Ethanol Gelation Test Research Articles

Blood Coagulation, Fibrinolysis and their Inhibitors Studies on Patients with Cancer

Fourteen parameters concerning coagulation, fibrinolysis and their inhibitors were measured in the 170 patients with various malignancies, including 68 cases of stomach cancer, 22 lung cancer, 21 colon and rectum cancer, 14 renal and urinary tract cancer, 9 primary hepatic cancer, 8 uterus and ovary cancer, 4 breast cancer and 19 others.Their parameters were platelet count, plasma fibrinogen level, prothrombin time, thrombotest, hepaplastintest, serial thrombin time, fibrinolytic split products, plasma plasminogen, ethanol gelation test, protamin sulfate test, cryofibrinogen, serum antithrombin III, serum α1 antitrypsin and serum α2 macroglobulin. Cases treated with anti-tumor agents were excluded in this study.Increased FDP in 52% (86 cases), prolonged serial thrombin time in 80%, positive paracoagulation tests and decreased antithrombin III were suggestive of increased activity of coagulation and fibrinolysis. The term of intravascular coagulation and fibrinolysis syndrome (ICF syndrome) was applied to the condition of increased FDP over 20μg/ml.And on the basis of fibrinogen (normal 200-400mg/dl) ICF syndrome was divided into 3 groups as following.Group I; hyperfibrinogenemiaPlatelet was increased, antithrombin III was decreased, plasminogen was normal or increased and paracoagulation tests tended to be positive. This conditions was defined as over compensated ICF syndrome, which was 16% (14 cases) of total ICF syndrome.Group II; normal fibrinogenemiaPlatelet was normal, antithrombin III was decreased, plasminogen was normal and paracoagulation test were less frequently positive. This condition was compensated ICF syndrome, which 59% (51 cases).Group III; hypofibrinogenemiaPlatelet was decreased, also antithrombin III and plasminogen were decreased. Paracoagulation tests were more frequently positive. This condition was defined as decompensated ICF syndrome, which was 25% (21 cases).Marked decrease of fibrinogen, platelet antithrombin III and plasminogen was observed in some patients in this group. DIC with bleeding tendency clinically observed belongs to this group.We concluded that it was significant to devide ICF syndrome due to malignancy into 3 groups on the basis of blood coagulation and fibrinnolysis for the management of patients with malignancy.

Studies on abnormalities of blood coagulation, fibrinolysis and their inhibitors in neoplasmas of blood and blood forming organs

A hemorrhagic tendency is a common feature in patients with leukemia and may occur in other forms of hematological malignancy. Bleeding in these diseases is generally associated with thrombocytopenia, but cases in which the hemorrhagic tendency was ascribed to pathological fibrinolytic activity or DIC have been described.From this point of view, the investigation on hemostatic abnormalities was carried out in 32 patients with acute leukemia (11 AML, 16 APL, 2 AM0L and 3 ALL), 31 with malignant lymphoma (stage III and IV), 13 with multiple myeloma and 20 with myeloproliferative disorders (10CML, 4CLL, 1 myelofibrosis, 3 primary thrombocythemia and 2 polycythemia vera).The diagnosis of DIC in neoplasmas of blood and blood forming organs is to be attentive because the finding in basic diseases themselves accompanied with thrombocytopenia.We studied F. VIII activity (F. VIII act.), F. VIII-related antigen (F. VIII ant.) and the ratio of F. VIII act./F. VIII ant. with other 18 hemostatic parameters: platelet count, PTT, prothrombin time, thrombotest, hepaplastintest, F. I, F. V, and F. VII were measured for coagulation system, STT (30'), FDP (HIT), plasminogen (single radial immunodiffusion method) and paracoagulation test (protamine sulphate test, ethanol gelation test and cryofibrinogen) for fibrinolytic system. Antithrombin III, alpha 1 antitrypsin and alpha 2 macroglobulin were also measured for their inhibitors by single radial immunodiffusion method.We observed a poor correlation between F. VIII act, and F. VIII ant. levels in neoplasmas of blood and blood forming organs in which the elevation of the F. VIII ant. was significantly higher than that of the F. VIII act.In most cases of DIC, F. VIII act, was various, but F. VIII ant. increased significantly, and therefore, the ratio of F. VIII act./F. VIII ant. was markedly low. A discrepancy between F. VIII act. and F. VIII ant. may be explained by consumption of F. VIII act. and accumulation of F. VIII ant., destruction of F. VIII act. and degradation of F. VIII ant. caused by plasmin, differences in the degree of catabolism of two components and so on.A discrepancy between F. VIII act. and F. VIII ant. disappered with improvement of DIC. It is suggested that the ratio of F. VIII act./F. VII ant. may be one of the most valuable parameters, not only for diagnosis but also prognosis of DIC.

