[Review: delta-storage pool disease]. / Mise au point : la maladie du pool vide plaquettaire.

Platelet storage pool disease is a qualitative platelet disorder associated with variable degrees of reduction in the numbers and contents of dense granules (delta-granules). Electron microscopy is the major tool for biological diagnosis. Patients presenting with this platelet disorder generally show a mild bleeding syndrome.

Ultrastructural and anti-proteinase activity modifications of human alpha 2-macroglobulin induced by zinc and other divalent cations.

Native tetrameric alpha 2-macroglobulin molecules (alpha 2M) can be converted into a population of dimers by incubation with various divalent cations such as Zn, Cd, Mg, Cu, Ni, Co. This dissociation is completed within 30 min at 37 degrees C. These dimers have a characteristic shape and a size of about 16 X 8 nm, and appear to be the half of the native alpha 2M molecule which has a clear tetrameric structure as seen in the electron microscope. At room temperature or below, dimers obtained with 5 to 100 mM Zn++ can reassociate in long linear polymers which display a regular chain-like arrangement and a helical periodicity. The structural characteristics of this polymer are described. The trypsin inhibitory capacity of Zn++-treated alpha 2M has been studied in an attempt to correlate its Zn++-induced conformational changes with its functional modifications.

Modulation of human endothelial cell proliferation and migration by fucoidan and heparin.

Fucoidan is a sulfated polysaccharide extracted from brown seaweeds. It has anticoagulant and antithrombotic properties and inhibits, as well as heparin, vascular smooth muscle cell growth. In this study, we investigated, in the presence of serum and human recombinant growth factors, the effects of fucoidan and heparin on the growth and migration of human umbilical vein endothelial cells (HUVEC) in culture. We found that fucoidan stimulated fetal bovine serum-induced HUVEC proliferation, whereas heparin inhibited it. In the presence of fibroblast growth factor-1 (FGF-1), both fucoidan and heparin potentiated HUVEC growth. In contrast, fucoidan and heparin inhibited HUVEC proliferation induced by FGF-2, but did not influence the mitogenic activity of vascular endothelial growth factor (VEGF). In the in vitro migration assay from a denuded area of confluent cells, the two sulfated polysaccharides markedly enhanced the migration of endothelial cells in the presence of FGF-1. Finally, a weak inhibitory effect on cell migration was found only with the two polysaccharides at high concentrations (> or = 100 micro/ml) in presence of serum or combined with FGF-2. All together, the results indicated that heparin and fucoidan can be used as tools to further investigate the cellular mechanisms regulating the proliferation and migration of human vascular cells. Moreover, the data already suggest a potential role of fucoidan as a new therapeutic agent of vegetal origin in the vascular endothelium wound repair.

Respective roles of antithrombin III and alpha 2 macroglobulin in thrombin inactivation.

In order to investigate the mechanism of thrombin inactivation in the presence of both antithrombin III (AT III) and alpha 2-macroglobulin (alpha 2 M), thrombin and the inhibitors have been purified from human material and thrombin inactivation studied using purified reagents either alone or added to defibrinated plasma. Comparison of clotting and amidolytic activities of residual thrombin allowed to measure the amount of thrombin bound to alpha 2 M. In a purified reagent system as well as in plasma, part of exogenous thrombin is bound to alpha 2 M. The amount of bound thrombin is related to alpha 2 M concentration. Conversely, previous plasma alpha 2 M depletion by immunoabsorption increases the consumption of heparin-cofactor activity by exogenous thrombi. Thus AT III and alpha 2 M compete for thrombin inactivation. This finding could be of practical interest in clinical situations associating high plasma alpha 2 M levels and a decrease of AT III concentrations.

Fucoidan, as heparin, induces tissue factor pathway inhibitor release from cultured human endothelial cells.

Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, has antithrombotic properties, the mechanism of which is not yet completely understood. Tissue factor pathway inhibitor (TFPI), which regulates the tissue factor-dependent pathway of blood coagulation, is released from the endothelium by heparin, a mechanism contributing to its antithrombotic activity. In this study, we demonstrated that fucoidan, as heparin, induces TFPI release from cultured human umbilical vein endothelial cells (HUVEC). The TFPI accumulation in the HUVEC supernatants depends on the incubation time and polysaccharide concentration. After 30 to 60 minutes of incubation, TFPI concentration (total antigen level) was twice higher in the presence of both polysaccharides than in their absence. After one hour of incubation, in the presence of increasing concentrations of each polysaccharide, an optimal stimulation was observed for 0.5 microg/ml of fucoidan and 5 microg/ml of heparin, as evidenced by a raise of the basal TFPI level: a 2-fold increase for the total antigen and a 3-fold increase for the free antigen. These data suggest that TFPI released from vascular endothelial cells may contribute to the antithrombotic effect of fucoidan.

Venous antithrombotic and anticoagulant activities of a fucoïdan fraction.

Fucoïdans catalyse thrombin inhibition by antithrombin (AT) and heparin cofactor II (HCII); their affinity for each serpin varies according to the seaweed species from which they are extracted, as well as their chemical composition and molecular weight. We extracted a homogeneous fucoïdan fraction from Ascophyllum nodosum, a brown seaweed, and tested its anticoagulant and antithrombotic activities. At a fucoïdan concentration of 3.75 micrograms/ml, thrombin inhibition mediated by AT showed an apparent second-order rate constant (kapp) of 2 x 10(8) M-1 min-1, compared to 1.5 x 10(6) M-1 min-1 for the uncatalyzed reaction. The kapp value of thrombin inhibition via HCII was 1.17 x 10(9) M-1 min-1 at a fucoïdan concentration of 50 micrograms/ml, compared to 1.72 x 10(5) M-1 min-1 for the uncatalyzed reaction. In a Wessler model of venous thrombosis, the fucoïdan fraction, injected intravenously to rabbits 10 min before thrombosis induction, exhibited antithrombotic activity: 1.8 mg/kg was the dose which inhibited F Xa-induced thrombus formation by 80% (ED80), compared to a heparin ED80 of 0.1 mg/kg. At this ED80 the antithrombotic effect of the fucoïdan persisted longer than that of heparin (30 min versus 15 min). The thrombin clotting time (TCT) was significantly prolonged (73 s versus control 29 s, compared to 53 s with heparin) 10 min after a fucoïdan bolus infusion giving a plasma fucoïdan concentration of 14.6 +/- 2.7 micrograms/ml. The bleeding time was slightly increased after fucoïdan infusion at the ED80. Fucoïdan extracted from marine flora thus shows promise as an antithrombotic drug.

Modulation of vascular human endothelial and rat smooth muscle cell growth by a fucosylated chondroitin sulfate from echinoderm.

Fucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC50 =10 +/- 5 microg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 microg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 +/- 7.2%) and 27% (126.9 +/- 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 microg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 +/- 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 +/- 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form. +98% (198.2 +/- 25%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.

Mechanism of thrombin inhibition by antithrombin and heparin cofactor II in the presence of heparin.

The kinetics of thrombin inhibition by antithrombin (AT) and heparin cofactor II (HC II) were analysed as a function of the heparin concentration, from 10(-9) to 10(-4) M. The initial concentrations of inhibitor (l) and thrombin (E) were set at equimolar levels (CI = CE = 10(-8) M). The experimental data indicate that the reaction of thrombin inhibition was second-order both in the absence and in the presence of heparin, and that the apparent rate constant increased at heparin concentrations ranging from 10(-9) to 10(-6) M and decreased at higher concentrations. The data fit with the kinetic model established by Jordan et al. [J. Biol. Chem. 1979, 254, 2902-2913] for the catalysis of the thrombin-AT reaction by a low-molecular-weight heparin fraction. In this model, heparin (H) binds quickly to the inhibitor (I) and forms a heparin-inhibitor complex (HI), which is more reactive than the free inhibitor towards thrombin, leading to the formation of an inactive inhibitor-thrombin complex (I*E) and the release of free heparin, in a second step which is rate limiting. KH,I, the dissociation constant of HI, and k, the second-order rate constant of free thrombin inhibition by HI, were found to be 3.7 x 10(-7) M and 1.3 x 10(9) M-1 min-1, respectively, for AT, compared to a KH,I of 2.0 x 10(-6) M and k of 6.4 x 10(9) M-1 min-1 for HC II. These data indicate that heparin-HC II complex reactivity is greater than that of the heparin-AT complex towards thrombin, whereas heparin affinity is stronger for AT. At heparin concentrations higher than 10(-6) M, the decrease in the reaction rate was in keeping with the formation of a heparin-thrombin complex (HE), whose inactivation by the heparin-inhibitor complex (HI) is slower than that of the free protease.

