[Acquired alpha-thalassemia as early sign for myelodysplastic syndrome (refractory anaemia) with secondary haemochromatosis]. / Alpha-thalassémie acquise révélatrice d'un syndrome myélodysplasique de type anémie réfractaire avec hémochromatose secondaire.

We report the case of a 59 year old man presenting a regenerative microcytic hypochromic anaemia. The investigations revealed the presence of haemoglobin H, suggesting abnormalities in the alpha-globin chains synthesis. Alpha-thalassemia was thus suspected. The patient had no personal or familial history. The association with aniso-poïkilocytosis and a marked iron overload (ferritinemia > 1,500 microg/L) suggested a myelodysplastic syndrome, which was confirmed with a bone marrow aspiration. The pattern was consistent with the Acquired alpha-Thalassemia-Myelodysplastic Syndrome (ATMDS). About a hundred cases are listed worldwidely and collected in an international registry. The causes of ATMDS are ignored, but recent reports indicate that the ATRX gene may be implicated in the pathogenesis. ATRX is a chromatin-associated protein, involved in the transcription of several genes. The alpha globin genes could be one of the targets of the ATRX protein.

[Incubated osmotic fragility test does not exclude red blood cell membrane disorders! About a case of hereditary elliptocytosis]. / Une résistance globulaire osmotique normale n'exclut pas une membranopathie érythrocytaire ! A propos d'un cas d'elliptocytose héréditaire.

We report a case of hereditary elliptocytosis in an infant diagnosed a few months after the birth, in a context of regenerative normocytic normochromic anaemia. The investigations, including incubated osmotic fragility, erythrocytic enzymes study and haemoglobin electrophoresis, were not contributive. Only the persistence of elongated (or cigar-shaped) erythrocytes on blood smears was noted. Hereditary elliptocytosis was confirmed by specialized investigations (rheological study and erythrocytic membrane proteins electrophoresis). Investigations in the mother were realized and led to the discovery of a similar biological pattern. Hereditary elliptocytosis is a red blood cell membrane disorder due to the defect in cytoskeleton proteins (spectrin or 4.1), leading to the loss of deformability properties of erythrocytes. This disorder is considered as rare; however, its incidence is probably underestimated because most cases are pauci- or asymptomatic and the discovery is often fortuitous. The absence of detection of this defect by incubated osmotic fragility should not discard the hypothesis of erythrocytes membrane disorders. The persistent observation of elongated erythrocytes on blood smear must encourage the biologist to evocate a hereditary elliptocytosis.

Preparation and characterization of heparin-loaded polymeric microparticles.

Microparticles containing heparin were prepared by a water-in-oil-in-water emulsification and evaporation process with pure or blends of biodegradable (poly-epsilon-caprolactone and poly(D,L-lactic-co-glycolic acid)) and of positively-charged non-biodegradable (Eudragit RS and RL) polymers. The influence of polymers and some excipients (gelatin A and B, NaCl) on the particle size, the morphology, the heparin encapsulation rate as well as the in vitro drug release was investigated. The diameter of the microparticles prepared with the various polymers ranged from 80 to 130 microns and was found to increase significantly with the addition of gelatin A into the internal aqueous phase. Microparticles prepared with Eudragit RS and RL exhibited higher drug entrapment efficiency (49 and 80% respectively) but lower drug release within 24 h (17 and 3.5% respectively) than those prepared with PCL and PLAGA. The use of blends of two polymers in the organic phase was found to modify the drug entrapment as well as the heparin release kinetics compared with microparticles prepared with a single polymer. In addition, microparticles prepared with gelatin A showed higher entrapment efficiency, but a significant initial burst effect was observed during the heparin release. The in vitro biological activity of heparin released from the formulations affording a suitable drug release has been tested by measuring the anti-Xa activity by a colorimetric assay with a chromogenic substrate. The results confirmed that heparin remained unaltered after the entrapment process.

The virucidal effect of platelet concentrates: preliminary study and first conclusions.

