Prothrombin evaluation as obtained by kinetics studies of antigen-antibody reaction in a laser nephelometer.

Laser nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetics of the reaction between antigen and antibody. We studied the kinetics of the reaction between prothrombin and an antiprothrombin antiserum using several prothrombins namely: Prothrombin Padua, prothrombin Molise, which are two congenital dysprothrombinemias, cirrhotic, coumarin or normal prothrombins. Different behaviors in the kinetics of the reactions were shown even when the concentration of prothrombins was about the same in all plasma tested. These differences were analyzed by means of a computer (Apple II 48 RAM) programmed to solve four unknown equations (Rodbard's equation). From the data so obtained one can see that when voltages at the beginning and at the end of the reaction are in all cases about the same, a clear difference in the time required to reach half the maximum value of the voltage can still be demonstrated. This parameter, which is expressed in minutes, is longer in coumarin and prothrombin Molise than in controls. On the contrary it is shorter in prothrombin Padua and has about the same value of controls in the cirrhotic patient. Moreover the time at which the maximum rate is obtained is longer in coumarin and prothrombin Molise than in controls and shorter in liver cirrhosis and prothrombin Padua. In conclusion data obtained show that coumarin prothrombin behaves in a different way from cirrhotic prothrombin and also that there is a different behaviour between the two congenital dysprothrombinemias.

The role of laser nephelometer in the study of abnormal clotting factors: characterization of two abnormal antithrombins (AT III Padua and AT III Padua2).

The immunologic concentration of two abnormal antithrombins III (AT III), namely antithrombin III Padua (AT III Padua) and antithrombin III Padua2 (AT III Padua2) and the kinetics of the reaction of these two ATs III with an anti AT III antiserum was investigated by means of a laser nephelometer. The immunologic concentration of these two AT III both in presence (0.2 IU/mL) or absence of heparin was normal. On the contrary, the analysis of kinetics behavior demonstrated that AT III Padua is radically different from pooled normal plasma both in presence or in absence of heparin. This was not the case for AT III Padua2, which showed no difference from pooled normal plasma regardless of the presence or absence of heparin. Both abnormal antithrombins III reached the plateau of the reaction at about the corresponding value of pooled normal plasma, indicating a normal antigen level. These experimental data were analyzed by means of a computer (Apple II 48 RAM) programmed to solve a four unknowns equation (Rodbard's equation). This analysis showed that the time needed to reach half of the maximum voltage, i.e., the parameter C, which is expressed in minutes, is clearly longer in the case of AT III Padua samples (heparinized or not) as compared with pooled normal plasma. Moreover, the time at which the maximum rate was reached was also longer. On the contrary, in the case of AT III Padua2 there is no difference from pooled normal plasma. These data confirm the view that a different kind of defect is present in these two AT III abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Prothrombin antigen evaluation by means of laser nephelometry in health and disease.

Prothrombin antigen concentration was evaluated by means of laser nephelometry in 10 patients on coumarin therapy, in 17 patients with cirrhosis of the liver, and in four patients with congenital hypo- or dysprothrombinemias. The average values obtained were 46.4, 37.7, and 35.6%, respectively, for anticoagulated, cirrhotic, and congenitally abnormal patients. These values correlated well with those obtained by means of electroimmunoassay (Laurell) and immunodiffusion (Mancini) methods. Similarly, satisfactory results were obtained in eight normal subjects. Multiple evaluations at different incubation times, also allowed the authors to construct kinetic curves of the interaction between antigen and antibody. However, an abnormal kinetic curve was demonstrated only for coumarin-treated patients.

Differences between normal antithrombin III and antithrombin III Padua as determined by laser nephelometry.

Laser nephelometry was used to characterize an abnormal antithrombin III (AT III Padua) in comparison with normal antithrombin. Heparinized (0.2 IU/ml) or non-heparinized AT III Padua plasma reacts with Laser nephelometry antithrombin III antiserum in a different way compared with pooled normal plasma. There is in fact a slower antigen-antibody reaction during the first 40-45 min both in AT III Padua heparinized and non-heparinized plasmas, compared with pooled normal plasma; then the kinetics overlap. On the contrary the concentrations of antithrombin III Padua in percent correlate well with those obtained by Mancini's and Laurell's methods. These data indicate that Laser nephelometry is suitable for AT III antigen determinations and may also supply useful information for the characterization of abnormal clotting factors.