Heparin-Neutralizing Activity, Total Progressive Antithrombin and the Ethanol Gel Test in Acute Chest Pain

92 patients admitted to a cardiac monitoring unit with chest pain were investigated. The mean heparin-neutralizing activity of the 34 patients with severe myocardial infarction was found to be significantly different from the mean of 70 normal controls. However, there was considerable overlap between individual observations and the normal range. Only 5 of the 34 patients with severe myocardial infarction had antithrombin levels outside the normal range. In patients with a severe myocardial infarct tested on the 2nd and 3rd day after infarction there was s significant increase in the number of positive ethanol gel tests compared with the other patients studied.

Identification of soluble fibrinogen fibrin monomer complexes by non-enzymatic polymerisation in the tissue.

In states of plasmic hypercoagulability and consumption coagulopathy ethanol favours the non-enzymatic polymerization of circulating soluble fibrinogen fibrin monomer complexes (FFMC) in vitro. The ethanol-gelation test of Godal and Abildgaard makes use of this phenomenon, called paracoagulation. The present studies show that it is also possible to visualize soluble FFMC by means of ethanol-gelation. In the electron microscope, FFMC, polymerized non-enzymatically by ethanol in the spleen, are characterized by plump or slender mycelioid fibrillar precipitates that show a uniform rhythmic transverse striation, a period-coincidental filamentary arrangement and an average periodicity of 23 nm. The ultrastructure demonstrates these ethanol-induced filaments to be in vitro-polymerized fibrin monomer derivatives. Paracoagulation with ethanol allows the identification of soluble FFMC in the tissue prior to the formation of highly polymerized fibrin-rich microthrombi, the established equivalents of the DIC-syndrome. The electron microscope studies also show the existence of a second type of fibrillary structure in the tissue polymerized by ethanol. This second type lacks the characteristic periodicity of fibrin and the period-coincidental arrangement of the filamentary structures, but is characterized by closely packed or chain-like aligned, irregularly sized spherical bodies. There is some evidence that these spherical bodies in vitro represent non-enzymatically polymerized complexes of fibrin monomers and fibrin degradation products (FDP), the equivalent of a limited local or generalized fibrinolysis in vivo.

Content of soluble fibrin in plasma of patients after myocardial infarction, with carcinomas and consumption coagulopathy

Soluble fibrin or fibrin monomer was isolated from plasma samples by affinity chromatography on insolubilized agarose coupled fibrinogen. The separated soluble fibrin was quantified by the staphylococcal clumping test. The results of quantitation of soluble fibrin in plasma of patients with myocardial infarction, of females with genital carcinomas and of patients with consumption coagulopathy initiated by different causes are presented. In some of the patients with infarction and the females with the genital tumors, where no change of the usual coagulation parameters were observed except the sometimes positive ethanol gelation test, the plasma fibrin concentration increased to 6 mg% above normal. The highest values up to about 6 – 14 mg% above the basal level were detected, when the clotting analysis showed signs of consumption coagulopathy in the patients. From the data presented the method of affinity chromatography seems to be suitable to evaluate the soluble fibrin concentration in plasma with a stimulated coagulation system.

