Clauss Method Research Articles

Fibrinogen date: Congenital hypodysfibrinogenemia associated with decreased binding of tissue-plasminogen activator

A new case of congenital hypodysfibrinogenemia associated with a thrombotic tendency (thrombosis of the peroneal artery, the pulmonary artery and the deep veins of the lower extremities), is reported. Clotting time using Reptilase was significantly prolonged. The low levels of plasma fibrinogen were demonstrated using both thrombin time (Clauss) method (45.0 mg/ml) and radial immunodiffusion technique (106.1 mg/ml). The fibrinogen is also characterized by a defective polymerization of fibrin monomers. Furthermore, the important functional property of this fibrinogen is that the patient's fibrin adsorbs approximately 31-38% of added tissue-plasminogen activator (t-PA) at the time of fibrin formation, whereas control fibrin can adsorb about 79% of added t-PA. This result could provide an explanation for the thrombotic tendency in this patient. This variant fibrinogen appears to have unique characteristics and has been designated as "Fibrinogen Date," according to the city where the proband has lived.

Tumor necrosis factor (TNF) is induced in mice by Candida albicans: role of TNF in fibrinogen increase

One intraperitoneal dose of Candida albicans (10(8) CFU) caused a chronic (longer than 2 months), significant elevation of plasma fibrinogen levels (Clauss method) in mice of strain C3H/HeN. Even a small dose (10(6) CFU) resulted in a significant increase in fibrinogen level for 5 days following injection, whereas other blood parameters (leukocytes, erythrocytes, platelets, hematocrit, hemoglobin, blood urea nitrogen, aspartate aminotransferase, albumin, alkaline phosphatase, antithrombin III, glucose, calcium, and total protein) measured by standard methods were normal. Blood taken during this period was negative for C. albicans. The role of tumor necrosis factor (TNF) in C. albicans infections was investigated by measuring the fibrinogen response after the administration of C. albicans or recombinant mouse TNF-alpha. Both challenges resulted in an elevated fibrinogen level. When polyclonal antibodies to mouse TNF-alpha were given prior to challenge with C. albicans or mouse TNF-alpha, the fibrinogen increase was significantly inhibited. C. albicans injections were found to significantly elevate endogenous TNF levels in mice (enzyme-linked immunosorbent assay). It was concluded that C. albicans induces TNF in the mouse. Furthermore, these data give evidence which supports a relationship between TNF and the fibrinogen increase induced by C. albicans.

Specific assays of hemostasis proteins: Fibrinogen

Fibrinogen levels are considered a useful indicator in several pathological conditions and recent epidemiological studies have indicated a relationship between fibrinogen levels and increased risk of cardiovascular disease. An accurate measurement of this protein is therefore recommended and the Italian Committee for Standardization of Methods in Hematology and Laboratory has carried out a collaborative study to determine accuracy, precision and comparability of results obtained by six different methods, i.e., 1. Blombäck and Blombäck method, 2. clotting assay according to von Clauss, 3. radial immunodiffusion according to Mancini et al., 4. total amount of clottable fibrinogen by means of turbidimetric assay according to Ellis and Stransky, and 5. with ChromotimeSystem, 6. prothrombin time (PT)-derived fibrinogen assay on ACL coagulometer. The most accurate resulted the von Clauss method, but only if calibrated with an internal standard; in fact, when the manufacturer's tables are used, the method proved to be highly inaccurate. The best precision, both intra- and between-laboratory, was obtained by the PT-derived test on ACL. On the basis of this still incomplete evaluation of the CISMEL study data, we can conclude that: i. some methods used in clinical laboratories give accurate results only after adequate calibration; ii. a reference standard pool may be a valid tool for calibration and for a better between-laboratory comparability; iii. a predilution of the samples with high fibrinogen levels seems indicated; iv. automation markedly increases the precision of methods.

Predictive value of factors of the hemostatic system in screening procedures for coronary artery disease.

Thromboembolic events play a major pathogenetic role in the final occlusion of atherosclerotic vessels. May such a catastrophic event be predicted or an increased risk be indicated by analyzing the hemostatic system in plasma? A hugh literature exists about disturbances of the platelet, coagulation and fibrinolytic systems after atherosclerotic events such as myocardial infarction or stroke. This data, however, has no predictive significance. In contrast, the epidemiological studies in healthy persons have shown fibrinogen to be a potent risk predictor which is independent from other risk factors such as age, cholesterol or cigarette smoking. Results on factor VII:C are still controversial. Ongoing studies have included further factors of the hemostatic system. A second approach to elaborate the predictive power of hemostatic factors is to follow up patients with overt atherosclerotic disease and to correlate baseline hemostatic tests with event recurrences. The ECAT Angina Pectoris Study performed in 19 European clinical centers will present its prospective results by 1990. Some correlations of risk factors at baseline point to the many interrelationships among each other and to the need of careful statistical management of such data. It is reasonable to include fibrinogen in the recently developed coronary risk scores. So far, thrombin clotting time procedures, such as the Clauss method, appear to be appropriate for the purposes of risk prediction.