Despite the increased safety of blood components, achieved through improved donor selection and testing, transfusion recipients remain at risk of transfusion-associated diseases. Transfusion of cellular blood components has been implicated in transmission of viral, bacterial and protozoan diseases. Investigators have studied a myriad of processes for pathogen depletion and/or inactivation. No successful treatments, apart the leukodepletion, have already been identified for red cells and platelets. And more, several evidences indicate that platelets play a key role in host defence against infection. High levels of pathogens were added to single-donor platelet concentrates (PC) containing 3 to 5 10(11) platelets in 300 ml. The infectivity of each pathogen was measured with established biologic assays. The following levels of pathogen inactivation were achieved : >10(2.63) plaque-forming units (PFU) per ml of adenovirus 5 (ADV5), >10(5.6) PFU per ml of Poliovirus 1 (P1) and >10(4.1) PFU per ml of vaccinia virus (VaV). In conclusion, the PC show a potential virucidal effect. This inactivation process has been found with bacteria and still remains unknown for viruses.

Preparation and in vitro evaluation of heparin-loaded polymeric nanoparticles.

Nanoparticles of a highly soluble macromolecular drug, heparin, were formulated with two biodegradable polymers (poly-E-caprolactone [PCL] and poly (D, L-lactic-co-glycolic-acid) 50/50 [PLAGA]) and two nonbiodegradable positively charged polymers (Eudragit RS and RL) by the double emulsion and solvent evaporation method, using a high-pressure homogenization device. The encapsulation efficiency and heparin release profiles were studied as a function of the type of polymers employed (alone or in combination) and the concentration of heparin. Optimal encapsulation efficiency was observed when 5000 IU of heparin were incorporated in the first emulsion. High drug entrapment efficiency was observed in both Eudragit RS and RL nanoparticles (60% and 98%, respectively), compared with PLAGA and PCL nanoparticles (<14%). The use of the two types of Eudragit in combination with PCL and PLAGA increased the encapsulation efficiency compared with these two biodegradable polymers used alone; however, the in vitro drug release was not modified and remained low. On the other hand, the addition of esterase to the dissolution medium resulted in a significant increase in heparin release. The in vitro biological activity of released heparin, evaluated by measuring the anti-Xa activity by a colorimetric assay, was conserved after the encapsulation process.

A stable reagent system for screening and identifying red blood cell irregular antibodies: application to commercial antibodies.

OBJECTIVE: Development of a new solid-phase system for screening and identifying irregular red cell antibodies. MATERIALS AND METHODS: Red blood cell membranes were prepared by a semi-automated procedure in which the hemolysate solution was passed through a hollow-fiber system. The membranes were fixed to the solid phase (microtiter plates) by centrifugation and incubated with 8% fat-free milk. Antibodies added to the microtiter plate were detected by anti-human antibodies adsorbed onto yellow latex particles. RESULTS: The system had good sensitivity (titer <1); 97% of anti-D samples were detected. The detection system was stable for 6 months at 4 degrees C. CONCLUSION: This stable-antigen solid-phase system readily detects and identifies red cell antibodies that are important in transfusion.

In vitro study of the protective effect of trehalose and dextran during freezing of human red blood cells in liquid nitrogen.

Two nonpermeant cryoprotectants, the disaccharide trehalose and the polymeric carbohydrate (dextran, 40 kDa), were assessed as substitutes for glycerol in the cryopreservation of human red blood cells (RBC). The agents were evaluated by measuring the percentage of RBC recovery (total of free hemoglobin after freezing) and by evaluating the erythrocyte state after freezing. Ninety percent of the red cells were recovered after freezing in 30% (w/v) dextran in liquid nitrogen, which is very close to the recovery obtained in 35. 5% (w/v) glycerol (92%). The activities of pyruvate kinase and glucose-6-phosphate dehydrogenase of RBCs frozen and thawed with dextran were not modified, and the 2,3-diphosphoglycerate was reduced by 26%, but remained within normal values. ATP was reduced by 56%. The erythrocyte membrane integrity, evaluated by its osmotic fragility, was not altered, and the RBCs protected by dextran retained their normal discoid shape without the formation of microvesicles. The 24-h hemolysis of the washed red cells after storage at 4 degrees C was 7%. These results suggest that dextran protects red blood cells during freezing in liquid nitrogen, but that some effort is still needed to limit the drop of ATP concentration. One of the main advantages of dextran is that it does not penetrate the RBCs and requires less washing than glycerol.

Blood group antigen survival on freeze-dried erythrocytes and ghosts after isolation.

