Influence of three types of automated coagulometers on the international sensitivity index (ISI) of rabbit, human, and recombinant human tissue factor preparations--a multicenter study.

Five tissue factor reagents and three types of automated instruments for prothrombin time (PT) determination were studied in an international multicenter collaborative exercise. The purpose of this work was to determine the international sensitivity index (ISI) for each combination of reagent and instrument against the international reference preparation RBT/90. Each type of instrument was used by 3 or 4 centers to assess the interlaboratory variation of the ISI. The interlaboratory variation of the ISI for each combination of reagent and instrument ranged between 0.4% and 7.8% coefficient of variation. For three reagents, the mean ISI values for ACL (nephelometric) and STA (mechanical) were practically identical, but the mean ISI values for MLA (photo-optical) were at least 10% higher. For two other reagents prepared from rabbit tissue, the mean ISI values increased in the order ACL, STA, MLA. The widest range of mean ISI values was noted with one rabbit tissue factor reagent: 1.68 for ACL and 2.00 for MLA. Exclusion of patient specimens with INR <1.5 and INR >4.5 determined by the international reference preparation resulted in a slight decrease of the mean ISI. The interlaboratory variation of the International Normalized Ratio (INR) was assessed from the results obtained with common lyophilized and deep-frozen plasmas. The use of instrument-specific ISI values resulted in reduced interlaboratory variation of the INR. It is recommended that thromboplastin manufacturers provide instrument-specific ISI values.

Control of oral anticoagulation in patients with the antiphospholipid syndrome--influence of the lupus anticoagulant on International Normalized Ratio. Groupe Méthodologie en Hémostase du Groupe d'Etudes sur l'Hémostases et la Thrombose.

The recommended therapeutic range of International Normalized Ratio (INR) for oral anticoagulant treatment in patients with the antiphospholipid syndrome remains controversial. As a part of this controversy, it has been suggested that lupus anticoagulants (LA) could interfere with the determination of prothrombin time, thus questioning the validity of monitoring the treatment of these patients using INR. To clarify this point, we compared the values of INR obtained in the plasmas of two groups of patients, one without LA (n = 47), and the other with LA (n = 43). INR were determined using 8 different thromboplastin reagents on the same automated coagulation instrument. Chromogenic factor X, which is supposed to be insensitive to the presence of LA, was also measured. The results are the following: provided INR was calculated using calibrated reference plasmas, there was no significant difference between INR values obtained with the 8 reagents, both in the non-LA and in the LA groups (CV: 5.9 and 6.7%. respectively). Closer examination revealed that INR results obtained with one reagent (the recombinant thromboplastin Innovin) diverged from those of the 7 others, leading to an overestimation of INR, to a very large extent in some instances. However this effect was restricted to a subset of the patient population with LA (6 out of 43). Finally, the relationship between INR (average value obtained using the 8 reagents) and factor X was identical in non-LA and in LA patient groups. We conclude that, provided the reagents which display the LA interference are identified and excluded for this purpose, the INR system is valid for monitoring oral anticoagulant treatment in patients with LA.

Long-term French experience in INR standardization by a procedure using plasma calibrants.

The International Normalized Ratio (INR) has not lowered the interlaboratory differences in prothrombin time (PT) values to the extent expected, mainly because of the instrument-dependency of the International Sensitivity Index (ISI) and other factors (eg, accurate determination of the ISI, the normal value used in the PT ratio). The procedure (PPC) using plasma calibrants (reference lyophilized plasmas with assigned activity) has been evaluated since 1977 in nine French national external quality assessment surveys (NEQAS) involving approximately 4,000 laboratories and numerous local thromboplastin technique combinations. The PPC was compared with the conventional procedure (using the manufacturer's ISI), and the efficiency of antivitamin K-calibrated (AK Cal) plasmas from patients receiving oral anticoagulants vs artificially depleted plasma calibrants was also evaluated. The PPC efficiently standardized PTs with AK Cal plasmas, reducing interlaboratory variability (eg, coefficient of variation, 12% to 6% for survey 92 D) and reagent-instrument effects. However, AK Cal plasmas have drawbacks, such as limited supply, cost, and batch-to-batch variability. The artificially depleted plasma calibrants were less efficient, but usable if carefully prepared. The value of this simple procedure is that local practices are considered in the determination of PT, thus correcting for coagulometer effects and avoiding use of the manufacturer's ISI and need for a normal control plasma. These large-scale French surveys have demonstrated the validity of PPC through 15 years of experience and have shown that it offers the best compromise available in PT standardization.

