[Novel criteria for assessing red blood cell viability in blood banks]. / Nouveaux critères d'évaluation de la viabilité des hématies destinées à la transfusion.

UNLABELLED: In light of recent results on the mechanism of programmed cell death of human red blood cells (RBC), the aim of the present study was to solve the enigma of the rapid clearance of transfused RBCs. MATERIALS AND METHODS: We describe new criteria of RBC viability founded on the use of flow cytometry. They were applied, in association with the classical ones: ATP and hemolysis measurements, to RBCs stored in SAGM medium for 42 days. RESULTS AND CONCLUSIONS: Application of an original method of flow cytometric quantitation of in vitro erythrophagocytosis showed that an important proportion of stored RBCs were phagocytized although the following classical signals for phagocytosis were absent, i.e.: desialylation, phosphatidylserine exposure in the outer leaflet of the RBC membrane, loss of CD47 receptor, an antiphagocytosis signal. In addition, ATP was still present and hemolysis was very low. This enigma was solved by the use of scanning electron microscopy, which showed the disappearance of discocytes and the presence of an important proportion of spheroechinocytes, which are the phagocytable forms of RBCs. The mechanism of this dramatic morphological transformation remains to be elucidated.

Two red blood cell units collected in SAG-M additive solution with the ALYX component collection system.

The first protocol available for the new ALYX component system (Baxter Healthcare Inc.) allows automated collection of two Red Blood Cell (RBC) units from one donor. The primary objective of our evaluation was to assess donor safety, comfort and to check the quality of blood products collected. 30 procedures were performed on eligible donors according to French best donation practices. Eligibility criteria were defined in order to ensure a post donation hemoglobin concentration of 11 g/dL minimum. Pre donation ferritin level was also checked. 360 ml of absolute RBC were collected from each donor. Donors physiological parameters and haematological profile were measured immediately before and after donation. Adverse events and donors were observed during the procedure and followed daily during 5 days after donation. Hemolysis in RBC was followed until of shelf life (<0.8% on 42 days storage). The evaluation of different parameters during storage show no difference if we compare with the manual technique. The concentration of hemoglobin is good and all ou concentrates are conform. No serious adverse effects were reported during and after donation. All donors confirmed they would agree to donate 2 RBC units again with this system. We have seen a good quality of RBC products. This evaluation indicates that 2 RBC donation is feasible on the ALYX system, comfortable and safe for eligible donors.

The estimation of whole blood viscosity by a porous bed method.

A significant impediment in determining the relative contribution of whole blood viscosity to the pathogenesis of cardiovascular and cerebrovascular disease has been the lack of an uncomplicated method to measure whole blood viscosity. To address this problem, a simplified porous bed viscometer has been developed to measure whole blood viscosity. Whole blood is passed through a porous bed of branching channels with a mean pore diameter of 69.6 +/- 20.2 microns and an estimated mean shear rate of 19.6 seconds-1. The effects of sample collection, sample storage, and temperature are described. The mean whole blood viscosity of 242 healthy persons was 22.7 +/- 5.3 seconds, which, when corrected to centipoise using Darcy's equation, corresponds to an apparent viscosity of 5.7 +/- 1.3 cp. There was a significant difference in the whole blood viscosity of normal men and women related to their different packed cell volumes. Platelets and granulocytes influenced whole blood viscosity in proportion to their contribution to the total packed cell volume. Fibrinogen levels did not significantly influence measured whole blood viscosity, which is consistent with the disaggregating conditions and the mean shear rate of the instrument. The porous bed viscometer is a convenient means to measure whole blood viscosity and it should be useful as a screening test for clinical and epidemiologic studies.

Toxicity of HPA-23 (ammonium-21-tungsto-9-antimoniate) for normal human myeloid progenitor cells (GM-CFU) in vitro.

HPA-23 is a competitive inhibitor of the RNA-dependent DNA polymerase (reverse transcriptase) of the human immunodeficiency virus (HIV). It may therefore potentially benefit patients with HIV infection. This study aimed at defining the haematopoietic toxicity of this drug and particularly its effects on the normal human granulocyte-macrophage progenitor cells (GM-CFU). Our in vitro studies, in semi-solid agar, have shown an inhibitory effect of increasing concentrations of HPA-23 on colony and cluster formation. This effect is probably dose-dependent. An almost complete inhibition of colony formation was observed at doses of more than 20 micrograms/ml. Regarding cluster formation, a similar although much more progressive inhibitory effect was found. Our experimental data should be extrapolated with caution to clinical situations. However, they must be kept in mind for optimal design of HPA-23 therapy in HIV infected patients.

