Search

Background: The quality of red blood cells (RBCs) stored in red cell concentrates (RCCs) is influenced by processing, storage and donor characteristics, and can have a clinical impact on transfused patients. To evaluate RBC properties and their potential impact in a transfusion setting, a simple in vitro-transfusional model has been developed., Materials and Methods: Transfusion was simulated by mixing a washed RBC pool from two male-derived RCCs stored at 4°C with a pool of 15 male-derived fresh frozen plasma (FFP) units, representing the recipient, at a hematocrit (HCT) of 30% ("control" setting) or 5% (alternative model). The mixtures were incubated at 37°C, 5% of CO 2 up to 48 h. Different metabolites, hemolysis and microvesicles (MVs) were quantified at several incubation times and RBC-morphology changes and deformability after incubation. For each model, biological triplicates have been investigated with RCCs at storage days 2 and 43., Results: The 5%-HCT model restored the 2,3-DPG level and maintained the ATP level. Furthermore, glucose consumption and corresponding lactate production were increased in the 5%- vs the 30%-HCT condition. Lower hemolysis was observed with 5%-HCT, but only at day 2. However, morphological analysis by digital holographic microscopy (DHM) revealed a decreased fraction of discocytes at 5% rather than at 30% of HCT at storage day 2 but at day 43, the trend was inverted. Concordantly, RBCs incubated at 5% of HCT were more deformable than at 30% at day 43 (p<0.0001)., Discussion: Higher metabolic activity of RBCs in the 5%-HCT condition was promoted by a higher glucose availability and limited cell-waste accumulation. The conditions of the new proposed model thus enabled rejuvenation of RBCs and maintained them in a physiological-close state in contrast to the 30%-HCT model. It may be used as a first approach to evaluate e.g., the impact of donor and recipient characteristics on RBC properties.

Glycol ethers are solvents used in a plethora of occupational and household products exposing the users to potential toxic effects. Several glycol ethers derived from ethylene glycol induce hematological toxicity, such as anemia in workers. The exposure effects on blood cells of glycol ethers derived from propylene glycol are unknown in humans. The aim of our study was to evaluate blood parameters indicative of red blood cell (RBC) hemolysis and oxidative stress in participants exposed to propylene glycol (propylene glycol monobutyl ether (PGBE) and propylene glycol monomethyl ether (PGME)), two extensively used propylene glycol derivatives worldwide. Seventeen participants were exposed 2 h in a control inhalation exposure chamber to low PGME (35 ppm) and PGBE (15 ppm) air concentrations. Blood was regularly collected before, during (15, 30, 60, and 120 min), and 60 min after exposure for RBC and oxidative stress analyses. Urine was also collected for clinical effects related to hemolysis. Under the study conditions, our results showed that the blood parameters such as RBCs, hemoglobin concentration, and white blood cells tended to increase in response to PGME and PGBE exposures. These results raise questions about the possible effects in people regularly exposed to higher concentrations, such as workers., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average ( n = 3), as well as from microvesicles ( n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), α-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas α-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) ( p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBCs.

Background and Objectives: Red blood cells (RBCs) suffer from lesions during cold storage, depending in part on their ability to counterbalance oxidative stress by activating their antioxidant defence. The aim of this study was to monitor the antioxidant power (AOP) in erythrocyte concentrates (ECs) during cold storage., Materials and Methods: Six ECs were prepared in saline-adenine-glucose-mannitol (SAGM) additive solution and followed during 43 days. The AOP was quantified electrochemically using disposable electrode strips and compared with results obtained from a colorimetric assay. Haematological data, data on haemolysis and the extracellular concentration of uric acid were also recorded. Additionally, a kinetic model was developed to extract quantitative kinetic data on the AOP behaviour., Results: The AOP of total ECs and their extracellular samples attained a maximum after 1 week of storage prior to decaying and reaching a plateau, as shown by the electrochemical measurements. The observed trend was confirmed with a colorimetric assay. Uric acid had a major contribution to the extracellular AOP. Interestingly, the AOP and uric acid levels were linked to the sex of the donors., Conclusion: The marked increase in AOP during the first week of storage suggests that RBCs are impacted early by the modification of their environment. The AOP behaviour reflects the changes in metabolism activity following the adjustment of the extracellular uric acid level. Knowing the origin, interdonor variability and the effects of the AOP on the RBCs could be beneficial for the storage quality, which will have to be further studied., (© 2017 International Society of Blood Transfusion.)

