Separation of blood microsamples by exploiting sedimentation at the microscale.

Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical technologies enable the analysis of volumes down to a few tens of microliters. Despite the availability of analytical methods, the difficulty in obtaining high-quality and standardized microsamples at the point of collection remains a major limitation of the process. Here, we detail and model a blood separation principle which exploits discrete viscosity differences caused by blood particle sedimentation in a laminar flow. Based on this phenomenon, we developed a portable capillary-driven microfluidic device that separates blood microsamples collected from finger-pricks and delivers 2 µL of metered serum for bench-top analysis. Flow cytometric analysis demonstrated the high purity of generated microsamples. Proteomic and metabolomic analyses of the microsamples of 283 proteins and 1351 metabolite features was consistent with samples generated via a conventional centrifugation method. These results were confirmed by a clinical study scrutinising 8 blood markers in obese patients.

New approaches for measuring coagulation.

Although specific assays of coagulation factors are essential for diagnostic purposes they only give partial information about an individual's haemostatic state. This can be better assessed by various global tests, and recent developments and evaluations of five such tests are described in this symposium: the PFA-100; waveform analysis; thrombin generation; overall haemostasis potential; thrombelastography. Each test has advantages in various applications, but the thrombin generation test and waveform analysis have been found most useful in haemophilia, whilst the PFA-100 is helpful in von Willebrand's disease.

Fixed dosage of low-molecular-weight heparins causes large individual variation in coagulability, only partly correlated to body weight.

BACKGROUND: Low-molecular-weight heparins (LMWHs) are routinely given without the control of their effect on coagulation. The endogenous thrombin potential (ETP) is a sensitive detector of the heparin effect. QUESTION: What is the interindividual variation in TG after a fixed dose of LMWH in normal volunteers, is it explained by variation in weight? METHODS: Subcutaneous (s.c.) injection, in 12 healthy volunteers, of 9000 aXa-units of unfractionated heparin (UFH) and of three heparins with narrow MW distribution around 10.5, 6.0 and 4.5 kD. Measurement of anti-thrombin (aIIa) and antifactor Xa (aXa)-activities and ETP at 11 time points over 24 h. RESULTS: The coefficient of variation (CV) of the AUCs of aXa- and aIIa-activities is 50% for UFH and 22-37% for LMWHs. Because of the hyperbolic form of the dose-response curve, the CV of the inhibition of the ETP is lower: 32% for UFH and 13-21% for the LMWHs. Fixed dosage of LMWH caused under-dosage in 10-13% of the samples and over-dosage in 5-11%. High or low response is an individual property independent of the type of heparin injected and only partially explained by variation in body weight. CONCLUSION: Optimized individual dosage of LMWH is possible through recognition of high and low responders, which requires one measurement of the heparin concentration or, preferably, the heparin effect on the ETP, 2-5 h after a first injection.

Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time.

Heparin can be quantified with antifactor Xa and IIa tests (aXa, aIIa) but the anticoagulant power of heparin depends upon plasma properties as well as upon heparin concentrations and thus differs between subjects. Measuring the effect, as with the activated partial thromboplastin time (APTT) therefore is clinically more relevant. Here we investigate the use of the endogenous thrombin potential (ETP) for this purpose. In 12 volunteers 9000 IU of four heparins of different mol. wt distributions were injected. Samples were taken at 11 time points between 0 and 24 h. With the exception of the 0 and 24-h time points, heparin could be demonstrated by its aIIa and aXa activity in virtually all samples. The APTT showed the effect of this heparin in 34% of the samples; the ETP in 80%. This is partly due to the wide margins of the normal values, caused by large interindividual variation [coefficient of variation (CV) approximately 12% for the APTT, approximately 17% for the ETP]. The intraindividual variation is much smaller (CV approximately 4% for the APTT, approximately 5% for the ETP). Relative to the baseline value of the individual, the heparin effect was recognized by the APTT in 55% of the cases and by the ETP in 98%. There were no large differences between the different types of heparin.

Fibrin polymerization is crucial for thrombin generation in platelet-rich plasma in a VWF-GPIb-dependent process, defective in Bernard-Soulier syndrome.