Multicenter study of an erythro-aggregometer: quality control and standardization.

There is a need for quality assurance procedures in hemorheology, especially for clinical and pharmacological studies, which require reliable and well-calibrated instruments to be interpretable and comparable. Preliminary investigations allowed preparation of standardized SM (normal NS and hyperaggregable HS), and checking of storage conditions (in accordance with the guidelines of the SSC of ISTH) of RBC in nutritive SAG mannitol for at least 2 or 3 weeks with subsequent washings and resuspension in SM. In our study, we compared erythro-aggregometers of the same brand in 6 laboratories, using the same red blood cells (RBC) and suspending media (SM) for each series of tests. EA was measured by laser backscattering with determination of aggregation time (AT), partial dissociation threshold (PDT) and aggregation index (AI). Prior to the study, devices were set up on the same day with the same standardized blood, and calibration data were then analyzed. Within-assay precision (WAP) was assessed on 3 days for the 2 types of SM (n = 18 x 2). Between-assay precision (BAP) was assessed on 6 occasions, once every month (n = 6 x 2 x 6). Accuracy was studied with 3 series of RBC resuspended in 10 SM of "unknown" aggregability. Good agreement was observed between 5/6 centers for the 3 parameters of EA. WEP was good: CV of AT ranging from 1.4 to 2.5% for the NS and from 1.4 to 2.4% for the HS. In each center, BAP was slightly lower than WEP (CV: 8-11.8% for the NS and 3.8-4.7% for the HS over the 6-month study), with no drift, except for one center. Precision was always better with the HS than with the NS which seemed a better tool to assess it. As to accuracy, non-significant differences were generally found between centers, with similar data to the reference values. This work also stressed the importance of the RBC parameter itself in rheological data. For the first time, a protocol for standardization of EA has been developed and evaluated, permitting the Quality Control of this technique.

Validation protocol of analytical hemostasis systems: measurement of anti-Xa activity of low-molecular-weight heparins.

A standard validation protocol adapted to the chromogenic assay of anti-Xa activity of low-molecular-weight heparins was used in a multicenter study to assess its suitability for comparing and evaluating analytical hemostasis systems. The protocol included: familiarization with the system (repeatability); assessment of limits of linearity, detection limits, and cross-contamination; and validation (reproducibility and accuracy of measurements of treated patients' plasmas). We calibrated the systems with the same range of lyophilized plasmas daily and evaluated repeatability and reproducibility by using a single batch of lyophilized plasmas at three anti-Xa activities. The two automated systems tested [SB 300 (Gilford) and ACL (IL)] and the two semiautomated systems [ST 888 (D. Stago) and Chromotimer (Behring)] gave similar mean values. Dispersion of results was lower with the automated systems than with the semiautomated ones, especially at low anti-Xa activities, a tendency that also was observed for reproducibility. Because each analytical system gave linear results for activities as great as 1000 IU/L, suitable sample dilution is advisable for higher anti-Xa activities. Accuracy was greater in the automated systems. We conclude that this protocol is feasible and is applicable to validation of other analytical hemostasis instruments, in particular the latest generation of fully automated instruments.

Inhibition of thrombin generation by heparin and LMW heparins: a comparison of chromogenic and clotting methods.