Purpose: Erythrocyte concentrates (ECs) represent the most transfused labile blood products. They are stored at 4°C in additive solutions for up to 56 days. Protein oxidation is a marker of oxidative stress and cysteine residues, whose oxidations are required for physiological cell functions, are highly prone to such modification., Experimental Design: Five ECs from independent donations were followed. Soluble protein extracts were prepared at days 6, 27, and 41, and cysteines were alkylated, reduced, and labeled with infrared dyes. Samples were mixed two by two (day 6 as reference) and analyzed by 2D-DIGE. Detection of labeled cysteines allows quantitative comparison of oxidative status. Spots of interest were analyzed by proteomics., Results: Thirty-two spots containing 43 proteins were classified as increasing, decreasing, or exhibiting a peak of expression during storage. Proteins having catalytic and antioxidant activities were particularly affected during storage, for example, peroxiredoxin-1 and DJ-1 were reversibly oxidized and catalase was irreversibly oxidized. These proteins could be used to evaluate different storage strategies to maintain proper protein function during the overall storage period., Conclusions and Clinical Relevance: This redox-DIGE approach brings new quantitative data on oxidized proteins in stored red blood cells. As previously reported on carbonylation, the oxidative damages differently affect protein functions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Transfusion of red blood cells (RBCs) is a daily medical procedure. Erythrocyte concentrates (ECs) can be stored up to 56 days at 4 °C in saline additive solution mainly composed of adenine and sugar. Such nonphysiological conditions induce the occurrence of storage lesions, such as alterations of metabolism, protein oxidation, and deterioration of rheological properties. Their accumulation tends to decrease the main EC therapeutic property, that is, the oxygenation capacity. Protein carbonylation is a marker of oxidative stress and aging, and its occurrence during RBC storage was earlier characterized as a time-dependent and cellular compartment dependent modification., Experimental Design: Three ECs from independent donations were followed. The carbolynome was here characterized in soluble and membrane extracts (n-dodecyl β-D-maltoside-based extraction buffer) of RBCs stored for 6, 27, and 41 days, through biotin hydrazide derivatization, biotin-avidin affinity purification, SDS-PAGE separation, and LC-MS/MS analyses., Results: A total of 142 and 20 proteins were identified as carbonylated in soluble and membrane extracts, respectively. Particularly, a time-dependent evolution of 26.8% of the soluble carbonylome was observed., Conclusions and Clinical Relevance: Affected cellular mechanisms involve antioxidant defenses, metabolism pathways, and proteasomal degradation. To better store RBCs those functions have to be preserved, which opens new routes of investigation in transfusion medicine., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Microvesicles (MVs), or microparticles, are a complex, dynamic and functional part of cells. Red blood cell (RBC)-derived MVs are naturally produced in vivo (during normal aging processes or in several diseases) as well as ex vivo during cold storage of RBCs, or in vitro by ATP depletion or treatment with Ca(2+) and calcium ionophore. All these MVs are equivalently classified according to their size and/or surface markers. Nevertheless, their content in proteins can differ and a few differences in terms of lipid raft proteins, notably stomatin and flotillin-2, have been reported. Based on two-dimensional gel electrophoreses, the present study highlights the differences between MVs induced during storage of RBCs (storage-MVs) and MVs stimulated by Ca(2+) entry (Ca-MVs). Upon treatment, Ca-MVs are formed following a clear recruitment of Ca(2+)-binding proteins (sorcin, grancalcin, PDCD6) and particularly annexins (4 and 5). Therefore, it emerges that different molecular pathways are available to produce similar MVs by disturbing the membrane/cytoskeleton interactions. Interestingly, these differences provide non-negligible pieces of information on the parent cells, and the mechanisms and modes of actions involved in the formation of MVs. In addition to biophysical characterization, protein analysis is important to classify these cellular corpuscles and evaluate their potential impacts in diseases or transfusion medicine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Red blood cell-derived microparticles (RMPs) are small phospholipid vesicles shed from RBCs in blood units, where they accumulate during storage. Because microparticles are bioactive, it could be suggested that RMPs are mediators of posttransfusion complications or, on the contrary, constitute a potential hemostatic agent., Study Design and Methods: This study was performed to establish the impact on coagulation of RMPs isolated from blood units. Using calibrated automated thrombography, we investigated whether RMPs affect thrombin generation (TG) in plasma., Results: We found that RMPs were not only able to increase TG in plasma in the presence of a low exogenous tissue factor (TF) concentration, but also to initiate TG in plasma in absence of exogenous TF. TG induced by RMPs in the absence of exogenous TF was neither affected by the presence of blocking anti-TF nor by the absence of Factor (F)VII. It was significantly reduced in plasma deficient in FVIII or F IX and abolished in FII-, FV-, FX-, or FXI-deficient plasma. TG was also totally abolished when anti-XI 01A6 was added in the sample. Finally, neither Western blotting, flow cytometry, nor immunogold labeling allowed the detection of traces of TF antigen. In addition, RMPs did not comprise polyphosphate, an important modulator of coagulation., Conclusions: Taken together, our data show that RMPs have FXI-dependent procoagulant properties and are able to initiate and propagate TG. The anionic surface of RMPs might be the site of FXI-mediated TG amplification and intrinsic tenase and prothrombinase complex assembly., (© 2012 American Association of Blood Banks.)

Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins-which modulate proteasome activity, stability, localization, or substrate uptake-rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.

In the past decades, transfusion medicine has been driven by the quest for increased safety against transfusion-transmitted infections, mainly by better donor selection and by the development of improved serological and nucleic-acid-based screening assays. Recently, pathogen reduction technologies became available and started to be implemented in several countries, with the primary goal to fight against bacterial contamination of blood products, a rare but dramatic event against which there was no definitive measure. Though pathogen reduction technologies represent a quantum leap in transfusion safety, the biomedical efficacy of platelet concentrates (PCs) treated with various pathogen reduction techniques has been recently questioned by clinical studies. Here, a gel-based proteomic analysis of PCs (n=5), Intercept-treated or untreated, from pooled buffy-coat (10 donors per PC) at Days 1, 2 and 8, shows that the Intercept process that is the most widespread pathogen reduction technique to date, has relatively low impact on the proteome of treated platelets: the process induces modifications of DJ-1 protein, glutaredoxin 5, and G(i)alpha 2 protein. As for the impact of storage, chloride intracellular channel protein 4 (CLIC4) and actin increased independently of Intercept treatment during storage. Whereas alteration of the DJ-1 protein and glutaredoxin 5 points out an oxidative stress-associated lesion, modification of G(i)alpha2 directly connects a possible Intercept-associated lesion to haemostatic properties of Intercept-treated platelets. This article is part of a Special Issue entitled: Integrated omics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