Defective prothrombin consumption has been reported in the proband case of Bernard-Soulier syndrome (BSS). There is no consensus, however, on whether the formation of platelet procoagulant activity (PPA) is impaired in BSS and, if so, whether this is due to the lack of GPIb-V-IX-dependent binding of thrombin or of von Willebrand factor (VWF). We show thrombin generation (TG) in platelet-rich plasma of BSS (BSS-PRP) to be defective provided that fibrin remains present in the reaction mixture and that the giant platelets are not damaged by frequent subsampling. In BSS-PRP addition of (thrombin-free) fibrin did not increase TG as in normal PRP, supporting our previous hypothesis that the interaction of fibrin, VWF and GPIb triggers PPA development. Fibrin formed during the lag phase of TG by a snake venom enzyme which only removed fibrinopeptide A induced an immediate burst of TG, that was inhibited by a monoclonal antibody against GPIb (6D1) that abolishes ristocetin-induced binding of VWF to platelets. Inversely, inhibition of polymerization decreased TG and the residual activity was insensitive to 6D1. We conclude that polymerizing fibrin interacts with VWF so as to activate GPIb.

The ionic contrast medium ioxaglate interferes with thrombin-mediated feedback activation of factor V, factor VIII and platelets.

Clinical observation shows that radiographic contrast media (CM) may influence thrombus formation. In the search for the underlying mechanism, we have shown that the ionic CM ioxaglate is a potent inhibitor of thrombin generation in platelet-poor and platelet-rich plasma, whereas the influence of the non-ionic contrast medium iodixanol is minimal. Ioxaglate boosts the inhibitory effect of the platelet GPIIb/IIIa antagonist abciximab and the effects of ioxaglate and heparin are additive. Ioxaglate inhibits the clotting of fibrinogen and the activation of factors V and VIII, and of platelets by thrombin. It does not inhibit hydrolysis of small chromogenic thrombin substrates, nor does it influence the heparin-catalyzed inactivation of thrombin by antithrombin. We assume therefore that ioxaglate interferes with the binding of macromolecular substrates to the anionic exosite I of thrombin. The biological correlation to the observed antithrombotic effect of ioxaglate is then to be found in the inhibition of thrombin generation via inhibition of thrombin-mediated feedback activations.

The inhibition of blood coagulation by heparins of different molecular weight is caused by a common functional motif--the C-domain.

BACKGROUND: Heparins in clinical use differ considerably as to mode of preparation, molecular weight distribution and pharmacodynamic properties. OBJECTIVES: Find a common basis for their anticoagulant action. METHODS: In 50 fractions of virtually single molecular weight (Mr), prepared from unfractionated heparin (UFH) and four low-molecular-weight heparins (LMWH), we determined: (i) the molar concentration of material (HAM) containing the antithrombin binding pentasaccharide (A-domain); (ii) the specific catalytic activity in thrombin and factor Xa inactivation; (iii) the capacity to inhibit thrombin generation (TG) and prolong the activated partial thromboplastin time (APTT). We also calculated the molar concentration of A-domain with 12 sugar units at its non-reducing end, i.e. the structure that carries antithrombin activity (C-domain). RESULTS: The antithrombin activity and the effects on TG and APTT are primarily determined by the concentration of C-domain and independent of the source material (UFH or LMWH) or Mr. High Mr fractions (>15 000) are less active, probably through interaction with non-antithrombin plasma proteins. Anti-factor Xa activity is proportional to the concentration of A-domain, it is Ca2+- and Mr-dependent and does not determine the effect on TG and APTT. CONCLUSION: For any type of heparin, the capacity to inhibit the coagulation process in plasma is primarily determined by the concentration of C-domain, i.e. the AT-binding pentasaccharide with 12 or more sugar units at its non-reducing end.

The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability.