A Monoclonal Antibody-Based Qunatitative Enzyme Immunoassay for the Determination of Plasma Fibrinogen Concentartions

The most commonly used fibrinogen assays in the clinic are clotting rate assays, e.g. the Clauss method. Such functional assays may be disturbed by e.g. heparin, anticoagulant fibrinogen degradation products (FgDP) and in the case of a dysfibrinogenemia. Immunological methods would not suffer from these interferences. However, immunological assays for fibrinogen, which do not measure FgDPs, do not exist. To set up such an enzyme immunoassay (EIA) we developed two monoclonal antibodies. The first monoclonal antibody (G8) has its epitope in the carboxyl-terminal 150 amino acid stretches of the fibrinogen A alpha-chains. G8 is used to coat the wells of microtitration plates, and is the capture antibody in this EIA. The second antibody (Y18) has been described by us previously (Blood 1985; 66: 503). It is directed against fibrinopeptide A, covalently bound to the alpha-chains i.e. against the amino-terminal stretches of the A alpha-chains. Y18 is conjugated with horse-radish peroxidase, and used as tagging antibody. The EIA does not react with, and is not interfered by FgDP such as purified fragments X and Y, up to a concentration of 800 micrograms/ml. An FgDP mixture such as generated by Streptokinase treatment of plasma does not respond. Fibrin degradation products (whole blood lysate) up to 800 micrograms/ml do not interfere nor do heparin, EDTA or oxalate. The time-to-result of the EIA is only 45 minutes. Some patient plasmas yielded dose-response curves which are not parallel with the calibration curve of the EIA. An explanation for this phenomenon could not be given.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological study of intravenous anisoylated plasminogen streptokinase activator complex in acute myocardial infarction.

An anisoylated plasminogen streptokinase activator complex (APSAC) has been administered as a bolus intravenous injection of 30U to 14 patients with acute myocardial infarction. Systemic effects on coagulation and fibrinolysis were studied. In 1 patient, the treatment produced no biological modification, which could be explained by an increased streptokinase resistance in this patient, apparent from the sample collected before treatment. In the other patients, as expected, fibrinogen, plasminogen, alpha 2-antiplasmin and factors V and VIIIc fell dramatically, while there was an increase in serum fibrinogen degradation product concentrations. In addition, plasma fibrin derivatives increased during APSAC therapy, both in patients who had occluded or patent coronary arteries. Discrepancies were found between methods used to measure fibrinogen: with the Ellis and Stransky method, concentrations were higher than with the Clauss method; and for plasminogen, automated methods using different analysers may give higher results in some patients.

A functional photometric assay for plasma fibrinogen

We have developed a photometric assay for fibrinogen concentration. The sample is mixed with a snake venom enzyme (Batroxobin) and fibrin formation is recorded turbidimetrically at 334 nm. Reaction conditions are such that a linear increase in absorbance is obtained over a concentration range of fibrinogen from 80–700 mg/dl. Higher or lower ranges can be measured by adjusting the sample volume. Calibration is performed with a single standard. Precision and accuracy are comparable with the usual clinical chemical methods. Experiments with plasma samples digested with streptokinase showed that interference of fibrinolytic split products (FSP) is less than using the Clauss method. Large amounts of FSP in the sample, however, delay the increase in absorbance. This phenomenon can be utilized for the additional estimation of the FSP-content of the sample. Results obtained with the present method corresponded well with those obtained using the method of Clauss.

Gerinnungsveränderungen bei Kindern mit akuter myeloischer Leukämie: Thrombineffekt oder Proteolyse?

Sequential coagulation tests were carried out in 13 children with acute nonlymphoblastic leukemia (ANLL) treated with the German cooperative protocols BFM 78 and 82. The test program included a PTT, Quick's Prothrombin time, Thrombin time, Fibrinogen (Clauss method and RID), coagulation factors II, V, VII, AT III, antiplasmin, plasminogen and FDP. Severe coagulation changes could be demonstrated in ANLL patients with FAB type M2 (myeloblastic leukemia with maturation) and FAB type M5 (monocytic leukemia). Usually they were found already at the time of diagnosis and improved during induction therapy. A variety of coagulation changes were observed, resembling classical DIC, typical hyperfibrinolysis or atypical proteolysis. It is allowed to question the concept of DIC as the typical coagulation disturbance in children with ANLL.