During blood banking, erythrocytes undergo storage lesions, altering or degrading their metabolism, rheological properties, and protein content. Carbonylation is a hallmark of protein oxidative lesions, thus of red blood cell oxidative stress. In order to improve global erythrocyte protein carbonylation assessment, subcellular fractionation has been established, allowing us to work on four different protein populations, namely soluble hemoglobin, hemoglobin-depleted soluble fraction, integral membrane and cytoskeleton membrane protein fractions. Carbonylation in erythrocyte-derived microparticles has also been investigated. Carbonylated proteins were derivatized with 2,4-dinitrophenylhydrazine (2,4-DNPH) and quantified by western blot analyses. In particular, carbonylation in the cytoskeletal membrane fraction increased remarkably between day 29 and day 43 (P<0.01). Moreover, protein carbonylation within microparticles released during storage showed a two-fold increase along the storage period (P<0.01). As a result, carbonylation of cytoplasmic and membrane protein fractions differs along storage, and the present study allows explaining two distinct steps in global erythrocyte protein carbonylation evolution during blood banking. This article is part of a Special Issue entitled: Integrated omics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Summary: Microparticles are small phospholipid vesicles of less than 1 µm released into the blood flow by various types of cells such as endothelial, platelet, white or red blood cells. They are involved in many biological and physiological processes including hemostasis. In addition, an elevated number of microparticles in the blood is observed in various pathological situations. In the context of transfusion, erythrocyte-derived microparticles are found in red blood cell concentrates. Their role is not elucidated, and they are considered as a type of storage lesion. The purpose of this review is to present recent data showing that erythrocyte-derived microparticles most likely play a role in transfusion medicine and could cause transfusion complications.

Background: The storage of blood induces the formation of erythrocytes-derived microparticles. Their pathogenic role in blood transfusion is not known so far, especially the risk to trigger alloantibody production in the recipient. This work aims to study the expression of clinically significant blood group antigens on the surface of red blood cells microparticles., Material and Methods: Red blood cells contained in erythrocyte concentrates were stained with specific antibodies directed against blood group antigens and routinely used in immunohematology practice. After inducing erythrocytes vesiculation with calcium ionophore, the presence of blood group antigens was analysed by flow cytometry., Results: The expression of several blood group antigens from the RH, KEL, JK, FY, MNS, LE and LU systems was detected on erythrocyte microparticles. The presence of M (MNS1), N (MNS2) and s (MNS4) antigens could not be demonstrated by flow cytometry, despite that glycophorin A and B were identified on microparticles using anti-CD235a and anti-MNS3., Discussion: We conclude that blood group antigens are localized on erythrocytes-derived microparticles and probably keep their immunogenicity because of their capacity to bind specific antibody. Selective segregation process during vesiculation or their ability to elicit an immune response in vivo has to be tested by further studies.

Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking.

Cell proliferation and differentiation are under the control of cytokines and growth factors. Different signaling pathways are involved in the transmission of a specific signal through successive phosphorylation and dephosphorylation of proteins leading to gene transcription necessary for growth and differentiation. The cytokines and growth factors activate the Stat family of transcription factors. The Jak-Stat pathway is essential for cytokine signal transduction. Dysregulation of this cascade might lead to uncontrolled hematopoiesis. Studies have been carried out to examine the functionality of this pathway in cells from patients with acute leukemia. Members of the Stat protein family (Stat1, Stat3 and Stat5) are constitutively activated in cells collected from some acute leukemias suggesting dysregulation of the Jak-Stat pathway. Evidence of the existence of constitutively activated spliced variants of Stat3 and Stat5 proteins are described. The mechanisms of such activation remain to be clarified.

A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.

Four spuriously lowered WBC counts due to in vitro leukoagglutination were reported from an automated cell counter (Coulter STKS). Leukocyte aggregates (3 to 50 cells), detected in the peripheral blood smears, included different cell types, normal (neutrophils, eosinophils, monocytes, lymphocytes) or abnormal (lymphoma cells). The phenomenon was associated with either a spurious leukoneutropenia or an underestimation of hyperleucocytosis. Leukoagglutination was extensively investigated in 3 cases : as shown in several reports, leukoagglutination may occur with various features, especially due to temperature and anticoagulant dependence. Our four cases reflected this variability. Furthermore, one case was found both temperature-dependent and anticoagulant-independent, a pattern not yet described in the literature. A common STKS graphic pattern was found in our 4 cases, suggesting that hematology analyzers such as Coulter STKS may be useful to detect leukoagglutination. In conclusion, each leukoneutropenia and/or each suggestive graphic pattern must be controlled by means of a blood smear examination in order to rule out the possibility of in vitro leukoagglutination.

