 months in patients allocated to the quinine group, and 8 months in patients treated with chemotherapy alone (p = 0.01). In PGP negative patients, median KM survival was 14 months in patients allocated to the quinine group, and 14 months in patients treated with chemotherapy alone. Side effects of quinine mainly included vertigo and tinnitus that generally disappeared with dose reduction. Mucositis was significantly more frequently observed in the quinine group. No life threatening cardiac toxicity was observed. In conclusion, results of this randomized study show that quinine increases the CR rate and survival in PGP positive MDS cases treated with IC. The fact that quinine had no effect on the response rate and survival of PGP negative MDS suggests a specific effect on PGP mediated drug resistance rather than, for instance, a simple effect on the metabolism of Mitoxantrone and/or AraC.

We prospectively assessed autologous stem cell transplantation for consolidation treatment in a trial of intensive chemotherapy in high risk myelodysplastic syndromes (MDS). In this trial, patients aged 55 years or less with no HLA-identical sibling and achieving CR were scheduled to receive unmanipulated autologous bone marrow transplantation (ABMT) preceded by a consolidation chemotherapy course. Forty-two of the 83 patients aged 55 years or less included in the trial (51%) achieved CR. Three were allografted in CR. Twenty-four of the remaining 39 patients who achieved CR (62%) received ABMT (16 patients) or autologous peripheral blood stem cell transplantation (APSCT) (eight patients). Indeed, as bone marrow harvest was often insufficient, APSCT was subsequently proposed after mobilization by consolidation chemotherapy followed by G-CSF. The conditioning regimen combined cyclophosphamide and busulfan. ABMT and APSCT were performed 1-7 months (median 3) after CR achievement. Hematological reconstitution occurred in all patients and tended to be faster after APSCT than ABMT although not significantly. Three patients died from the procedure, nine relapsed after 2-26 months and 12 (50%) were still in CR after 8-55 months. In autografted patients, median Kaplan-Meier disease-free survival and survival were 29 and 33 months from the autograft, respectively. Thus, ABMT or APSCT can be performed in almost two-thirds of MDS patients who achieve CR with intensive chemotherapy. PBSC collection may yield higher numbers of stem cells than marrow collection in some cases, and could improve the percentage of MDS patients autografted in CR. Longer follow-up is required to determine if autograft will prolong CR duration in at least some patients.

We designed a randomized trial of IC with or without quinine, an agent capable of reverting the multidrug resistance (mdr) phenotype, in patients aged < 65 years with high risk MDS. Patients were randomized to receive Mitoxantrone 12mg/m2/d d2–5 + AraC 1g/m2/12h d1–5, with (Q+) or without (Q) quinine (30mg/kg/day). 131 patients were included. PGP expression analysis was successfully made in 91 patients and 42 patients (46%) had positive PGP expression. In PGP positive cases, 13 of the 25 (52%) patients who received quinine achieved CR, as compared to 3 of the 17 (18%) patients treated with chemotherapy alone (p=0.02). In PGP negative cases, the CR rate was 35% and 49%, respectively in patients who received quinine or chemotherapy alone (difference not significant). In the 42 PGP positive patients, median Kaplan-Meier (KM) survival was 13 months in patients allocated to the quinine group, and 8 months in patients treated with chemotherapy alone (p=0.01). In PGP negative patients, median KM survival was 14 months in patients allocated to the quinine group, and 14 months in patients treated with chemotherapy alone. Side effects of quinine mainly included vertigo and tinnitus that generally disappeared with dose reduction. Mucositis was significantly more frequently observed in the quinine group. No life threatening cardiac toxicity was observed. In conclusion, results of this randomized study show that quinine increases the CR rate and survival in PGP positive MDS cases treated with IC. The fact that quinine had no effect on the response rate and survival of PGP negative MDS suggests a specific effect on PGP mediated drug resistance rather than, for instance, a simple effect on the metabolism of Mitoxantrone and/or AraC.