A radioimmunoassay technique for the rapid measurement of human fibrinopeptide A

Fibrinopeptide A (FPA) has been measured in human plasma by a radioimmunoassay (RIA) modified as to give results available 28 or 8 hours after venipuncture. Rabbit antibodies were elicited against synthetic FPA coupled to thyroglobulin. The tracer was prepared by coupling FPA to desaminotyrosine and radioiodinated using the chloramine-T method. The labeled peptide was purified using a two-step method, which prevented label damage for 10 weeks. The shortening of the procedure was mainly achieved by reduction of the FPA extraction from plasma using a new equilibrium dialyser. The recovery of added FPA was 52 – 54%, the detection limit of the method 0.54 ng/ml. A further substantial shortening can be achieved by reduction of the incubation time from 24 to 21/2 hours, the results obtained with these two incubation times showing an excellent correlation when FPA is elevated above 2 ng/ml. In 24 normal individuals the FPA level was 1.01 ng/ml with an SD of 0.45. Grossly elevated levels were found in patients with various diseases in which the fibrinogen-fibrin conversion is accelerated. In patients with acute thrombosis, the FPA levels were markedly reduced 10 min after injection of 5000 U of Heparin. Infusions of the snake venom enzyme Defibrase in one patient produced high levels of FPA, associated with hypofibrinogenemia and negative ethanol gelation test. The rapid FPA assay is thus of particular interest in those clinical emergencies associated with a low plasma fibrinogen and a negative ethanol test.

The ethanol gelation test and thrombosis.

Intravascular fibrin formation takes place during thrombus formation or by activation of coagulation in circulating blood. Incorporation of fibrin in thrombi may be detected by usine 125I-labelled fibrinogen. Soluble fibrin may be present during thrombophilic states or during thromboembolic complications and diseminated intravascular coagulation (DIC) and may be detected and quantitated by various techniques such as N-terminal amino acid analysi, chromatographic procedures and so-called paracoagulation test, using ethanol or protamine sulfate. Finally, transformation of fibrinogen to fibrin may be indirectly assayed by quantitation of fibrinopeptide A released, by immunological technique. For routine purposes and especially in emergency situation, rapid and simple tests are required. In these circumstances, only the paracoagulation tests are at hand. Below, the present situation with regard to the ethanol gelation test (EGT) is outlined.

Marathon Run I: Effects on Blood Coagulation and Fibrinolysis

Blood coagulation and fibrinolysis were assessed in 13 Finnish amateur runners aged 31 to 48, and one 65-year old taking part in a non-competitive marathon (42.2 km). After the run the mean values of partial thromboplastin time showed a very significant shortening, whereas the mean values of the prothrombin time and of plasma fibrinogen were not significantly altered. The mean values of euglobulin lysis time were significantly shorter and the mean values of fibrin degradation products increased highly significantly. After the run, protamine sulphate was positive or strongly positive in all subjects, whereas the ethanol gelation test was negative in all runners; no cryofibrinogen was detected in any participant. Thus, running a marathon race affects the haemostatic balance and activates the fibrinolytic mechanism. The effects of training and physical fitness on the above parameters are discussed.