The morphological profiles of red blood cells (RBC) and platelets (Plt) derived from the distribution analysis given by the Coulter STKS were evaluated in two groups of patients suffering from non hematological (n = 293) and hematological (n = 257) conditions. The RBC and Plt flags were studied in terms of sensitivity of the morphological analysis and specificity and significance of each flag. When all RBC and Plt flags were considered, the percentages of false negatives and false positives in our subjects were found to be 4.7% and 13.4% respectively, with a global efficiency of 81.8%. The sensitivity of the alarm system was higher than 90% for all types of abnormality, except microcytosis (81%) and Howell-Jolly bodies (57% over a limited number of 7 cases). The specificity of the STKS response was found to be low except for anisocytosis (88.5%) and macrocytosis (86.1%). It was shown that the flags microcytosis and/or hypochromia and macrocytosis were poorly significant when they appeared in isolation (false positive rates of 86.6% and 84.2% respectively). Thus, these alarms could be eliminated from the review criteria. When considering only the flags anisocytosis, NRBCs, micro RBCs/RBC fragments, dimorphic RBC pop, PLT clumps and giant Plt, the percentage of false positives was 8.1%. However, it must be kept in mind that suspect leucocyte flags remained review criteria, resulting in a final false negative rate of 2.5% for RBC and Plt morphological abnormalities.

A 36-year-old patient was investigated for a lifelong history of epistaxis and delayed bleeding after minor surgeries. Deficiencies or abnormalities of the coagulation system, of platelet function, or of factor XIII and alpha-2-antiplasmin were excluded. Consistently, however, over a period of 7 years, a high basal euglobulin fibrinolytic activity was observed that was characterized by a high tissue-type plasminogen activator (t-PA) activity, normal t-PA antigen, and undetectable plasminogen activator inhibitor type-1 (PAI-1) antigen and activity. The high specific activity of t-PA (640,000 IU/mg) and the minimal amounts of t-PA/PAI-1 complexes detected by fibrin zymography suggest that in this patient all t-PA was active. This is in striking contrast to normal plasma, where the majority of t-PA is complexed to PAI-1. Thus, in this patient, a severe deficiency of PAI-1 is associated with a delayed type bleeding tendency. Our observation underscores the importance of plasma PAI-1 for the stabilization of the hemostatic plug.

A family with an antithrombin III variant (AT-III-Amiens) demonstrating abnormal heparin cofactor activity is described. Amplification and direct sequencing of genomic DNA by the polymerase chain reaction procedure permitted the identification of an Arg47----Cys mutation in exon 2 of the variant antithrombin III gene.

The hemorrhagic risk of an association of the low molecular weight (LMWH), Fraxiparine injected intravenously at the dose of 7.500 AXalCU or of unfractionated heparin (UFH) injected intravenously at the usual dose used during hemodialysis (3.750 +/- 1.280 IU + 1.000 IU after 2 hours of dialysis) to the subcutaneous administration once daily of a thromboembolism preventive dose of Fraxiparine (7.500 AXalCU) was evaluated on the modification of the following hemostasis parameters: thrombin time, activated partial thromboplastin time (APTT), anti Xa activity, in 13 uremic patients on hemodialysis. The association of intravenous and subcutaneous Fraxiparine prevented efficiently the clotting of the extracorporeal circulation without inducing a detectable antithrombinic activity. In contrast, the association of I.V. UFH to subcutaneous Fraxiparine induced a significant increase of the thrombin time and of the APTT, so explained by the activity of UFH. It is concluded that subcutaneous Fraxiparine at the thromboembolism preventive dose can be associated as well to I.V. Fraxiparine as to UFH without increasing the potential hemorrhagic risk. Nevertheless the association of SC and IV Fraxiparine 7.500 AXalC u seems preferable to the association of SC Fraxiparine with UFH.