Low-dose chemotherapy in myelodysplastic syndromes (MDS) gives overall limited results in high-risk MDS [1–3]. Intensive chemotherapy in MDS and secondary leukemias (i.e., acute myeloid leukemia, AML, following de novo or therapy-related MDS) gives lower complete remission (CR) rates and shorter CR duration than in de novo AML [4–11]. This may be due in part to a higher incidence of mdr gene expression in MDS than in de novo AML as mdr gene expression is associated in our experience, with very low CR rates with intensive chemotherapy [12]. These recent results suggested that the use of agents capable of reverting mdr gene expression could improve the CR rate in MDS and secondary leukemias.

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.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.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], p0.0001), evolution to pancytopenia (5.5 [2.8-11], p0.0001), myelodysplastic syndrome or acute leukaemia (19.1 [7.3-50], p0.001), age over 55 years at diagnosis (4 [2.4-6.9], p0.0001), need for additional treatment (2.1 [1.3-3.6], p0.003), and thrombocytopenia at diagnosis (2.2 [1.3-3.8, p0.003). Better survival was shown for patients in whom aplastic anaemia antedated PNH (0.32 [0.14-0.72], p0.02). 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).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.

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.

We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E- C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+- sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co- culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T- cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.

Three allogeneic bone marrow transplantation patients who exhibited a suppressive subset of T cells for in vitro hematopoiesis have been investigated to determine whether this T cell suppressive effect was genetically restricted. In the three cases, T cells separated by sheep red cell rosetting inhibited blood colony-forming units granulocyte-monocyte (CFU-GM) and burst-forming unit erythroid (BFU-E) growth from the patients and from the bone marrow donors who were HLA identical, but not from randomly chosen unrelated subjects. In one case, cocultures were performed between the patient T cells and the T-depleted cells from eight siblings and from the mother. A marked inhibition (30 to 60%) of CFU-GM and BFU-E growth was found in the relatives who shared a haplo-identical HLA-DR 5. The same degree of suppression was found with respect to whether the siblings were homozygous or heterozygous for the HLA-DR 5 antigen, and whether or not they shared common class I antigens. This inhibition was totally abolished when a monoclonal antibody against HLA-DR was added, whereas a monoclonal antibody against class I histocompatibility antigen had no effect. To additionally demonstrate that this inhibition was mediated by a single HLA-DR haplotype, T cells from the patient were co-cultured with cells from three normal unrelated individuals, one with a phenotypically identical DR and two with only one haploidentical DR. Inhibition was similarly found in the subject exhibiting complete DR identity, and the subject with only the DR 5 haploidentical phenotype. These results demonstrate that a unique subset of T cells present in allogeneic bone marrow transplants specifically suppress differentiation of hemopoietic progenitors that bear one phenotypically haplo-identical HLA-DR antigen.

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.

We have investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on iron uptake into human hematopoietic cell lines K562, U937, and HL-60. TPA inhibited both cell growth and iron uptake by these cell lines. This effect was rapid, which is typical of phorbol esters which are biologically active, and it occurred at very low concentrations of TPA. This effect of TPA was dependent upon an inhibition of the transferrin-binding capacity as estimated on intact cells. However, experiments with transferrin binding on cell samples dissolved in 1% Triton X-100 showed that TPA-treated cells exhibited a transferrin-binding capacity similar to that of control cells. On the basis of this result, it is suggested that TPA modified a part of transferrin receptors present in the cells; as a result of this modification, these receptors became unavailable for binding transferrin, but they remained physically present in the cell. Other compounds capable of inducing the differentiation of leukemic cells, such as dimethyl sulfoxide, butyrate, retinoic acid, and 1 alpha,25-dihydroxy-vitamin D3, did not acutely inhibit iron uptake. We also investigated the effect of TPA on transferrin receptors in a cellular system in which phorbol esters stimulate cell proliferation. At 16 X 10(-9) M, TPA markedly stimulated the proliferation of T-lymphocytes. However, in spite of this marked stimulation of cell proliferation, TPA-stimulated lymphocytes exhibited a transferrin-binding capacity much inferior to cells stimulated by other mitogens, such as phytohemagglutinin.