By using a "slow" fluorogenic thrombin substrate and continuous comparison to a simultaneously run calibrator, thrombin generation can be monitored automatically, on line, in clotting PPP or PRP at a throughput of up to 100 samples per hour. The resulting "Thrombogram" in PPP measures hypocoagulability (haemophilias, oral anticoagulants, heparins (-likes), direct inhibitors) and hypercoagulabilities (AT deficiency, prothrombin hyperexpression, prot. C and S deficiency, factor V Leiden, oral contraceptives). In PRP it is diminished in thrombopathies, in von Willebrand disease, by antibodies blocking GPIIb-IIIa or GPIb, or by antiplatelet drugs like aspirin and clopidogrel. Lupus anticoagulant both retards and increases thrombin generation. The thrombogram thus appears to be a broad function test of the haemostatic-thrombotic mechanism of the blood.

Vitamin K-dependent and vitamin K-independent hypocoagulant effects of dietary fish oil in rats.

In rats, dietary fish oil causes a plasma triglyceride-lowering as well as hypocoagulant effect. The latter is apparent from reduced levels of vitamin K-dependent coagulation factors and a decreased thrombin-forming potential of the coagulating plasma. Here, we describe that intervention with low levels of n-3 polyunsaturated fatty acids (n-3 PUFAs, about 2.5% of digestible energy, en%) resulted in no more than a small reduction in coagulation factors, when supplied as part of a high-fat diet relatively rich in vitamin K. Plasma triglycerides also remained unchanged. On the other hand, when feeding rats with low- or high-fat diets restricted in vitamin K, intervention with 3 en% of n-3 PUFAs acids (fish oil) caused only a lowering in triglycerides in combination with high fat. The fish caused a reduction in coagulation potential and levels vitamin K-dependent coagulation factors (prothrombin and factor VII) that was most prominent with the low-fat diet. Fish oil, in combination with low fat but not with high fat, reduced the vitamin K levels in the liver of the animals. In addition, regardless of the fat content, the vitamin K-independent coagulation factor V was decreased in the fish oil groups. Taken together, these results indicate that, in the rat, the hypocoagulant effect of a low dose of n-3 PUFAs is most apparent at low intakes of both vitamin K and fat, is not linked to the triglyceride plasma level, but involves modulation of both vitamin K-dependent and -independent coagulation factors.

The role of factor XI in thrombin generation induced by low concentrations of tissue factor.

Thrombin generation has been studied in the plasma of severely factor XI deficient patients under conditions in which contact activation did not play a role. In platelet-rich as well as platelet-poor plasma, thrombin generation was dependent upon the presence of factor XI at tissue factor concentrations of between 1 and 20 pg/ml i.e. approximately 0.01 to 0.20% of the concentration normally present in the thromboplastin time determination. The requirement for factor XI is low; significant thrombin generation was seen at 1% factor XI; at 10%, thrombin formation was nearly normalised. A suspension of normal platelets in severely factor XI deficient plasma did not increase thrombin generation. This implies that there is no significant factor XI activity carried by normal platelets, although the presence of factor XI and factor XI inhibitors in platelets cannot be ruled out.

The effect of DDAVP infusion on thrombin generation in platelet-rich plasma of von Willebrand type 1 and in mild haemophilia A patients.

In von Willebrand disease (vWD) type 1 and mild haemophilia A patients we studied the effect of an infusion of DDAVP (0.3 microg/kg body weight) on thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Baseline thrombin generation in PRP was diminished both in the haemophilia A and vWD patients. It was normal in vWD plasma when sufficient procoagulant phospholipids were present, either via adding phospholipid vesicles to PPP or via scrambling of the platelet membrane with ionomycin in PRP. In haemophilia A plasma, thrombin generation did not normalize by providing procoagulant phospholipids. Treatment with DDAVP temporarily restored thrombin generation in PRP to normal in both diseases. To investigate the individual roles of von Willebrand factor (vWF) and factor VIII, we also studied the effect of factor VIII infusion on thrombin generation in a severe haemophilia patient. It appears that at a fixed normal vWF concentration, <25% factor VIII is sufficient for normal thrombin generation in PRP. At a sufficient factor VIII concentration, however, thrombin generation is still lower than normal in vWD patients; approximately 40% of vWF is required for half-normal thrombin generation in PRP. It thus appears that vWF is also a clotting factor, in the sense that it is required for normal thrombin generation. This underlines the importance of the interaction between coagulation and the platelets in normal haemostasis. Thrombin generation in PRP appears to be a suitable test to reflect the combined function.