Anticoagulant activity of dextran derivatives. Part II: Mechanism of thrombin inactivation.

The mechanism of anticoagulant activity of dextrans substituted with carboxylic and benzylamide sulphonate groups is studied by various coagulation tests. These derivatives exhibit a heparin-like antithrombic activity which requires the presence of antithrombin III; however they are less effective than heparin on a weight basis. They also exert a direct antithrombic activity by an antithrombic III independent pathway; but this action is negligible compared to the thrombin inhibition observed in the presence of antithrombin III. Dextran derivatives have been prepared with antithrombic properties similar to those of heparin.

Interactions of poly(lactic acid) and poly(lactic acid-co-ethylene oxide) nanoparticles with the plasma factors of the coagulation system.

When surfactant-stabilized biodegradable poly(lactic acid) (PLA) particles are injected into rats, the rate of clearance from blood is fast. The rate can be strongly reduced by using particles made from diblock copolymers of PLA and poly(ethylene oxide) (PLA-PEO), resulting in an increased duration of contact with the components of the coagulation system. Thus, possible adverse effects such as activation of the coagulation cascade could occur. In this paper, the interactions of surfactant-stabilized PLA and PLA-PEO nanoparticle suspensions with the plasma factors of the coagulation system are presented. PLA suspensions stabilized by sodium cholate (PLA-Ch) interact with thrombin, factor V and calcium ions. Formation of complexes and aggregates is induced by addition of calcium ions to PLA-Ch suspensions in the presence or in the absence of plasma. On the contrary, PLA-PEO suspensions are remarkably inert towards the coagulation factors and calcium ions, even when cholate is present. Steric repulsion owing to the high surface density of PEO is sufficient to avoid strong interations with the proteins and formation of aggregates between particles.

Mechanism of thrombin inhibition by heparin cofactor II in the presence of dermatan sulphates, native or oversulphated, and a heparin-like dextran derivative.

The kinetics of thrombin inhibition by heparin cofactor II (HC II) in the presence of dermatan sulphates, native (DS), or oversulphated (DSS 1 and DSS 2) and a biospecific dextran derivative substituted with carboxymethyl, carboxymethyl-benzylamide and carboxymethyl benzylamide-sulphonate functional groups (CMDBS), has been studied as a function of the sulphated polysaccharide concentration. The initial HC II and thrombin concentrations were set at equimolar levels. Analysis of the experimental data obtained for DS, DSS1 and DSS2 was performed using a previously described model which allows computation of the dissociation constant (KPS,HC) of the polysaccharide-HC II complex and the rate constant of thrombin inhibition by the polysaccharide-HC II complex (k). A KPS.HC of 9.6 x'10(-7) M and a k of 4.5 x 10(9) M-1 min-1 were found for DS, whereas KPS,HC 2.1 x 10(-6) M, k 1.1 x 10(10) M-1 min-1 and KPS,HC 4.3 x 10(-7) M, k 1.4 x 10(10) M-1 min-1 were found for DSS1 and DSS2, respectively. Knowing that DSS1 has a sulphur content per disaccharide of 7.8%, compared with 11.5% for DSS2, these results indicate that the polysaccharide affinity for HC II is increased only in the case of DSS 2, whereas the oversulphation increases the reactivities towards thrombin of both complexes DSS1-HC II and DSS2-HC II. A better conformation of these complexes may favour a faster interaction with the protease. Unlike heparin, DS at concentrations higher than 10(-5) M does not modify the reaction rate of thrombin inhibition, a fact which can be explained by the absence of complex formation between DS and thrombin. The experimental data obtained for CMDBS fit a kinetic model in which the biospecific dextran derivative rapidly forms a complex with thrombin which is more reactive towards HC II than the free protease. The reaction rate remained unchanged for CMDBS concentrations equal to or higher than 10(-5) M, whereas CMDBS was found to interfere strongly with the fibrinogen-thrombin interaction. These data suggest that CMDBS has a strong affinity for the protease and no affinity for HC II. The computed dissociation constant of the CMDBS-thrombin complex (KPS,E) was 2.4 x 10(-7) M and the rate constant of the reaction of this complex with HC II (k) was 1.7 x 10(8) M-1 min-1. These findings indicate that CMDBS exerts its catalytic effect through a unique mechanism of action and may constitute a new class of anticoagulant drugs.