Extracorporeal photopheresis technical aspects.

Extracorporeal photochemotherapy is a monotherapy first developed by Edelson et al. in 1987. It is a therapy in which 8-methoxypsoralen (8-MOP) containing lymphocytes are exposed to a long wavelength ultraviolet radiation (UVA) in an extracorporeal system. The initial design of the treatment was based on the use of the UVAR system (Therakos) for both the collection and photoirradiation of the mononuclear cells. This machine is replaced now by XTS apparatus, which is fully automatic and has integrated the fluid logic module (the "heart" of the system). Another principle is used: the two independent steps technique. It consists in first collection of mononuclear cells by a continuous blood cell separator and secondly irradiation in an independent machine: UV-MATIC irradiator (VILBER LOURMAT). These three techniques are analyzed through numerous parameters: technical, biological, hematological data. In the future, we consider we need to precise our requirements for adequate UVA energy and 8-MOP concentration. We also have to define therapeutic dose of irradiated cells and so standardize the process.

Plateletpheresis concentrates produced with the COMTEC cell separator: the French experience.

The latest generation of cell separators such as Trima (Gambro), Amicus (Baxter) and AS-TEC 204 (Fresenius), allow the collection of leucocyte-reduced platelet concentrates without secondary filtration. Fresenius has recently developed the COMTEC cell separator whose performance has been evaluated by several teams in France. This new cell separator is an improved version of the Fresenius AS-TEC 204 cell separator, designed to allow more efficient platelet collections. This study reports on the experience of six French teams (from Bordeaux, Clermont-Ferrand, Creteil, Dijon, Lille and Nancy) who obtained 696 leucocyte-reduced plateletpheresis concentrates in the course of collection using the new Fresenius COMTEC cell separator. All healthy volunteer donors fulfilled French selection criteria for platelet apheresis. Donors were eligible if they had suitable venous accesses, if their bodyweight was *50 kg and if their pre-apheresis platelet count was >150 x 10(9) l(-1). Between 4606 and 5229 ml of blood were processed. The mean volume of the platelet concentrates was between 439 and 493 ml (mean 460 +/- 63 ml). The platelet yield was of the order of 5.18 +/- 1.02 x 10(11) with only one platelet concentrate below the norm of 2 x 10(11) platelets (0.91 x 10(11)). No plausible explanation for this was found. The residual leucocyte levels conform to current norms. The platelet concentrates contained less than 1 x 10(6) leucocytes per concentrate (mean 0.233 +/- 0.150 x 10(6) leucocytes) in more than 97% of the components produced with >95% statistical confidence. The efficacy of the cell separator (52.44 +/- 7.35%) is comparable to that of other separators. The Fresenius COMTEC cell separator makes it possible to obtain leucocyte-reduced platelet concentrates which comply with current standards both in terms of platelet content and residual leucocyte level.

Evaluation of platelet concentrates with the new Dideco blood cell separator: Excel.

The Excel Cell Separator produced by Dideco represents a good compromise between automation and Platelet efficiency. Platelet activation is a phenomenon that we can find with the concentrates of all cell separators. Although, the complement activation of Excel is extremely lower in comparison with the same data obtained using all the other cell separators, the 5-day storage is acceptable. It will be important to research the bacteriological controls. Like the pH of the platelet bags, because in certain cases it was too low. For the future it will be interesting to study the possibility to predict the quantity of platelets collected having a system that is adaptable with the donor parameters.

Study of parameters involved in specific immunoadsorption of apolipoprotein B.