The patients with inherited bleeding diathesis related to quantitative, structural, and/or functional abnormalities of von Willebrand factor (vWF) are said to have von Willebrand's disease (vWD). We report here the clinical and laboratory features of a 50-year-old woman with a life-long history of excessive bleeding. Her particular laboratory data are factor VIII (FVIII) deficiency, subnormal bleeding time, and the presence of all plasma and platelet vWF multimers in normal amounts. Infused with FVIII/vWF concentrate, she showed a persistent increase in FVIII that led us to discard hemophilia A carrier or "acquired hemophilia" diagnoses. vWF devoid of FVIII purified from normal and patient's plasma by immunoaffinity on anti-vWF monoclonal antibody (MoAb) was immobilized onto polystyrene tubes that were further incubated with purified normal FVIII. The bound FVIII was evidenced using radiolabeled anti-FVIII MoAb. The data showed that the patient's vWF, in contrast to vWF purified from normal plasma, was unable to bind FVIII. Furthermore, no inhibitor of FVIII/vWF interaction was evidenced in incubating purified normal vWF with the patient's plasma before the addition of FVIII and anti-FVIII MoAb. These results support the concept that the bleeding diathesis of this patient appears to be due mainly to her abnormal vWF preventing FVIII/vWF interaction. This abnormality, which is not yet described in present classification of vWD, could be considered as a new variant of vWD.

The hemorrhagic risk of an association of fractioned heparin (Fraxiparine) injected intravenously at the dose of 7500 AXaICU or of unfractionned heparin (UFH) injected intravenously at the usual dose used for a priming dose for hemodialysis (3750 +/- 1280 IU + 1000 IU after 2 hours of dialysis) to the subcutaneous administration of a thrombo-embolism preventive dose of Fraxiparine (7500 AXaICU) was evaluated on the modifications of the following hemostasis parameters: thrombin time, Activated Partial Thrombin Time (APTT), prothrombin time, anti Xa activity in 13 uremic patients on hemodialysis. The association of intravenous and subcutaneous Fraxiparine prevents efficiently the clotting of the extracorporeal circulation without inducing a detectable antithrombinic activity. In contrast, the association of I.V. UFH to subcutaneous Fraxiparine induces a significant increase of the thrombin time and of the APTT. This latter is exclusively explained by the activity of UFH. It is concluded that subcutaneous Fraxiparine at the thromboembolism preventive dose can be associated as well to I.V. Fraxiparine as to UFH without increasing the antithrombinic activity of the plasma.

Severe pulmonary embolism with thrombosis of the inferior vena cava was observed in a 16 year old girl with no risk factors and treated successfully by fibrinolytic therapy. Secondarily, despite heparino-therapy, upper limb venous thrombosis occurred. Investigation of the clotting factors in the patient and her family revealed a hereditary deficit of antithrombin III. The features of the haemotological diagnosis of this rare condition and the therapeutic implications are discussed.

A case of hereditary autosomal recessive thrombocytopenia is reported. Thrombocytopathy is associated with the thrombocytopenia. There is a contrast between the cytologic aspect of poor bone-marrow without any megakaryocyte and the histologic aspect of dense bone-marrow with a normal number of megakaryocytes. Myelofibrosis can explain this discrepancy. The life time of platelets being just a little shortened, the disease is probably due to a lack of bone-marrow production.

We report the case of a 66-year-old male patient with portal hypertension related to systemic mastocytosis. The liver was enlarged; microscopic examination showed portal mast cell infiltration and fibrosis. Portal hypertension was evidenced by splenomegaly, esophageal varices, and increased wedged-free hepatic venous pressure gradient. Arteriography showed that portal vein was patent. Portal hypertension could be the consequence of intrahepatic block due to mast cell infiltration and/or fibrosis of the liver.