Hemostatic studies in obstructive jaundice

Hemostatic studies were performed in 64 patients with obstructive jaundice. Elevated fibrinogen level, shortened “r” and “r+k” and increased “ma” in thrombelastogram, prolonged serial tbrombin time, elevated fibrin degradation products, positive ethanol gelation test, decreased plasminogen activator activity, increased antiplasmin activity and increased α1-antitrypsin level were observed. All of these changes were statistically significant when compared with normal controls. In addition, levels of antithrombin-III and plasminogen decreased, but no significance was observed statistically. These hemostatic data revealed the presence of hypercoagulable state with decreased fibrinolytic activity and several evidences of intravascular coagulation.In order to elucidate the cause of these characteristic patterns in hemostatic studies, the correlation between these hemostatic parameters and liver function tests was studied. Changes in prothrombin time, plasminogen and antithrombin-III were well correlated with albumin, γ-globulin and TTT which mainly reflect liver cellular damage. Accordingly, low levels of plasminogen and antithrombin-III in some cases of this series may be attributable not only to enhanced consumption, but also to decreased synthesis in liver. In addition, serum fibrinogen level which is the most important factor in this hypercoagulable state showed positive correlation with alkaline phosphatase and cholesterol which reflect the degree of biliary obstruction.Six out of 64 cases were diagnosed as disseminated intravascular coagulation (DIC). Five of 6 cases were complicated with biliary infection. In this series, bacteriological examination of bile was performed in 43 cases and gram negative bacilli were found in 32 cases. In addition, 22 cases eventually suffered from cholangitis.Recently we experienced a very interesting patient with massive bleeding tendency complicated with biliary infection. High antiactivator activity in a low molecular weight fraction was detected in the gel filtration pattern of Lysine Sepharose treated serum through G-200 Sephadex. From these observations it is suggested that this low molecular fraction may have an important role in the decreased fibrinolytic activity in obstructive jaundice.The present study revealed the presence of hyper-coagulable state in obstructive jaundice which may be easely leading to DIC.

The abscence of intravascular clotting in rat anaphylaxis.

Intravascular fibrin formation could not be detected in various phase of IgE mediated anaphylactic shock of rats, either by using an isotope technique or testing the plasma samples by the ethanol gelation test.

Impaired Anticoagulant Effect of Heparin in the Artificial Kidney: An Experimental Study

Bjo˚rnson, J. & Godal, H. C. Impaired Anticoagulant Effect of Heparin in the Artificial Kidney. An Experimental Study. Scand. J. clin. Lab. Invest. 36, 581–589, 1976.Dialysis of blood and plasma was performed in vitro, in a ‘mini-Kiil’ dialyser as well as in dialysis bags. A marked shortening of the thrombin-clotting time was observed, indicating fall in heparin anticoagulant effect. The concentration of heparin, however, as measured by polybrene titration, was substantially less reduced. Fibrin formation, as evidenced by the ethanol gelation test, occurred more often in the dialysed than in the control plasma. In conclusion, the discrepancy between concentration and anticoagulant effect of heparin could be partly explained by influx from the dialysate of calcium, magnesium, and acetate ions. The fibrin-polymerizing effect of these ions was confirmed by a shortening of the clotting time with Reptilase, a proteolytic enzyme not influenced by thrombin inhibitors such as heparin. In addition, liberation of platelet factor 4 may be responsible for some reduction in antithrombin activity of heparin. No evidence of heparin being dialysed or adhering to the dialysis membrane was found.

Coagulation studies and platelet function after somatostatin infusion.

The effect of short- and long-term somatostatin (GIF) administration on haemostatic function in man was investigated. The dosage programme applied in this study was 250 mug GIF as a bolus injection and 250 mug GIF/h by way of infusion. In five healthy volunteers a short-term (3h) treatment resulted in a statistically significant drop of platelet count and impairment of platelet aggregation at the end of infusion. However, these changes were within the physiologically normal range and disappeared after two hours on all subjects. Other parameters such as bleeding time, thromboplastin and partial thromboplastin time, fibrinogen, fibrin/fibrinogen split products, plasma factor XIII, ethanol gelation test were not affected. In two patients with gastric haemorrhage and persistent amylasaemia a 67 or 120-h treatment induced no remarkable haemostatic defect. By contrast, peptic ulcer bleeding in one patient stopped 60 min after starting the GIF infusion. These studies indicated that somatostatin administration in man at the dosage programme used neither results in clinical evidence indicating bleeding tendency nor does it influence laboratory parameters in an apparent way.