A therapeutic immunoadsorption system on immobilized and anti-apolipoprotein B as a plasma cholesterol lowering procedure was optimized. Several antibodies were compared and highest adsorption capacity was obtained with goat polyclonal antibodies. Optimum quantities of antibodies to be immobilized on the gel and quantities of apo-B to be applied to columns were determined. The amount of antibodies released from immunoadsorbents can be minimized by treatment with a 0.005% glutaraldehyde solution with an acceptable reduction rate of adsorption capacity. Each phase, adsorption and desorption respectively, were well-defined and synchronized so two columns could be used in parallel in an automated procedure. In these conditions, the immunoadsorption system can efficiently, specifically and safely remove cholesterol and has to be subjected to clinical trials.

[Concentration of platelets from apheresis: methods of preparation]. / Les concentrés de plaquettes d'aphérèse: méthodes de préparation.

Apheresis platelet concentrates are platelet suspension obtained by apheresis from a blood cell separator. These techniques are now 20 years old. They are best way to collect great quantities of platelets from only one donor. These blood cell separators (discontinuous or continuous flow), realise extracorporeal circuit in which the blood in mixed with an anticoagulant (mainly citrate). All the procedures used are automatic and adapted with the donor parameters. The platelet donor is between 18 and 60 years old and can give 5 times per year. The platelet concentration in the product is divided into 3 classes (1 = 2 - 4 x 10(11) - 2 = 4 - 6 x 10(11) - 3 > 6 x 10(11)). Each category has a special price. Actually, we can store these platelets only 5 days. In the future, we need new studies and new directions (additive solution, new plastic) to propose a better viability and a longer storage.

[New automatic test for the study of platelet response to variations in osmotic pressure: application in the evaluation of the viability of platelet concentrates]. / Nouveau test automatique d'étude de la réponse des plaquettes aux variations de pression osmotique: application à l'évaluation de la viabilité des concentrés plaquettaires.

Studying the osmotic resistance or swelling of platelets has often been suggested as a global test to assess the viability of those cells. A number of authors have also analysed the behaviour of platelets in hypotonic media by a variety of complementary methods (cell count, morphology, determinations of substances released, photometric measurement of aggregation induced by aggregating agents, etc). Most studies are currently based on the so-called "osmotic shock response" test, which measures according to time the light transmitted through platelet-rich plasma (PRP) after dilution in distilled water. In this study, the authors describe a new automated and reproducible test using slow dialysis to assess platelet osmotic resistance. The "Fragilimeter", a device initially described by the authors to characterise RBC fragility, has been adapted to the study of platelet osmotic behaviour. The variations in light transmission through a platelet suspension according to NaCl concentration are linked to the change in cellular volume and lysis and characterise the viability of the cells. The results obtained with normal platelets revealed the good reproducibility of the technique. The osmotic resistance is evaluated for two parameters: anticoagulant (citrate, EDTA) and cellular concentration. The test was applied to quality control of stored platelet concentrates for transfusion, prepared with different cell separators.

[Erythrocyte aggregation in patients with monoclonal gammopathies: effect of plasma exchange treatment]. / Agrégation érythrocytaire chez des patients atteints de gammapathies monoclonales: incidence du traitement par échanges plasmatiques.

Analyses of erythrocyte aggregation have been carried out in 15 patients with multiple myeloma or Waldenström's macroglobulinemia before and after plasma exchange. The volume of exchanged plasma was about 1.2-1.5 plasmatic mass. It was replaced by the same volume of a 4% solution of human albumin. Each treatment included at least 3 plasma exchanges at 48 hours intervals. The kinetic (aggregation times) and rheological (dissociation threshold) parameters of erythrocyte aggregation were determined before and immediately after each plasma exchange using a device based on the analysis of the backscattered light by a blood suspension. Before the first plasma exchange, erythrocyte aggregation measurements revealed a pronounced reduction in time aggregation and high values of dissociation thresholds indicating an hyperaggregation of red cells. The successive plasma exchanges produced very different changes in erythrocyte aggregation. For some patients, the aggregation parameters presented normal values from the first plasma exchange when similar changes were only noted after the second plasma exchange in other patients. Finally, in some cases, an hyperaggregation state was maintained after a series of three plasma exchanges. The influence of the nature and the concentration of monoclonal immunoglobulin as well as plasmatic fibrinogen are discussed.

[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.

[Hemorrheological changes in 50 monoclonal dysglobulinemias treated by plasma exchange]. / Variations hémorhéologiques au cours de 50 dysglobulinémies monoclonales traitées par échanges plasmatiques.