Inhibition of thrombin generation by heparin and low-molecular-weight (LMW) heparins is an important parameter which relates to their anticoagulant actions in vivo. Previous studies in our laboratory used a clotting assay for assessment of thrombin generation but other published studies have used a chromogenic method. We have therefore measured the inhibition of thrombin generation by unfractionated heparin (UFH) and LMW heparins by a modified chromogenic method using microtitre plates and compared the results with the clotting method. The degree of inhibition of thrombin generation when calculated from both peak thrombin concentrations and areas under the curve in the chromogenic assay was the same. When the activities of each heparin were expressed as EC80, i.e. concentrations required for 80% inhibition of thrombin generation, the EC80 were higher in the chromogenic assay than in the clotting system. However when the potencies of the LMW heparins were expressed as percentages of that of the UFH standard by parallel line analysis, the differences between clotting and chromogenic assay results were small and not statistically significant (P > 0.05). This study demonstrates the feasibility of measuring inhibition of thrombin generation by a modified chromogenic method using microtitre plates, and shows that the results with this method are similar to those obtained with the clotting method.

[Modifications of hemorheologic parameters in the course of hemodialysis in chronic renal insufficiency]. / Modifications de paramètres hémorhéologiques au cours de l'hémodialyse chez l'insuffisant rénal chronique.

The influence of haemodialysis (HD) was assessed in 72 patients, undergoing a thrice weekly routine HD for chronic renal failure (CRF). Some of them received human recombinant erythropoietin (Eprex). Measurements were performed before and after the HD session: the erythrocyte aggregation (EA) was carried out by a laser backscattering technique with determination of the aggregation time (AT) and of the dissociation thresholds. Plasma viscosity (PV) was evaluated in an automatic capillary viscometer. Fibrinogen (Fg) levels, haematological features (blood cell count), serum proteins, creatinine, and some other biochemical parameters, were also determined. Anaemia was a common feature. Our results compared to those of a control group, confirmed the erythrocyte hyperaggregation before HD which increased during HD. PV also elevated before HD, further increased after HD; the same finding was observed for Fg. Some of these results might be related to the haemoconcentration. Significant correlations were noted between AT and PV, AT and Fg with closer correlations after HD, suggesting a strong cohesion of RBC aggregates, which enhanced during HD. Correlations were highly significant between relative variations of AT and relative variations of PV, Fg, proteins and body weight, before and after HD. Special attention was given to the group of patients under long term treatment with Eprex compared to non-treated dialysed patients: no significant difference was found between both groups. Our results are in agreement with a blood and plasma viscosity syndrome due to increased EA and with a tendency, to thrombosis reported in those patients.

Interlaboratory evaluation of methods for the assay of Protein C in purified concentrates.

Determination of the quantity and activity of the Protein C molecule is of the utmost importance in highly purified concentrates prepared for replacement therapy. A multicenter study was undertaken to evaluate the comparability and accuracy of Protein C assays from commercial sources. Significant between-assay and interlaboratory differences were found for both functional and immunological assays. The interlaboratory variability is explained in part by the use of different control plasmas. The results also indicate the importance of the diluent used. This study emphasizes the need for standardized methods for determining the characteristics of Protein C concentrates.

[Comparative evaluation of unfractionated and low molecular weight heparins. Results of the French Etalonorme interlaboratory quality control]. / Evaluation comparative des héparines non fractionnées et de bas poids moléculaire. Résultats du contrôle de qualité interlaboratoire français Etalonorme.

Four recent interlaboratory surveys have been conducted by the French National "Etalonorme" Group of Quality Control in Haematology, involving about 3,800 laboratories working in coagulation. They have been concerned with human lyophilised plasma samples heparinized in vitro either with unfractionated heparins (UFH), or with low molecular weight heparins (LMWH) (Fragmin 89 D, and Fraxiparin 90 A). The doses added simulated therapeutic situations or were on the border-line of over- or underdosage: UFHs 89 B3/B4, 0.14 and 0.22 IU/ml; 89 C3/C4, 0.3 and 0.4 IU/ml respectively; LMWHs: D3-A3, 0.6; and D4-A4, 1.2 IU/ml. Automated partial thromboplastin times (APTT) were prolonged at these high doses of LMWH, due to noticeable residual anti-IIa activity. A relationship between APTT and heparinaemia was observed with both types of heparin; sensitivities of cephalin reagents to heparin were also noted, but they were different for UFH compared to LMWH. Dispersion on APTT results was still high (CV: 12-23% for the UFH samples, 12-19% for the four LMWH samples). Dispersion on heparin determinations was higher for UFH than for LMWH, even for lower anti-IIa activity, but they involved more heterogeneous assays. The anti-Xa activity for LMWH samples was mainly determined using clotting assays (70% of the laboratories) with larger dispersions (CV: 32-37%), than those observed on amidolytic assays (CV: 28%); however, these amidolytic assays were performed in 90% of the laboratories with only two reagents available from a single manufacturer. The clotting techniques, more than the colorimetric assays, seemed to underestimate the high heparinaemias.(ABSTRACT TRUNCATED AT 250 WORDS)

