Proteomics applied to transfusion plasma: the beginning of the story.

'Safe blood' is and has always been the major concern in transfusion medicine. Plasma can undergo virus inactivation treatments based on physicochemical, photochemical or thermal methodologies for pathogen inactivation. The validation of these treatments is essentially based on clottability assays and clotting factors' titration; however, their impact on plasma proteins at the molecular level has not yet been evaluated. Proteomics appears as particularly adapted to identify, to localize and, consequently, to correlate these modifications to the biological activity change. At the crossroads of biology and analytical sciences, proteomics is the large-scale study of proteins in tissues, physiological fluids or cells at a given moment and in a precise environment. The proteomic strategy is based on a set of methodologies involving separative techniques like mono- and bidimensional gel electrophoresis and chromatography, analytical techniques, especially mass spectrometry, and bioinformatics. Even if plasma has been extensively studied since the very beginning of proteomics, its application to transfusion medicine has just begun. In the first part of this review, we present the principles of proteomics analysis. Then, we propose a state of the art of proteomics applied to plasma analysis. Finally, the use of proteomics for the evaluation of the impact of storage conditions and pathogen inactivation treatments applied to transfusion plasma and for the evaluation of therapeutic protein fractionated is discussed.

[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.

[Ethics, motivations of plasmapheresis donors]. / Ethiques, motivations des donneurs d'aphérèse plasmatique.

For 50 years, the French Blood Transfusion Service has been developing ethical concepts of anonymous, voluntary and non-profit donation. The subject of this work is the study of ethical aspects and motivations of plasmapheresis donors. Three hundred donors were questioned on these subjects. The questionnaire was created after analysing the semi-orientated interviews of ten donors. The donors are male, aged over 35, with relatively high social and professional backgrounds. The main reason given for the first donation is the request by a relative or another person. With regards to further donations, solidarity with patients is mentioned as a reason. Even if the majority of the donors are aware of the ethical aspects of donation, over 50% of them accept to give plasmapheresis regardless of these principles. Plasmapheresis donors are primarily motivated by solidarity reasons. Thereby, they are fully active in the evolution of society.

Chromatographic purification and properties of a therapeutic human protein C concentrate.

Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency.

Programmed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria.

Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.

Improved storage of erythrocytes by prior leukodepletion: flow cytometric evaluation of stored erythrocytes.

In vivo phagocytosis of senescent red blood cells (RBCs) by macrophages occurs 120 days after their release into the circulation. It depends on two sequential signals that trigger phagocytosis: (1) desialylation of membrane glycoconjugates with the exposure of the penultimate beta-galactosyl residues and (2) exposure of phosphatidylserine in the membrane outer leaflet. Leukodepleted and nonleukodepleted RBCs were compared using flow cytometric procedures to determine whether the in vitro deterioration of RBCs during storage might be attributable to an identical mechanism of desialylation induced by leukocyte neuraminidases, resulting in exposure of beta-galactosyl and subsequently phosphatidylserine residues - signals of senescent RBCs. Without prior leukodepletion, stored RBCs showed an increased population of senescent RBCs (using light scatter measurements), extensive desialylation with the exposure of beta-galactosyl residues (using specific fluorescein isothiocyanate [FITC]-lectins), significant exposure of phosphatidylserine in the outer leaflet of the RBC membrane (using FITC-annexin V), and extensive in vitro phagocytosis (using PKH-26-labeled RBCs). There were minimal changes observed with the leukodepleted RBCs. These results lead to the conclusion that leukocyte enzymes, including neuraminidases, are definitive contributers to the desialylation of RBCs during storage and to the exposure of phosphatidylserine residues. These deleterious effects resulting from highly active leukocyte enzymes are preventable by prior leukodepletion of the stored RBCs. Previously developed flow cytometric procedures to detect in vivo "RBC senescence" have been applied and proved to be reliable criteria to monitor the viability of stored RBCs.

[Blood donation. Job description of the function of a physicion in a district subdivision]. / Don du sang. Fiche de définition de fonction d'un médecin d'arrondissement de collecte.

Following the 1995 national reorganization of the transfusion system, the Nord Pas de Calais blood transfusion center has modified its blood collection organization, with the creation of four districts divided into three or four subdivisions. This change was part of Quality Assurance implementation in the center. This article provides the job description for a physician in charge of such a subdivision as well as the method chosen to design this description (inspired by the diagram of Hijman). This job was conceived as an association of human skills knowledge with the strategy of the blood center. In addition to his medical activity, the district subdivision physician becomes a manager and an organizer. The method used highlights the participation of all disciplines involved in blood donation.

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.

[Bacterial contamination of platelet concentrates by Propionibacterium acnes]. / Contamination bactérienne de concentrés de plaquettes à Propionibacterium acnes.

In this study, three incidents of platelet contamination by Proprionibacterium acnes and an investigation of the transfusion process have been reported, which occurred at the Nord-Pas-de-Calais Blood Center over a period of several months. P. acnes is a bacterium that is present in the cutaneous flora; it does not produce any toxin, and is rarely considered as a pathogenic agent; its occurrence is widespread, in particular in those regions that are rich in sebum (face, back, scalp), and it is extremely apparent during adolescence. The three incidents occurred following the transfusion of a pool of leucodepleted platelet concentrates obtained from immunodeficient patients. The clinical outcome was in all cases positive. It was considered that the bacterial contamination of platelet concentrates could reflect insufficient skin disinfection at the site of the venipuncture and a minimal bacterial risk involving the blood collection procedure.

Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review.

Human red blood cells (RBCs) have a life-span of 120 days in circulation, after which they are phagocytized by resident macrophages. Extensive studies have been undertaken by many investigators in order to elucidate the cellular and molecular mechanisms of the erythrophagocytosis. The critical questions addressed by physiologists, clinicians and biochemists are: 'which of the many traumatic blemishes that appear on the erythrocyte surface as it winds its way through the circulation is the primary signal for clearance of the effete RBC from the circulation?', or 'What is the critical signal that it, and it alone, will activate the resident macrophage to adhere to and engulf it?'. Numerous, and often conflicting, hypotheses have been proposed. Each investigator focusing on but one of the many modifications that afflict the cell surface of the ageing erythrocyte, viz changes in either or both the carbohydrate or peptidic moieties of glycoproteins; abolishment of the pre-existing asymmetry in the lipid bilayer with the exposure of phosphatidylserine residues; or alterations in spectrin, to mention but a few. Many of these investigators also have invoked an intermediary role for auto-immune antibodies that recognise the change(s) on the erythrocyte surface and thereby serve as opsonins as a prelude to the erythrophagocytosis. The objective of the present review is to evaluate the data in support of the various hypotheses, and to submit some of our own recent observations involving the use of flow cytometric procedures that: i) provide evidence that the cell surface sialic acid serves as a determinant of the life-span; ii) characterise the senescent erythrocyte population that is specifically captured and phagocytized by macrophages (utilising the rapid and sensitive procedure we developed for quantification of in vitro erythrophagocytosis); and finally iii) provide evidence for the existence of an alternative pathway that is independent of immunoglobulins.

Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages.

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.

Four epitopes on tumor necrosis factor-alpha defined by murine anti-tumor necrosis factor-alpha monoclonal antibodies.

Eight murine anti-TNF alpha monoclonal antibodies (mAb) were produced after immunization of BALB/c mice with rhTNF alpha. Six of these mAbs were able to neutralize cytotoxic activity of TNF alpha against L929 cells. Two other mAbs had no neutralizing effect. Epitope mapping studies were performed by ELISA and by using a BIAcore system (Pharmacia). The described mAbs were allowed to define 4 different epitopes on TNF alpha. Three of them were involved in the binding of TNF alpha with its receptor (cytotoxic neutralization of TNF alpha). Another epitope was defined by non-neutralizing mAbs.

[Techniques of security of plasma transfusion]. / Les techniques de sécurisation du plasma thérapeutique.

Viral inactivation of fresh frozen plasma (FFP) is the main condition of a safe administration in its few remaining indications. Different techniques have been developed such as "quarantine", solvent-detergent plasma treatment, pasteurization or, more recently, photosensitizing agents adjunction. None of these techniques is entirely satisfactory and further clinical studies are needed. Meanwhile, FFP use must be restricted to some well known congenital ou acquired pathologies where it cannot be substituted.

Preparation and properties of a therapeutic inter-alpha-trypsin inhibitor concentrate from human plasma.

Inter-alpha-trypsin inhibitor (ITI) is a serine protease inhibitor found in human plasma. Its antiprotease activity is due to bikunin which is effective in various types of experimental shock and pancreatitis. Therefore ITI, which releases bikunin by proteolytic cleavage, could be of therapeutic interest. A method for the large-scale isolation of ITI from human plasma is described. ITI was purified from the prothrombin complex concentrate (PCC) by diethylaminoethyl-Sepharose fast-flow chromatography followed by a chromatographic step on immobilized heparin designed to remove C4, factor X and protein C. With this procedure, which was performed under mild conditions, a homogeneous preparation of native ITI was obtained, as demonstrated by electrophoretic and chromatographic analyses. ITI maintained its biological activity, as exhibited by its specific antitryptic activity of 420 +/- 65 IU/g. In order to decrease or eliminate the risk of transmission of viral disease due to lipid-enveloped viruses, the process incorporated a solvent-detergent treatment. Animal studies on the final product revealed no adverse side-effects in terms of toxicity, thrombogenicity or hypotension. This preparation appears suitable for therapeutic evaluation in animal experimental models.

[Validation of rheological properties of fibrin glue: Biocol-Human Thrombin]. / Validation des propriétés rhéologiques d'une colle de fibrine: le Biocol-Thrombine Humaine.

Rheological properties of one of the main fibrin glues, "Biocol-Thrombine Humaine", prepared by the Regional Blood Transfusion Center of Lille (France) were measured with a CSL controlled stress rheometer using cone-plates and parallel plates. The parameters followed were the dynamic viscosity of the fibrinogen concentrate, the gelification time, the breaking point in a destructive test and the development of elasticity of the fibrin clots according to time or frequency. The results showed that the fibrinogen concentrate of "Biocol-Thrombine Humaine" has a flow curve near that of Newtonian flow, and that its dynamic viscosity at 20 degrees C is about 100 mPa.s. The gelification time of the fibrinogen-thrombin mixture is very short (under 10 seconds). The breaking point is high, showing a great strength of the fibrin clots. The development of elasticity after mixing is very quick. It is also independent of the frequency shearing. The validation of these rheological tests provides data supporting their use in the assessment of the physical properties of fibrin glue in place of the currently used test based on the gluing of mouse skin.