Mixed cryoglobulinemia are divided in two types: type II is the association of polyclonal IgG and a monoclonal IgM and type III is the association of polyclonal IgG and polyclonal IgM. In 70 p. 100 of cryoglobulinemia, a cause may be found (hematologic, auto immune disorder, infections, hepatic disorders), in some cases the recognition of the cause is delayed. In 30 p. 100 of cases, no cause can be identified and the cryoglobulinemia is considered as essential. Main clinical finding are purpuric skin rash, urticaria, arthralgia, motor sensitive polyneuropathy, diffuse proliferative glomerulo nephritis. Biological signs associate anemia, rheumatoïd factor and hypocomplementemia. Mixed essential cryoglobulinemia (the Meltzer and Franklin syndrome) is characterized by a systemic vasculitis involving small and middle size vessels.

Efficacy of CY 222 for providing anticoagulation during hemodialysis was evaluated in three successive trials by rating quality of blood restitution (degree of coagulum formation in extracorporeal circulation) and by assay of fibrinopeptide A. Its safety was assessed by measurement of manual compression time necessary to ensure hemostasis of puncture points at end of session. Details of the first preliminary study were: 60 sessions in 11 chronic uremia patients; CY 222: 75, 150 and 300 A-Xa IC U/kg + 1,000 A-Xa IC U/h, then 150 and 300 A-Xa IC U/kg without continuous injection, compared with standard heparin (SH) at the usual dosage for each patient (60 +/- 13 IU/kg). Results showed CY 222 at 150 U/kg + 1,000 U/h to possess the same efficacy as SH and to give a shorter compression test time: for 150 U/kg the efficacy was satisfactory, although less than with SH, and compression times were shorter (interest in patients at risk of hemorrhage). For 300 U/kg, efficacy was superior (improved restitution and lower FPA level at end of seance: 6 ng/ml instead of 15 ng/ml.p less than 0.002) and compression times were identical. The second study to evaluate optimal dosage of CY 222 in chronic hemodialysis (CHD) involved: 10 patients; CY 222: 200 A-Xa IC U/kg and 250 A-Xa IC U/kg by single-dose injection. Results failed to demonstrate any significant difference in evolution of FPA levels, but restitution was better at 250 U/kg. In addition, investigation of the effect of rinsing of ECC with standard heparin before the session showed that its suppression did not alter effectiveness but improved tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Following the occurrence in two siblings of parvovirus B19-related acute transient erythroblastopenia with specific IgMs, hereditary spherocytosis (HS) was diagnosed for the first time in the two patients as well as in three other members of the family.

The low molecular weight heparin CY 222 (CHOAY) has been compared to unfractioned heparin (UFH) in patients on chronic haemodialysis and haemofiltration at various doses as regards it biological activity (measured by Activated Partial Thromboplastin Time and by anti-Xa activity) and its clinical effect on clot formation in the blood lines and bleeding at the puncture sites or recent wounds. Compared to UFH, CY 222 has a greater anti-Xa activity for a shorter APTT. This biological difference is of clinical advantage since clotting in lines is comparable or less than with UFH whereas compression time at puncture sites is shorter and recently bleeding wounds in 28 patients did not bleed again. The long half life of CY 222 allows its use as a single priming dose of 300 anti-Xa U/kg in haemodialysis and 450 anti-Xa U/kg in haemofiltration.

The filterability of total blood and erythrocytes has been investigated in 36 children from 2 to 14 years old by the filtration method developed by Reid. Red blood cells being obtained by centrifugation, were washed three times and resuspended in saline. Filterability was recorded as the filtration time of 1 ml of total blood or red cells suspension. The filtration time of total blood was: 49.10 +/- 14.5 s/ml; that of resuspended cells was: 18.19 +/- 6.16 s/ml. These results have been correlated with age, fibrinogen, white blood cells, glycaemia, haematocrit, and compared with the results obtained in 42 adults whose filtration times were: 33.04 +/- 7.40; and red cells' time: 13.99 +/- 2.95. Statistical analysis shows that the difference is very significant and not correlated with the investigated factors. Erythrocyte filterability may be influenced by still unknown factor.