Determination of glufosinate ammonium and its metabolite (AE F064619 and AE F061517) residues in water by gas chromatography with tandem mass spectrometry after ion exchange cleanup and derivatization.

An analytical method for the determination of glufosinate ammonium and its principal metabolites, AE F064619 and AE F061517, in water of two different hardnesses (5 and 30 DH, French hardness) has been developed and validated. Samples were spiked at different levels (0. 05 and 0.5 microgram/L) and were purified by column chromatography on ion-exchange resins. After derivatization with glacial acetic acid and trimethylarthoacetate mixture, the derivatives were quantified by using capillary gas chromatography with an ion-trap tandem mass spectrometric detector. Analytical conditions for MS/MS detection were optimized, and the quantification was carried out on the areas of the most representative ions. The limit of quantification was validated at 0.05 microgram/L for each compound. The mean recovery value and the relative standard deviation (n = 20) were 92.0% and 17. 8% for glufosinate ammonium, 90.2% and 15.8% for AE F064619, and 89. 7% and 12.7% for AE F061517.

Determination of glyphosate and aminomethylphosphonic acid residues in water by gas chromatography with tandem mass spectrometry after exchange ion resin purification and derivatization. Application on vegetable matrixes

An analytical method for the determination of glyphosate and its principal metabolite, aminomethylphosphonic acid (AMPA), in water of different hardnesses (5, 20, and 30 degrees DH, french hardness) has been developed. Samples were fortified at different levels (0.05, 0.1, 1, and 5 microg/L) and were purified by column chromatography on ion-exchange resins. After derivatization with TFAA/HFB mixture, the derivatives were quantified by using capillary gas chromatography with an ion-trap tandem mass spectrometric detector. Analytical conditions for MS/MS detection were optimized, and the quantification was carried out on the sum of areas of the three most representative ions: m/z 283, 223, and 181 for AMPA and m/z 440, 321, and 261 for glyphosate. The limit of quantification was demonstrated to be at 0.05 microg/L for each compound. The mean recovery value and the relative standard deviation (n = 65) were 93 and 12% for AMPA and 95 and 13% for glyphosate.

Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition comparison with other mammalian secreted phospholipases A2.

Human secreted group IIA phospholipase A2 (hGIIA) was reported to inhibit prothrombinase activity because of binding to factor Xa. This study further shows that hGIIA and its catalytically inactive H48Q mutant prolong the lag time of thrombin generation in human platelet-rich plasma with similar efficiency, indicating that hGIIA exerts an anticoagulant effect independently of phospholipid hydrolysis under ex vivo conditions. Charge reversal of basic residues on the interfacial binding surface (IBS) of hGIIA leads to decreased ability to inhibit prothrombinase activity, which correlates with a reduced affinity for factor Xa, as determined by surface plasmon resonance. Mutation of other surface-exposed basic residues, hydrophobic residues on the IBS, and His48, does not affect the ability of hGIIA to inhibit prothrombinase activity and bind to factor Xa. Other basic, but not neutral or acidic, mammalian secreted phospholipases A2 (sPLA2s) exert a phospholipid-independent inhibitory effect on prothrombinase activity, suggesting that these basic sPLA2s also bind to factor Xa. In conclusion, this study demonstrates that the anticoagulant effect of hGIIA is independent of phospholipid hydrolysis and is based on its interaction with factor Xa, leading to prothrombinase inhibition, even under ex vivo conditions. This study also shows that such an interaction involves basic residues located on the IBS of hGIIA, and suggests that other basic mammalian sPLA2s may also inhibit blood coagulation by a similar mechanism to that described for hGIIA.

The thrombogram: monitoring thrombin generation in platelet-rich plasma.

A method is described in which thrombin activity in clotting plasma can be monitored through the continuous measurement of the fluorescent split-product of the substrate Z-Gly-Gly-Arg-AMC. The signal is not impaired by turbidity; therefore proper measurement is not disturbed by the occurrence of a clot or the presence of platelets and direct measurement in platelet rich plasma is possible.