Blood coagulation changes during effective thrombolysis using urokinase and heparin

The efficacy and the safeness of a new urokinase dosage regimen (loading dose 250,000 IU, initial maintenance dosage 2,000 IU/kg/h in combination with heparin) was studied in ten cases of deep vein thrombosis with regard to the changes of the blood coagulation and fibrinolytic enzyme system. The coagulation analyses demonstrated a pronounced activation of the fibrinolytic system with a statistically significant (p < 0.05) shortening of the euglobulin clot lysis time and increase of the FDP. The fibrinogen concentration ranged from 50 – 100 mg/dl already after 12 – 36 hours. Plasminogen was reduced by 63%, α 2-macroglobulin by 32% and factor VIII:C by 42% (p < 0.05 each). The decrease of fibrinogen (Clauss method) related well to the method of Ratnoff and Menzie, the reduction of plasminogen and the shortening of the euglobulin clot lysis time. According to our data, a sufficient plasma fibrinogenolytic activity may permit on its own an assessment of an adequate therapy with urokinase and the requirements of dose adjustment. The dosage regimen applied here proved effective and readily controllable. Relevant side reactions were not observed.

A New Congenital Abnormality of Human Fibrinogen

SummaryA dysfibrinogenemia (fibrinogen Bethesda II) was recognized in a Caucasian family with no history of bleeding or thrombosis. Thrombin and Reptilase times were prolonged, and there was a discrepancy between the fibrinogen levels determined by the fibrin tyrosine content and the Clauss method. Other coagulation and fibrinolytic measurements yielded normal values. The half-life of homologous and autologous 125I-fibrinogen in the propositus was approximately 20% shorter than the mean (3.70 ±0.45 days) of normal individuals. Fibrinogen Bethesda II exhibited normal migration during electrophoresis in polyacrylamide gel (in the presence of sodium dodecyl sulfate) or cellulose acetate, normal ultracentrifugal behavior, a normal chromatographic elution pattern and normal levels of hexose and sialic acid. Upon immunoelectrophoresis of whole plasma, the arc was more anodal than that produced by the fibrinogen of normal plasma. Functionally, fibrinogen Bethesda II prolonged the thrombin and Reptilase times of normal plasma and fibrinogen. The rate at which fibrinopeptides were released by thrombin or ancrod was similar to that for normal fibrinogen, and the amounts eventually reached the same levels, but a slight delay was consistently observed in the early stages of the reaction. Aggregation of fibrin monomers prepared with either thrombin or Reptilase was markedly abnormal, whereas the crosslinking (as assessed by polyacrylamide gel electrophoresis) did not differ from that of normal fibrin.

The Use of Laboratory Determinations in the Control of Streptokinase Therapy

The determination of the thrombin time was found to be overly sensitive to circulating fibrinogen and fibrin degradation products, and, therefore, inadequate as an aid in the control of the thrombolytic state induced by the administration of streptokinase therapy. Similarly, the method described by Clauss tended to yield inordinately low values for fibrinogen in the thrombolytic state. Use of either the prothrombin time or the prota- mine-thrombin time, or both, along with the thrombin time, afforded a more comprehensive and dependable evaluation of the abnormal hemostatic balance. The measurement of fibrinogen by means of turbidimetric (protein- precipitation) methods which do not involve conversion to fibrin in the plasma of patients undergoing treatment with streptokinase was superior in precision to the Clauss method.

Fibrinogen Bethesda: a congenital dysfibrinogenemia with delayed fibrinopeptide release.

A dysfibrinogenemia (fibrinogen Bethesda) was detected in a 9 yr old male of Mexican-English extraction who had a lifelong history of mild bleeding diathesis. The prothrombin and partial thromboplastin times were moderately prolonged; the thrombin and Reptilase times were markedly prolonged. The plasma fibrinogen level was normal by conventional methods but was markedly reduced by the Clauss method. Results of all other tests for clotting factors, fibrinolysis, antithrombin levels, clot stabilization, and fibrin(ogen) degradation products were normal. The patient's plasma and fibrinogen inhibited the clotting of normal plasma or fibrinogen by thrombin. Family studies revealed that the propositus' mother and two siblings exhibited these abnormalities to a lesser degree and indicated an autosomal dominant inheritance. Fibrinogen Bethesda was similar to normal fibrinogen in the following respects: metabolic turnover time (measured in the propositus' mother); immunodiffusion, ultracentrifugal, electrophoretic (on cellulose acetate or polyacrylamide gel), and chromatographic (on DEAE-cellulose) characteristics; sialic acid content; and aggregation of fibrin monomers. By contrast, fibrinogen Bethesda gave an abnormal immunoelectrophoretic pattern especially when whole plasma (as opposed to purified fibrinogen) was examined, and it showed a pronounced decrease in the rate of fibrinopeptide release by thrombin. This decrease, which was shown to involve both fibrinopeptides A and B, distinguishes fibrinogen Bethesda from previously reported dysfibrinogenemias.