[Multicenter study on purified protein C concentrates and defined plasma levels]. / Etude multicentrique sur les dosages de concentrés de protéine C purifiée et de plasmas à taux définis.

A variety of protein C assays are available as commercial kits. A collaborative study was undertaken to evaluate the performance of protein C assays. Various samples including calibrated plasmas and high purity concentrates destined to therapy were distributed among five laboratories. This comparison of protein C assays indicates that protein C levels measured by different functional or immunological assays and by five laboratories are very close for calibrated plasmas but not for high purity concentrates. The selection of the standard and the dilution buffer for protein C concentrates have important implications for the interpretation of the results. Dilution of purified protein C concentrates in protein C deficient plasma which restaure a total protein level similar to that of normal plasma improve the accuracy of functional protein C assays.

[Comparative evaluation of 3 automated optical coagulation detection systems]. / Evaluation comparée de trois automates de coagulation à détection optique.

The Organon Teknika-General Diagnostics Coag A Mate X2 (CAM X2), Ortho Koagulab 40A, MLA Electra 700 (E 700), were simultaneously evaluated following the same protocol, on the same samples. Compared with the former photo-optical-based automated devices, these newer advanced instruments present a high throughput in fibrin endpoint detection, permitting fibrinogen determination with reliable results; they also present a higher degree of automatism, greater flexibility in use and easy mastering, allied to elaborated expression of results. Statistical comparison shows equivalent performance characteristics. Repetabilities are excellent for Prothrombin Time (CV: 1-2.5%) with 5 thromboplastins and for the prothrombin complex factors assays: they are also good for Activated Partial Thromboplastin Time (APTT) with 4 reagents (CV: 0.5 to 3%) provided that kaolin be not used on the KLO and E700; the same limitation exists for specific factors assays. Fibrinogen assays display good results (CV: 3-4% and 6-8%) respectively for times of 11 sec. (/=/ 2.4 g/l) and 23 sec. (/=/ 1 g/l). Standard linearities are generally excellent whatever the test. The correlation with results obtained on other devices is good (0.96 less than R less than 1). The most fully automated is the KLO with reliable system of sample delivery, automatic calibration and quality control function. The E700 is the slowest instrument, especially for APTT and the least adapted to large homogenous series, but its original loading system with binary coded cuvettes makes it interesting for emergencies and mixed tests. Controls and adjustments of the pumptubing are particularly required on the CAM X2 which is, on the other hand, the quickest instrument with its 4 photo-optical cells, well adapted to handling a large series of samples.

[Prophylactic and therapeutic use of a low molecular weight heparin fraction, CY 216]. / Utilisation prophylactique et thérapeutique d'une fraction d'héparine de bas poids moléculaire: CY 216.