To screen and identify irregular antibodies, whatever the technique used, fresh erythrocytes (RBCs) are needed to set up the panel. Solid-phase tests using dried blood cells are available, but the technique is based on the adherence of sensitized RBCs, which have a short life span. We have checked antigen survival on membranes with a saline test and an antiglobulin test for two methods to preserve the antigen substrate: freeze-drying of RBCs and preparation of RBC membranes. The different antigens of the ABO, Rhesus, Kell, P, Lewis, MNSs, Lutheran, Duffy, Kidd and Li systems are well recognized on the membranes after isolation and on freeze-dried cells. Demonstration of antigen survival leads us to consider using membranes or freeze-dried cells in new immunological tests.

Liquid preservation of polymorphonuclear leukocytes: effect of various additives on chemotaxis preservation.

Adequate storage of polymorphonuclear leukocytes would allow an easier in vitro study of their structure and their functions, an easier study of polymorphonuclear leukocyte diseases (e.g. chronic granulomatous disease) and an easier use of polymorphonuclear leukocytes as a clinical tool (e.g. for localizing infections). Unfortunately, polymorphonuclear leukocytes are nearly impossible to preserve, even in short-term storage. This study proposes a model for the study of polymorphonuclear leukocyte storage in a synthetic medium: Plasmion. For storage over a period of 24 h, we found that supplementation with each of the following additives: bicarbonate buffer, glucose, adenosine triphosphate (ATP), ascorbic acid, nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), alpha-tocopherol acetate, amikacin and ampicillin, significantly improves (p less than 0.05) one or several functions of the polymorphonuclear leukocytes. When samples were stored for 48 h, we found that the addition of bicarbonate buffer after 24 h significantly improves the maintenance of several functions of polymorphonuclear leukocytes, in particular chemotaxis. Preservation for 96 h was achieved by making additions of supplements on each day of storage, with a chemotaxis maintenance of 83% at 24 h, 59% at 48 h, 46% at 24 h and 20% at 96 h. In conclusion, by using the Plasmion medium, and adding the above-mentioned compounds on each day of storage, chemotaxis can be satisfactorily maintained over 4 days.

[Experimental study of a model for granulocyte storage]. / Etude expérimentale d'un modèle de conservation des polynucléaires neutrophiles.

An efficient granulocytes preservation would allow an easier study of their structure and functions, of granulocytes functional diseases (e.g. chronic granulomatous disease) and an easier use of granulocytes as a clinical tool (e.g. for localizing infections or for transfusions). Unfortunately, granulocytes are nearly impossible to preserve, even in a short-term storage (less than 24 hours). This study proposes an experimental model which could improve in vitro granulocyte functions maintenance. Bicarbonate buffer, glucose, adenosine triphosphate, vitamins C and E, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, amikacin, and ampicillin supplementation significantly (p 0.05) improve maintenance of one or several granulocyte functions during storage.

[Storage of granulocytes]. / Conservation des polynucléaires neutrophiles.

Granulocytes transfusions set numerous problems and are less and less used. The very short maintenance of the granulocyte functions (chemotaxis) limits their use are a clinical tool (e.g. for localizing infections and for transfusions). Neither granulocyte collection by centrifugation methods (continuous or discontinuous flow) nor glucocorticoid premedication significantly alter granulocytes functions (in particular chemotaxis). Liquid preservation methods give better results than cryogenic methods. Optimal storage parameters are a temperature of 22 degrees C and a pH level close to 7.4. Chemotaxis is the most sensitive indicator of granulocyte damages in connection with storage. Bicarbonate buffer, glucose, proteins and reducing agents supplementation significantly improve chemotaxis maintenance. Cryogenic methods are less studied and less used. They allow storage of only little amounts of granulocytes. In conclusion, efficient granulocyte storage (over several days) would allow an easier use of these cells in many fields, but unfortunately, no solution is yet available.

Effect of hirudin on the chemotactic properties of alpha-thrombin.

A study by an agarose technique of the chemotactic power of human alpha-thrombin on polymorphonuclear leukocytes reveals that this enzyme has a chemotactic activity. Hirudin, which inhibits the coagulant properties of thrombin, suppresses these chemotactic properties. This effect could be explained by the binding of hirudin to the structural domain of alpha-thrombin involved in its chemotactic activity.

[Hemoglobin niosomes. II. In vitro interactions of plasma proteins and phagocytes]. / Niosomes d'hémoglobine. II. Interactions in vitro avec les protéines plasmatiques et les phagocytes.