Rheological examinations were performed before and after plasma exchanges in 50 patients with monoclonal dysglobulinaemia. The parameters investigated were blood and plasma viscosity, erythrocyte filtrability, erythrocyte aggregation and kinetics of agglutination. The results showed reduction of blood and plasma viscosity and improvement in agglutination kinetics under plasma exchanges. Rheological examinations therefore seem to be useful to follow up blood disorders, notably the hyperviscosity syndrome. They can be performed prophylactically, leading to early plasma exchanges to prevent clinical deterioration.

[Plasma exchange in the treatment of Behcet's disease. Preliminary study apropos of 7 cases]. / L'utilisation des échanges plasmatiques dans le traitement de la maladie de Behçet. Etude préliminaire à propos de 7 observations.

Seven patients with Behcet's syndrome were given plasma exchanges (PE). They all had severe forms, essentially ocular, which had been previously treated by steroids and/or immunosuppressors for a number of years without effect on the course of the disease. The PE was associated with classical medical therapy in all cases. Four patients had daily PE on three consecutive days. Poor results were obtained in all cases with this technique. Five patients had monthly PE and a clinical improvement was observed in 4 cases, usually with a reduction in the number of exacerbations. All therapy failed in one patient who continued to have severe exacerbations. From this short series, PE would appear to be useful in chronic forms of the disease incompletely controlled by medical therapy. In these cases, PE should be repeated. However, a longer follow-up is necessary to confirm these results.

Development and evaluation of an automatic method for the study of platelet osmotic response.

Study of the osmotic resistance to hypotonic medium of platelets has often been suggested as a global test to assess the viability of these cells in transfusion or to study modification during haematological pathologies. A number of authors have analysed the behaviour of platelets in hypotonic media by a variety of methods (cell count, determinations of substances released, morphology, etc.), but most studies are currently based on the so-called "Hypotonic Shock Response" test (HSR). In this study, the authors describe a new automated and reproducible apparatus, called fragilimeter, using slow dialysis to assess platelet osmotic resistance. The variations in light transmission through a platelet suspension according to ionic strength are linked to the change in cellular volume and lysis and characterise the osmotic behaviour of the cells. The results revealed the good reproducibility and sensibility of the technique. This apparatus allows also the realisation of the "HSR" test.

[Cervical Pyoderma gangrenosum: treatment by plasma exchanges]. / Pyoderma gangrenosum cervical: traitement par échanges plasmatiques.

The authors report one case of Pyoderma gangrenosum whose unusual cervical localization was particularly bothering. Besides, the patient, an 18-year-old girl, had clinical symptoms of Crohn's disease confirmed by radiological investigations and bowel pathology. A benign monoclonal gammapathy and an auto-immune haemolytic anaemia were also found. After four plasma exchanges Pyoderma healed rapidly, Crohn's disease stabilized and biological abnormalities disappeared. Comments and review of the literature are reported. It appears that such a case is quite exceptional, as far as clinical, biological data and therapy (plasma exchange) are concerned.

[Erythrocyte concentrates]. / Les concentrés érythrocytaires.

Among the blood products distributed by blood banks red cell concentrates are mostly prescribed, for treating anaemia. We now have a relatively large choice of specially prepared red cell concentrates, which enables us to satisfy more accurately each patient's particular requirements. In order to select the best suited and safest product, the risk associated with the patient's disease as well as the side effects of red cell transfusion should be evaluated.

HTLV-I infection in the South West Indian Ocean islands, particularly in La Réunion and the Seychelles.

Data on HTLV-I are scarce in several Southwest Indian Ocean islands except for La Réunion and The Seychelles. The two cases of HTLV-I have been confirmed by Western-Blot in La Réunion, among blood donors. In Seychelles (87 400 inhabitants in 2012), where blood donors and some other cases are screened, HTLV-I was confirmed with a line immune assay in 43 persons and at least 10-20 patients are known to have tropical spastic paraparesia or adult T-cell lymphoma associated with HTLV-I. In the south-west Indian Ocean, a possibly important other issue may be co-infection of HTLV-1 with the Strongyloides stercoralis roundworm, which is endemic in all countries of the region and which can sometimes lead to severe symptomatic infestation.