Treatment of acute lymphoblastic leukemia (ALL) in children with effective regimens including induction with vincristine, cytoxan, asparaginase, and prednisone followed by prolonged maintenance with various drug combinations results in complete remission (CR) rates of approximately 90%, with more than 50% of the patients being possibly cured [1]. Use of similar regimens in patients over 15 has, however, been far less successful [2]. During the last decade, considerable improvement has been achieved in the treatment of adult ALL using more intensive regimens, and several protocols including more aggressive induction with anthracyclines followed by intensive consolidation and multidrug maintenance over several years have yielded therapeutic results similar to those obtained in children [3–6] and possibly similar to those achieved by allogeneic bone marrow transplantation [7] with approximately 50% long-term survivors. These regimens, however, are very toxic and may not be suitable for all patients.

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.

Red blood cells from normal subjects and subjects with heterocellular hereditary persistence of fatal haemoglobin and beta-thalassaemia were fractionated according to density by centrifugation on a discontinuous gradient of Stractan II. F-cells were studied by immunofluorescence and their proportion was evaluated in each separated population. This approach has permitted to show that F-cells were preferentially distributed among high density erythrocytes. This phenomenon reflects a peculiar characteristic of F-cells.

Between September 78 and May 81, 13 adults (mean age 27± 7 years) with acute myeloid leukemia (AML) in 1st remission (n=11) or chronic myeloid leukemia (CML) in 2nd chronic phase, were given donor buffy coat cells, Cyclophosphamide (60 mg/kg×2), and low dose total body irradiation (TBI) as conditioning regimen for allogeneic bone marrow transplantation (BMT) from an HLA identical sibling donor.

We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E-C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+-sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co-culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T-cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.

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.

The effect of phospholipases and of inhibitors of phospholipases on the binding of 125I-Trf to human hematopoietic cell lines was investigated. The results showed that incubation of the cells with phospholipases or inhibitors of phospholipases elicited a significant reduction of the Trf binding. This phenomenon was related to a reduction of Trf binding sites without affecting the affinity of the receptors for Trf. These results suggest that the phospholipids of the cell membrane may modulate the binding of transferrin to its receptor.

In order to study the cellular regulation of i and I antigen expressions n erythroid cells, burst-forming-unit erythroids were grown in methylcellulose from the blood of normal human adults. Erythroid colonies were harvested on days 9, 11 or 14 of culture. Individual colonies or pooled colonies were subsequently cytocentrifuged and then i or I antigens were studied by immunofluorescence using a monoclonal anti-i or I antibody. Labelled cells were firstly located, their erythroid origin and their stage of maturation were subsequently ascertained after May-Grünwald-Giemsa staining. This approach shows that: (1) a marked heterogeneity occurred from one colony to another, most of the colonies being i-negative while the others contained a mixed population composed by a minority of i-positive erythroid cells; (2) the proportion of both i colonies and i cells decreased with the aging of culture; (3) i antigen was preferentially expressed on immature erythroblasts; however, immaturity per se was not sufficient to determine i expression; (4) I antigen expression was also related to the process of erythroid maturation but in an inverse way; its expression was higher in mature erythroblasts than in immature ones.

In 164 patients with Hodgkin's disease staged between 1973 and 1979 the response to the 3 initial cycles of multiagent chemotherapy was evaluated as a prognosticator of survival. Treatment of localized disease (Stages I, II, III1) consisted of 3 cycles of chemotherapy followed by subtotal nodal irradiation, including the splenic area in non splenectomized patients. Treatment of extended disease (Stage III2 and IV) consisted of 6 cycles followed by low-dosage radiotherapy of initial bulky disease. Five-year actuarial survival was 88% in Stage I, 80% in II, 100% in III1, 45% in III2 and IV. Chemotherapy-induced complete remission after 3 cycles (CH leads to CR) was associated with a favorable prognosis. Five-year survival of Stage III2 and IV patients was better in those who reached CH leads to CR than in those who did not (75% versus 25%; P less than 0.01). This relationship between CH leads to CR and five-year survival was confirmed in patients with localized disease, as shown in Stage II patients (respectively 97% versus 63%; P less than 0.05). Therefore the response to initial chemotherapy provides a new prognostic factor that may serve to delineate a "high-risk" group of patients. The latter deserve aggressive therapy while those in the favorable group would benefit from a less aggressive combined regimen that would minimize long-term complications.

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.