We have studied the in vitro interactions versus some blood components of the hemoglobin niosomes whose preparation and physicochemical and oxyphoric properties have been published in a precedent paper (this journal, 1989, No. 7, p. 192). This work was devoted to the research of 1) Agglutination phenomena with ABO blood group substances, plasma, some of its components and three plasma expanders, finally main erythrocytic phenotypes. 2) Adsorption of plasma proteins by immunoelectrophoresis. 3) Effects of niosomes on blood coagulation by thromboelastography. 4) Interactions between niosomes and phagocytes by electron microscopy, chemotactic migration, oxygen consumption, superoxide generation and oxydases function. These assays allow to observe and conclude that: 1) The agglutination phenomena are almost constant except with red blood cells. The agglutinates are dissociable by shaking. The agglutination appears to be nonspecific of a niosome component but is not observed with "classical" DPPC-chol-DCP liposomes. 2) Albumin and eventually transferrin are adsorbed at the surface of niosomes but without destabilizing them. 3) The vesicules show no important effects on coagulation factors, the enhancement of clotting time appearing essentially the consequence of blood dilution. 4) Niosomes phagocytosis is important but all the measurements fail to show any cellular metabolism activation: cell oxygen consumption, oxygenated metabolites generation and oxydases activity are not enhanced whatever the "electric" charge or the niosomes/phagocytes ratio used.

[Hemoglobin niosomes. I. Preparation, functional and physico-chemical properties, and stability]. / Niosomes d'hémoglobine. I. Préparation, propriétés physicochimiques et oxyphoriques, stabilité.

Hemoglobin niosomes. I, Preparation, functional and physical properties and stability. Hemoglobin niosomes (non ionic liposomes) obtained from L'Oréal synthetic lipids by solvents vaporization appear as unilamellar spherical red vesicles, isolated from each other and with heterogeneous size (0.5 to 4 microns). Their suspensions show a visible spectra superimposable to that of free hemoglobin which is incorporated at a rate of 0.3 to 0.5 g per lipid gram. Vesicles are permeable to oxygen and the hemoglobin dissociation curve can be modified similarly to non-encapsulated hemoglobin. Niosomes appear physically stable while hemoglobin undergoes a progressive oxidation to methemoglobin reaching 30% after 5 months at 4 degrees C. Even in the absence of dicetylphosphate (DCP), niosomes possess a negative charge confering to them an electrophoretic mobility. 5% DCP allows to obtain a zeta potential near to that of erythrocytes. The niosomes suspensions are more viscous than red blood cells but their rheological behavior is similar and the vesicles may have some deformability.

Effects of indomethacin and diclofenac on some functions of polymorphonuclear neutrophils.

The effects in-vitro of two non-steroidal anti-inflammatory drugs, diclofenac and indomethacin, have been studied on human polymorphonuclear functions. Adhesivity and chemotaxis were decreased by the two drugs, but in a manner that varied with the attractant used. As shown by the nitro blue tetrazolium slide test, all cells remained active but the production of oxygen metabolites by opsonized zymosan was decreased in the presence of diclofenac.

[Disparity between the level of activity and immunochemical determination of terminal deoxynucleotidyl transferase in leukemia]. / Discordances entre la mesure d'activité et la détermination immunochimique de la terminal déoxynucléotidyl transférase dans les leucémies.

Terminal deoxynucleotidyl Transferase (TdT) is used in diagnostic investigation and in remission surveillance of leukaemias. We have compared the results obtained by enzyme activity measurement and by immunoperoxydase technic. Discrepancies between the two assays exist in 19% of the cases; they are due in particular to the anti-TdT nature who limits its specificity, and they restrict the TdT immunochemistry determination interest.

Ultramicromethod for measuring the activity of terminal deoxynucleotidyl transferase.

We describe an ultramicromethod for measurement of terminal deoxynucleotidyl transferase activity. The materials used are as proposed by Hösli (Clin Chem 1977;23:1476-84) for antenatal diagnosis. The technique is based on the methods of Beutler and Kuhl (Am J Clin Pathol 1978;70:733-7) and Yasmineh et al. (Clin Chem 1980; 26:891-5). The procedure makes it possible to reduce the blood sample to 2 mL at the most and to reduce the cost of the measurement very considerably. The technique gives results similar to those given by the conventional techniques.