Heparin-like functionalized polymer surfaces: discrimination between catalytic and adsorption processes during the course of thrombin inhibition.

Thrombus formation on blood-contacting artificial surfaces is a major problem. Antithrombogenic polymer surfaces have been obtained either by heparin binding, or by grafting sulphonate and/or amino acid sulphonamide groups on insoluble polystyrene. In addition to their capacity to adsorb thrombin, such surfaces were shown to be able to catalyse its inhibition by antithrombin III (AT), i.e. they are endowed with heparin-like activity. The results were mainly obtained by using clotting assays. In many cases, delineating adsorption and catalytic processes by such assays is not possible when evaluating anticoagulant polymer surfaces. To overcome this problem, the kinetics of thrombin adsorption and inhibitions by AT and heparin cofactor II (HC) in the presence of such surfaces have been measured by using an assay performed with a thrombin-specific chromogenic substrate. A simple kinetic model of thrombin consumption is proposed. The relevant calculations, carried out with the help of a computer program, lead to determination of relative second order rate constants of thrombin adsorption and inhibitions by AT and HC in the presence of the polymers. In addition to thrombin adsorption, polystyrene surfaces bearing only sulphonate groups catalyse inhibition by AT, whereas polystyrene surfaces bearing either aspartate, glycinate or isophthalate sulphonamide groups catalyse both inhibitions by AT and HC.

Plasmatic antiproteinase activity enhancement by insoluble functionalized polystyrene surfaces.

Antithrombogenic functional polymer surfaces have been obtained by grafting heparin or by substituting insoluble polystyrene with sulphonate and/or amino acid sulphamide groups. Their heparin-like properties have been related to their catalytic effects on the antithrombin III - thrombin complex formation. Amongst these antithrombogenic surfaces, this study demonstrates that some insoluble amino acid sulphamide derivatives of polystyrene strongly potentiate heparin cofactor II, in addition to antithrombin III. In contrast, an insoluble polystyrene sulphonate and, to a lesser extent, an insoluble heparin copolymer, are better catalysts of antithrombin III. It is hypothesized that such different behaviours result from different conformations of the species adsorbed onto the surfaces. The conclusions support the possible use of such amino acid sulphamide groups to prepare antithrombogenic surfaces in contact with blood.

Anticoagulant properties of a fucoïdan fraction.

Fucoïdans are a family of high molecular weight sulphated polysaccharides in the Mr range 8 x 10(5) -10(6), widely dispersed in brown seaweed cell wall. When extracted from several brown algae, they exhibit anticoagulant properties. The chemical degradation of a crude extract, from Pelvetia canaliculata, was undertaken to obtain a low molecular weight polysaccharide (Mr 20,000 +/- 5,000) with the purpose of a possible clinical use. Its anticoagulant potency was investigated through the inhibition of factor IIa and factor Xa in the presence of antithrombin III or heparin cofactor II. The degraded fucoïdan revealed a potent antithrombin activity: studied in an antithrombin III depleted plasma or in the presence of purified heparin cofactor II, the fucoïdan was as efficient as heparin and dermatan sulphate on heparin cofactor II potentiation, at the same concentration by weight. In whole plasma or in the presence of the purified inhibitor, an anti-factor IIa activity mediated by antithrombin III was detected (30 times less potent than for heparin, on a weight to weight basis). In contrast, no anti-factor Xa activity was detected in the presence of the degraded fucoïdan, under the same experimental conditions. These fucoïdans, by-products of alginates preparation in the food and cosmetologic industries, are obtained easily. Thus, they may represent a cheap and easy source of a new type of anticoagulants.

