Functional assay of protein S in 70 patients with congenital and acquired disorders.

A functional assay for the selective measurement of the active form of protein S in plasma, based on the prolongation of an APTT, was previously developed. This assay is sensitive, reproducible and specific, not affected by other clotting factors including FVIII. This method was applied to the measurement of protein S activity in congenital and acquired disorders. Results of protein S activity were compared to those of total and free antigen measured by ELISA. In 30 controls, there was an excellent correlation between protein S activity and free antigen. In patients with inflammatory disease, protein S activity and free antigen were normal, despite high levels of both C4b-binding protein and total protein S antigen. In dicoumarol-treated patients, protein S activity was lower than free antigen due to the presence of acarboxylated forms. Surprisingly, in liver cirrhosis, free antigen was only slightly decreased whereas protein S activity was significantly reduced. In 23 patients with congenital deficiency, protein S activity was consistently decreased, from less than 5% to 60% and showed good correlation with the free antigen. This functional assay allows the rapid diagnosis of congenital or acquired deficiency of protein S.

Determination of plasminogen activator inhibitor (PAI) activity of human plasma after dilution in a PAI-depleted plasma.

A simple and discriminating assay for the determination of the fast-acting plasminogen activator inhibitor (PAI) activity in human plasma is described. The method is based on the inhibition of purified tissue plasminogen activator (tPA) by plasma diluted with a PAI-depleted plasma and the subsequent measurement of residual tPA in the presence of CNBr-fibrinogen fragments, purified plasminogen and a plasmin sensitive chromogenic substrate CBS 10.65. This assay does not require any acidification step, and allows PAI determination directly on plasma. Since dilutions are made in PAI-depleted plasma, all the serine-protease inhibitors, except PAI, are kept constant in their effect on the assay. Thus, any detectable degree of inhibition can only be ascribed to PAI. Under these conditions, parallel titration curves of tPA are obtained in plasma and the values of PAI are reproducible when measured at different dilutions. The PAI levels of 31 normal volunteers ranged from 0.3 to 8.7 IU/ml (mean: 3.5 IU/ml). After venous occlusion, variations of PAI were associated with the release of tPA. A marked increase of PAI levels was observed in the post-operative period and in pregnancy. In this case both PAI-1 and PAI-2 related activities were measured. Due to its simplicity, the assay can be easily used for the screening of patients with thrombotic diseases.

[Rapid determination of protein C activity]. / Dosage rapide de l'activité de la protéine C.

The diagnosis of Protein C (PC) congenital deficiency is of first importance because it leads, more frequently than Antithrombin III deficiency, to serious thromboembolic accidents in young patients, even when PC levels are slightly decreased (40 p. cent up to 60 p. cent). In order to measure PC activity, the authors developed a new method using the activator from Agkistrodon C. Contortrix snake venom and the synthetic chromogenic substrate CBS 65-25. Results obtained on plasma from normal individuals, congenital and acquired deficiencies, are comparable on the one hand, to those found with an ELISA method, and on the other hand with the clotting method, except for patients under anticoagulant therapy. This new rapid and sensitive method can be performed manually and is easily adapted on instruments used in clinical chemistry laboratories. This method is potentially available for routine use.

Fast functional protein C assay using Protac, a novel protein C activator.

A simple and rapid clotting method for the quantitative determination of protein C (PC) in plasma consists of the conversion of PC into activated PC (APC) by means of Protac, an activator protein isolated from Agkistrodon contortrix contortrix venom, of the subsequent degradation of factors V and VIII in PC immuno-depleted plasma by the generated APC and of the measurement of the prolongation of the activated partial thromboplastin time (APTT) which is proportional to the amount of PC in the sample. In 33 normal individuals a mean PC level of 97.1% of a normal pooled plasma was found. Comparison with an enzyme-immunoassay for PC in 33 patients with liver disease revealed a good correlation (r = 0.986). Patients under warfarin therapy (n = 34) had a mean PC level of 19.8%; a comparison with the immunological assay (mean value = 55.3%) in the same population suggested that the assay did not co-estimate acarboxy forms of PC. The assay proved to be insensitive to heparin concentration lower than 1 U/ml. Due to its simplicity, it should be suitable for diagnostic routine and monitoring of patients with abnormal PC level, even if under anticoagulation.

Assay of factor IX antigen using an enzyme immuno assay.

An Enzyme Linked immuno Sorbent Assay (ELISA) was developed for the measurement of factor IX:Ag. The results obtained in 90 control subjects, 56 hemophilia B patients and 40 patients under oral anticoagulant treatment were compared with factor IX:C and factor IX:Ag levels according to Electro Immuno-Assay (EIA). In healthy volunteers the mean factor IX:C level was 106.7 U/dl, the mean factor IX:Ag level was 96.8 U/dl according to EIA procedure and 101.5 U/dl according to ELISA method. Using ELISA, 10 hemophiliacs had no detectable antigen, and 13 had minute amounts of IX:Ag. All these patients are classified as B-. Among the others 21 had reduced antigen and are considered as BR and 12 had normal level of IX:Ag and are B+. Patients taking oral anticoagulants had not only a decreased factor IX:C level but also a reduced factor IX:Ag level which is always lower when using EIA procedure in the presence of EDTA than according to ELISA method.

Heparin inactivation during blood storage: its prevention by blood collection in citric acid, theophylline, adenosine, dipyridamole-C.T.A.D. mixture.

An attempt was made to prevent heparin inactivation during the time that lapses between blood collection and laboratory assay. The blood collection in a combination of citric acid, theophylline, adenosine, dipyridamole - C.T.A.D. mixture - was found to reduce greatly the heparin loss during blood centrifugation and storage whatever the temperature for these two steps (centrifugation at 4 degrees C, 12 degrees C or 25 degrees C; storage at 4 degrees C or 20-25 degrees C). With this mixture the influence of centrifugation temperature upon the heparin loss appeared negligible. The heparin loss during the initial 5 hours storage at 20 degrees-25 degrees C was found to be 4.4 +/- 4% for a heparin concentration of 0.45 +/- 0.05IU/ml. The combination of the platelet active drugs with citric acid in C.T.A.D. mixture did not interfere either with the amidolytic heparin assay (anti IIa) or clotting tests such as activated partial thromboplastin time and calcium thrombin time. Thus the C.T.A.D. mixture appears to be useful in routine use because it is effective, cheap, stable, does not interfere with the tests currently used for monitoring heparin therapy and thus helps overcome the main cause of error in heparin assays.