Coumarin-induced abnormal factor IX: an immunological study in humans.

An excess of factor IX antigen or protein with respect to factor IX activity is present in coumarin treated patients. The average factor IX antigen value found in a group of 16 patients was 96.2 (S.D. = 24.46) whereas the average clotting activity was 19 (S.D. = 4.54) (p less than 0.001). In the electroimmunoassay system a normal peak or precipitate were seen in every instance. In haemophilia B--no peak or precipitate were seen. The coumarin induced abnormal factor IX shows a more anodic migration in the bidimensional immunoelectrophoresis system as compared with the normal counterpart. On the contrary, the factor IX protein present in haemophilia BM or in haemophilie B+ migrates as normal factor IX.

Apparent "inhibition" as tested by means of the dilution curve system in patients with clotting defect due to liver damage.

At equivalent Thrombotest basal values on undiluted non-contacted plasma, there is no difference between coumarin plasma and liver damage plasma in the dilution curve system. A hemophilia BM plasma with longer basal clotting times yielded a much flatter curve and a very large "inhibitory" effect (greater than 6 Units). A coumarin plasma with the same basal Thrombotest clotting times as Hemophilia BM plasma, namely 100 seconds, yielded a much steeper curve and a very low inhibition (0.7 Units). These findings are against the presence of inhibitors in coumarin or liver damage plasmas.

An immunological investigation of hemophilia B with a tentative classification of the disease into five variants.

23 patients with hemophilia B have been investigated by means of several immunological methods. 16 patients (69.9%) had no detectable factor XI antigen. Five had a normal factor IX antigen and the electrophoretic mobility of this abnormal factor IX was similar to that of its normal counterpart. One of these five patients had hemophilia Bm, since ox brain thromboplastin clotting time was severely prolonged. The remaining two patients had reduced or decreased factor IX antigen. Several patients showed a slight protongation of ox brain thromboplastin time due to an associated slight factor VII deficiency. On the basis of these results, a tentative classification of hemophilia B into five variants is proposed, namely: hemctor IX antigen; hemophilia Bra, or with reduced factor IX antigen; hemophilia Bm, or with normal factor IX antigen and severely prolonged ox brain thromboplastin; hemophilia B patients is feasible only by means of a battery of tests, namely:factor IX activity assay, factor IX antigen determination, ox brain thromboplastin clotting time, factor VII activity assay.

Congenital deficiency of factor XIII with normal subunit S and lack of subunit A. Report of a new family.

Two sisters born from a nonconsanguineous marriage were found to have congenital factor XIII deficiency. In the electroimmunoassay system, using an anti-subunit S antiserum, two distinct peaks or rockets were seen in normal plasma and serum whereas only one peak was present in the propositae plasma or serum. In the bidimensional immunoelectrophoresis system, using the anti-subunit S antiserum, two major peaks were seen in normal plasma whereas only one peak was seen in the propositae plasma. Using an anti-subunit A antiserum no peak or precipitate was seen in our propositae in the electroimmunoassay or in the bidimensional immunoelectrophoresis systems. Both the parents and the children of our two propositae showed a normal coagulation pattern. Therefore, the heredity appears to be autosomal recessive. These data indicate that the defect is characterized by a normal factor XIII subunit S (support) and a lack of factor XIII subunit A (activity).

An immunological investigation of factor VIII associated antigen in combined factor V and factor VIII deficiency.

The behavior of factor VIII associated antigen of three patients with combined factor V and factor VIII deficiency has been evaluated in several immunological systems. Factor VIII associated antigen resulted to be normal or higher than normal in all three patients in the radial immunodiffusion and in the electroimmunoassay systems. In the bidimensional electrophoresis system only one factor VIII precipitate was evident and such factor VIII precipitate showed the same electrophoretic mobility as normal factor VIII antigen. These findings firmly establish the fact that the factor VIII defect in congenital combined factor V and factor VIII deficiency is of the hemophilia type.