Hereditary spherocytosis and beta thalassaemia are rarely inherited together. We have studied a large family of Caucasian extraction in whom these two diseases segregate independently over four generations. The diagnosis rested on specialized laboratory findings and in a number of subjects on the measurement of alpha and beta globin chain synthesis. In addition, a viscometric method (osmotic gradient ektacytometry) was used to evaluate the rheological function of the erythrocytes. In patients inheriting both diseases, the results indicate that the clinical and biological expression of hereditary spherocytosis is modulated by the degree of imbalanced globin chain synthesis. The opposite properties of spherocytes (decreased surface/volume ratio, increased haemoglobin concentration) and thalassaemic red cells (increased surface/volume ratio, decreased haemoglobin concentration) may explain the antagonistic influence of each genotype.

Acute hypercalcaemia was observed in a 54 year old woman followed up over a five year period for a lymphocytic non-Hodgkin lymphoma, a complication which is rare in chronic lymphoproliferative disorders except multiple myeloma. This case is of interest from 3 points of view: haematological: the simultaneous occurrence of hypercalcaemia (4.80 mmol/l) and of a leukaemic phase (19,300 lymphocytes/mm3) with no signs of "transformation" of the haemopathy; physiopathological: increased osteoclastic bone resorption and a rise in serum parathormone (0.21 to 4.82 ng/ml); therapeutic: efficacy of oral ethane 1, hydroxy 1, 1 diphosphonate (20 mg/kg/day) in treating the hypercalcaemia.

Platelets functions are very often impaired in myeloproliferative disorders and acute leukemias. The abnormalities may either results from plasmatic factor affecting the platelet function or from an intrinsic platelet defect. The mechanism of the platelet defect is still a subject to discussion: it may be an abnormal response of the platelet to the aggregating agent or an impaired release of nucleotides.

Current evaluation of biological activity of low molecular weight heparins (LMWH) is dependent solely on technics determining Xa inhibition. Comparison of these technics was carried out during 2 studies of the use of CY 222 during hemodialysis. In the first study, 66 plasma samples from 11 patients treated with 200 or 250 U A Xa IC kg/i.v. at start of session (sample collections at 2-4 h) were tested using a chronometric technic (Hepaclot Stago-plasma diluted to 1/3) and 2 chromogenic technics on microplates using C.B.S. 31-39 substrates (Stachrom-modified Stago: incubation time 90" for a wide range) and S 2222 (Coatest-modified Kabi: incubation time 180"). In the second, 28 plasma samples from 7 patients (sample collection 2-4 h, TO following session; anticoagulation CY 222 10,000-15,000-20,000 U A Xa IC) were studied by 2 chronometric methods: Hepaclot (Stago) and Heptest (Hemachem). Standardization was with CY 222 in each case (results expressed as U A Xa IC). Mean blood heparin in the first study was 2.39 +/- 0.7 with Hepaclot, 2.50 +/- 0.55 with Stachrom and 1.94 +/- 0.37 with Coatest. Student's test failed to show any difference between results with Strachrom and Hepaclot (t = 1.48 NS) whereas the difference was very significant between Hepaclot and Coatest and Stachrom and Coatest (p less than 10(-9).(ABSTRACT TRUNCATED AT 250 WORDS)

Autosomal dominant hemorrhagic disorder, Willebrand's disease causes hemorrhagic complications during post-partum period. Nevertheless our case-report as well as others's have shown that these complications only occur when there is no spontaneous biologic improvement and can be prevented by prophylactic treatment with cryoprecipitates or anti-hemophilic A fractions. This risk requires thus a regular watch of the biologic aspects during pregnancy and before the delivery date in order to decide of a possible treatment, which will be absolutely necessary in case of obstetrical surgery.

Adult Still's disease is a newly described disorder that is of interest not only for the rheumatologist but also for the internist since fever or extra-articular symptoms can reveal this disorder, which appears to be identical to the Wissler-Fanconi's syndrome. We report a new case characterized by the occurrence of a constrictive pericarditis with clinical features of protracted fever, by the late appearance of a Sjögren's syndrome and by peculiar blood abnormalities: hyperleucocytosis up to 80 000 after splenectomy, transient Felty's syndrome, blood monocytosis and bone marrow mastocytosis.