Inactivation of thrombin by a fucosylated chondroitin sulfate from echinoderm.

A polysaccharide extracted from the sea cucumber body wall has the same backbone structure as the mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. These branches confer high anticoagulant activity to the polysaccharide. Since the sea cucumber chondroitin sulfate has analogy in structure with mammalian glycosaminoglycans and sulfated fucans from brown algae, we compared its anticoagulant action with that of heparin and of a homopolymeric sulfated fucan with approximately the same level of sulfation as the sulfated fucose branches found in the sea cucumber polysaccharide. These various compounds differ not only in their anticoagulant potencies but also in the mechanisms of thrombin inhibition. Fucosylated chondroitin sulfate, like heparin, requires antithrombin or heparin cofactor II for thrombin inhibition. Sulfated fucans from brown algae have an antithrombin effect mediated by antithrombin and heparin cofactor II, plus a direct antithrombin effect more pronounced for some fractions. But even in the case of these two polysaccharides, we observed some differences. In contrast with heparin, total inhibition of thrombin in the presence of antithrombin is not achieved with fucosylated chondroitin sulfate, possibly reflecting a less specific interaction. Fucosylated chondroitin sulfate is able to inhibit thrombin generation after stimulation by both contact-activated and thromboplastin-activated systems. It delayed only the contact-induced thrombin generation, as expected for an anticoagulant without direct thrombin inhibition. Overall, the specific spatial array of the sulfated fucose branches in the fucosylated chondroitin sulfate not only confer high anticoagulant activity to the polysaccharide but also determine differences in the way it inhibits thrombin.

Thrombin binding properties of insoluble modified polystyrene: Part II.

Antithrombin III (AT III) inhibits thrombin via an arginine-serine interaction. Insoluble polystyrene resins grafted with arginyl methyl ester have been synthesized, and their interaction with thrombin tested. One of these resins was selected for its high affinity for thrombin. In this paper we report the characteristics of this thrombin resin interaction. Using this substituted polystyrene resin as a support for affinity chromatography, we have compared the binding of thrombin with that of other proteins (prothrombin, Factor IXa, trypsin and AT III). It was found that 0.7 mg of highly purified human thrombin (2,100 U/mg) was bound to 1 g of resin. This could only be eluted at high ionic strength (1.5 M) and the amidolytic and clotting activities of the eluted thrombin remained unchanged. The binding of thrombin to the resin involves the active site of the enzyme but also other residues since, when DIP thrombin was used, the inactive enzyme could be eluted at lower ionic strength (1.0 M). This resin seems to be specific for thrombin because it does not bind the other serine-proteases (trypsin or Factor IXa), prothrombin (the inactive precursor of thrombin) or AT III. The arginyl residues of the resin are important for the specificity of the interaction with Factor IIa since prolyl residues are totally ineffective. Chromatography performed on such a resin is a very efficient method of purifying thrombin, and may be very useful for the removal of thrombin as a contaminant of plasma protein fractions.

Influence of the oversulfation method and the degree of sulfation on the anticoagulant properties of dermatan sulfate derivatives.