The aim of our work was to study in a population of high risk patients with hemorrhagic and or thrombotic disease, the preventive or therapeutic effect of a low molecular weight heparin fraction, CY 216 (Choay, France), particularly in surgery. CY 216 was given to 9 patients for the treatment of a thrombosis (pulmonary embolism, acute ischemia, deep venous thrombosis) and to 40 patients in prevention of thrombosis. In this second group, 28 had a high thromboembolic risk such as valvular prosthesis, cardiac arrythmia, coronary artery bypass, etc. For all the patients, CY 216 was injected sub-cutaneously twice or three times a day at the mean dose of 1.5 mg/kg/d, equivalent to 300 U anti-Xa Choay/24 h, and always injected 24 hours before surgery. The biological tests used were: blood cells count, platelet count, prothrombin time, activated partial thromboplastin time, heparinemia levels by two technics: anti-factor-Xa activity and anti-factor IIa activity. None thrombotic complication was observed in the 40 patients prophylactically treated and a constant improvement of thrombosis was noted for the 9 patients with thrombo-embolic disease. In 3 patients, bleeding complications were observed: for 2 patients, all the coagulation tests were normal and anti-Xa activities were less than 0.55 U/ml; in one patient, the bleeding time was prolonged (15 minutes Ivy Incision) and returned to normal when the CY 216 was stopped. Concerning the biology, there was no modification except for anti-Xa activity which mean was 0.30 U/ml (01-07). However, this test is unable to predict either thrombotic or hemorrhagic events.

[Antiprothrombinase anticoagulant and acquired prothrombin deficiency in childhood viral pathology. Spontaneous recovery]. / Anticoagulant antiprothrombinase et déficit acquis en prothrombine en pathologie virale infantile. Guérison spontanée.

A 2 year-old female developed an acute bleeding diathesis related to a profound, isolated and acquired prothrombin deficiency; evidence for a "lupus anticoagulant" was also demonstrated. This association of hypoprothrombinemia and "lupus anticoagulant", rarely reported, was previously considered to be rather specific for SLE. This case report demonstrates that these coagulation disorders may present as an acute form, in viral diseases of the child, with spontaneous and quick recovery. Specific characteristics of the biological coagulation defects, namely those related to the low factor II, are discussed.

[Success of treatment with a low-molecular-weight heparin fraction in a case of pulmonary embolism complicated by heparin-induced thrombopenia]. / Succès du traitement par une fraction d'héparine de bas poids moléculaire dans un cas d'embolie pulmonaire compliquant une thrombopénie induite par l'héparine.

A 40-year-old woman was admitted to Ambroise-Paré Hospital with a mitral stenosis and right ventricular failure. On the admission, heparino-therapy was started (Heparine Calcique Leo 30,000 IU/24 h); 11 days after, a thrombocytopenia (platelet count 60 . 10(9)/l) was observed and a few days later a pulmonary embolus was diagnosed. "In vitro", a heparin dependent plasma platelet aggregating factor was found (with Heparine Leo and all other standard ones tested) leading to the diagnosis of heparin associated thrombocytopenia; on the other this aggregating factor was not found with a low molecular weight (LMW) heparin fraction (Choay laboratory, Paris, batch CY216). We decided to stop standard heparin and to treat this patient with the LMW fraction (7,500 IU every 8 h SC) associated with oral antivitamin K. A rapid clinical improvement was observed and the platelet count rose progressively reaching 300 . 10(9)/l and has subsequently always been greater than 200 . 10(9)/l. The occurrence of heparin induced thrombocytopenia associated with thrombosis leads to heparin cessation; the treatment of the thrombotic episode seems to be improved by the use of LMW heparin associated with vitamin K antagonists.

In vivo interactions of autoantibodies to factor VIII with the factor VIII complex.

Two non-haemophilic elderly patients who had developed autoantibodies to factor VIII were studied over a period of 9 months to 5 years. Sequential measurements of antibody to factor VIII (anti-VII:C), factor VIII coagulant activity (VIII:C), factor VIII coagulant antigen (VIII:CAg), factor VIII-related antigen (VIIIR:Ag), and factor VIII ristocetin cofactor (VIII:WF) were performed. Before treatment, low VIII:C, normal or increased VIII:CAg and high VIIIR:Ag levels were found and were indicative of the presence of circulating immune complexes. Immunosuppressive therapy induced progressive correction of VIII:C and VIIIR:Ag values. High levels of VIII:CAg subsided in the patient who relapsed. It is suggested that antibodies to factor VIII bind and remove VIII:C from the circulation thereby inducing an increased synthesis of VIII:CAg which may be associated with an augmented release or production of VIIIR:Ag.