Three allogeneic bone marrow transplantation patients who exhibited a suppressive subset of T cells for in vitro hematopoiesis have been investigated to determine whether this T cell suppressive effect was genetically restricted. In the three cases, T cells separated by sheep red cell rosetting inhibited blood colony-forming units granulocyte-monocyte (CFU-GM) and burst-forming unit erythroid (BFU-E) growth from the patients and from the bone marrow donors who were HLA identical, but not from randomly chosen unrelated subjects. In one case, cocultures were performed between the patient T cells and the T-depleted cells from eight siblings and from the mother. A marked inhibition (30 to 60%) of CFU-GM and BFU-E growth was found in the relatives who shared a haplo-identical HLA-DR 5. The same degree of suppression was found with respect to whether the siblings were homozygous or heterozygous for the HLA-DR 5 antigen, and whether or not they shared common class I antigens. This inhibition was totally abolished when a monoclonal antibody against HLA-DR was added, whereas a monoclonal antibody against class I histocompatibility antigen had no effect. To additionally demonstrate that this inhibition was mediated by a single HLA-DR haplotype, T cells from the patient were co-cultured with cells from three normal unrelated individuals, one with a phenotypically identical DR and two with only one haploidentical DR. Inhibition was similarly found in the subject exhibiting complete DR identity, and the subject with only the DR 5 haploidentical phenotype. These results demonstrate that a unique subset of T cells present in allogeneic bone marrow transplants specifically suppress differentiation of hemopoietic progenitors that bear one phenotypically haplo-identical HLA-DR antigen.

In order to investigate whether the morphological abnormalities observed in congenital dyserythropoietic anemia type III (CDA III) have a cellular or an environmental origin; BFUE from the blood of a patient exhibiting a CDA type III were grown in vitro. The progeny derived from these BFUE were subsequently studied at light and electron microscopic level. Giant multinuclear erythroblasts which represent the most prominent finding of CDA III in bone marrow were also found in culture. Nuclear clefts found in vivo were also observed by electron microscopic studies performed on the erythroblasts growing in vitro. In each erythroid colony, morphologically normal and giant multinuclear erythroblasts were intermingled. This finding indicates that the two populations of erythroblasts derive from the same defective stem cell. The studies by indirect immunofluorescence of i antigen was preferentially expressed in the immature erythroblasts as in culture from normal subjects but not in the giant mature erythroblasts. This finding suggests that the excess of i antigen expression of CDA III in vivo is rather the indirect consequence of a stimulation of erythropoiesis than result of the disease.

Two sublines of the human leukemia cell line K562 including the original cell line and three clones have been investigated for their erythroid features. All of them produce embryonic and fetal hemoglobins, glycophorin A, spectrin and true acetylcholinesterase, but to a varying extent among the cell lines. The Hb and glycophorin contents were correlated in the different K562 cell lines, whereas acetylcholinesterase was independently expressed from these two other erythroid markers. Hb accumulation is enhanced by exposure of the cells to 100 microM hemin without a significant modification of the expression of the other erythroid markers. Butyrate greatly increased the activity of acetylcholinesterase, slightly enhanced the production of hemoglobin, but did not modify the expression of glycophorin and spectrin. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced an almost complete disappearance of glycophorin, reduced the synthesis of Hb by K562 cells and also abolished the action of hemin on Hb accumulation. Therefore, all the different K562 cell lines exhibit clear erythroid features including acetylcholinesterase. Butyrate or hemin did not induce terminal differentiation of K562 cells, whereas TPA significantly diminished the erythroid phenotype.

A double immunofluorescence technique was used to study the expression of I, i, H and A1 antigens on individual erythrocytes of neonates, adult controls and patients with various haematological disorders. I, i and H staining was heterogeneously distributed from cell to cell as was already demonstrated for A1 antigen. i staining occured in 41 to 64% of cord cells (11 to 32% were strongly fluorescent) but only in 0.2 to 2.3% of adult erythrocytes. I staining occurred in 54 to 97.5% of adult erythrocytes but only in 1 to 14% of those of cord blood. Patients' cells tended to mimic a foetal pattern of heterogeneity in that an increase of i stained cells occured associated with a decrease of A1 or H stained cells. It is suggested that the foetal-like red cells observed in patients with haematological disorders result from an expansion of a normal process of differentiation rather than clonal development of abnormal cells.

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.