Six oversulfated dermatan sulfate (DS) derivatives, differing in their tissue origin (porcine skin, bovine and porcine intestinal mucosa), and the oversulfation method of preparation, have been tested for their anticoagulant properties. In the first method, the SO3-trimethylamine complex is added to a DS sodium salt dissolved in formamide while it is added to a DS-benzethonium salt dissolved in dimethyl formamide in the second method. The rate of sulfation of these compounds ranged from 7.8 to 11.5 percent of sulfur on a weight basis, whereas it is 6% and 12% for natural DS and for heparin respectively. The anticoagulant potency was assessed by determining the catalytic effect of each glycosaminoglycan on the inhibition of thrombin added to (i) plasma (ii) purified heparin cofactor II(HC II) or (iii) purified antithrombin III(AT III). The catalytic effect on Factor Xa inhibition in the presence of AT III has also been investigated. The increased sulfation is found to enhance the antithrombin activity of the native dermatan sulfate whatever the method used, while the Factor Xa inhibition by AT III could be catalysed only by the most sulfated derivative obtained by the second method. The two derivatives which were less oversulfated, by the first oversulfation method, exhibit equal or even higher catalytic effects on thrombin inhibition when compared to the four other derivatives. The use of the first oversulfation method provides slightly oversulfated derivatives which exhibit strong anticoagulant properties and may constitute effective antithrombotic drugs with no bleeding tendency, a side effect perhaps related to a high rate of sulfation.

Antithrombic properties of insoluble modified polystyrene: Part I.

Chlorosulfonated polystyrenes grafted with arginyl methyl ester have been synthetized and characterized by elemental analyses. When suspended in plasma or in fibrinogen solutions, these insoluble polymers cause an increase in the thrombin clotting time. Consequently the antithrombin activity observed is not dependent upon to the presence of antithrombin III. Binding between the resin and thrombin can be demonstrated. An approximative value of the affinity constant is calculated: 2.3 X 10(6) 1/M. At high ionic strength, thrombin can be desorbed and exhibits normal coagulation properties. The prolongation of the thrombin time therefore can be attributed to the thrombin-resin interaction.

[Laboratory testing for venous thromboembolism]. / Bilan biologique de la maladie thromboembolique veineuse.

Evaluation of inherited thrombophilia in patients with venous thromboembolism includes testing for functional activity of antithrombin, protein C and protein S, and resistance to activated protein C (factor V Leiden), which can be assessed with plasma and DNA-based assays. The antiphospholipid syndrome is an acquired disorder related to the development of antibodies against phospholipid-protein complexes. Testing for the antiphospholipid syndrome includes measurement of antibodies to phospholipid-protein complexes by immunoassay or by detecting interference of anti-phospholipid antibodies in sensitive phospholipid-based assays. Other genetic risk factors have been listed, including a common polymorphism in prothrombin gene (3'-untranslated region) related to an increase of prothrombin level (> 115%) and a common polymorphism in the methylene tetrahydrofolate reductase (enzyme involved in homocysteine metabolism) gene related to a mild increase of homocysteine blood level. More recently high plasmatic levels of factor VIII (> 150%) or factor XI (> 120%), not related so far to a molecular defect, have been identified as risk factors for deep vein thrombosis. As a candidate gene, factor XIII gene polymorphisms are under investigation. Beside the acquired or genetic risk factors involved in thrombophilia, the gene-environment interactions are of importance in the onset of thrombosis.

[Multicenter study of commercial kits for the determination of human IgG subclasses, using the ELISA technique]. / Etude multicentrique des trousses commercialisées pour le dosage des sous-classes d'IgG humaines par technique ELISA.

We evaluated two commercially available sandwich type Elisa procedures for the measurement of IgG subclasses in human serum. Assay kits from The Binding Site and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service were tested in six laboratories. The performance of spectrophotometers, pipettes and dilutors were assessed at each center. Within-run precision was estimated according to the Valtec method (Société Française de Biologie Clinique). The overall coefficient of variation ranged from 4 to 50% depending on subclass and kit. We also evaluated the IgG2 and IgG4 specificity using four sera containing a monoclonal IgG2 or IgG4 (kappa or lambda type). Using total IgG and immunoelectrophoresis as a comparative technique, IgG2 kappa and IgG4 kappa were both underestimated, IgG2 lambda was overestimated while IgG4 lambda compared favorably. Polyclonal IgG subclasses were frequently overestimated in these sera suggesting cross-reactions with either monoclonal IgG or other polyclonal IgG. Antigen excess was investigated and not encountered with either kit. Our results demonstrate that these procedures are insufficiently accurate or precise for routine clinical use.