Abnormal coagulation and fibrinolysis in eclampsia: A clinical and laboratory correlation study

This study evaluated the existence of abnormally increased coagulation and fibrinolysis in 33 severely toxemic and eclamptic women by means of a combined hemotologic profile with clinical and morphologic correlations. The dominant findings were: different degrees of thrombocytopenia, abnormal levels of blood fibrinogen, prolonged thrombin time, and positive protamine sulfate test. Altered activated partial thromboplastin time and positive ethanol gelation test were slightly less frequent, and only few cases showed prolonged prothrombin time or early lysis of euglobulins. These abnormalities seemed to be more numerous and more pronounced in the worst cases of the series and their severity seemed to be associated with the age of the patient and the presence of previous underlying disease. These variously handicapped pregnant women exhibited worse hematologic hematologic abnormalities, and provided most of the fatal cases in the series. Finally, the main findings were discussed and commented upon.

Comparison between a Modified Ethanol Gelation Test and Protamine Sulfate Test. Experimental Studies

A comparative experimental study has been made to correlate the protamine sulfate test and a modified ethanol gelation test, based on clinical observations of the solubility of a gel formed at 20 degrees C (Godal and Abildgaard procedure) when it was transferred to a bath at 37 degrees C. Two different results were obtained: the gel remained insoluble at 37 degrees C or it became completely soluble, with intermediate degrees of partial solubility. Our studies indicate that this is due to the amount of fibrin monomers formed and the level of fibrinogen: the first are responsible for the insolubility of the gel and the second for its solubility. This furnishes us with useful information for diagnostic purposes. We found the protamine sulfate test more sensitive than the ethanol gelation test, and its sensitivity increased when fibrinogen level decreased. An insoluble gelation test is a sure indication of the presence of fibrin monomers, but a soluble gel calls for the protamine sulfate test to confirm this or the existence of high fibrinogen level.

Impaired anticoagulant effect of heparin in the artificial kidney. An experimental study.

Dialysis of blood and plasma was performed in vitro, in a 'mini-Kill' dialyser as well as in dialysis bags. A marked shortening of the thrombin-clotting time was observed, indicating fall in heparin anticoagulant effect. The concentration of heparin, however, as measured by polybrene titration, was substantially less reduced. Fibrin formation, as evidenced by the ethanol gelation test, occurred more often in the dialysed than in the control plasma. In conclusion, the discrepancy between concentration and anticoagulant effect of heparin could be partly explained by influx from the dialysate of calcium, magnesium, and acetate ions. The fibrin-polymerizing effect of these ions was confirmed by a shortening of the clotting time with Reptilase, a proteolytic enzyme not influenced by thrombin inhibitors such as heparin. In addition, liberation of platelet factor 4 may be responsible for some reduction in antithrombin activity of heparin. No evidence of heparin being dialysed or adhering to the dialysis membrane was found.

Coagulation profile and the ethanol gelation test with special reference to components consumed during coagulation.

In 753 consequtive blood samples (from the same number of patients) routinely examined for coagulation abnormalities, a positive ethanol gelation test was found in 160. In 7 samples with plasma fibrinogen below 150 mg/100 ml and a positive test, other laboratory signs of consumption coagulopathy (low thrombocyte count, low factor V, low antithrombin III, large amounts of FDP in serum and great discrepancies between Normotest and Thrombotest) were regularly observed. Also at normal or high fibrinogen levels, a positive ethanol gelation test was more frequently associated with low thrombocyte counts, low factor V and Normotest/Thrombotest discrepancy than was a negative test. In 46% of the samples with a positive test, fibrinogen was higher than 500 mg/100 ml (versus 4% in those with a negative test). In one-third of these samples, all other parameters gave normal results.

Heparin precipitable fraction (HPF) from dermatological patients. I. Characterization of the thrombin-clottable protein

A heparin precipitable fraction (HPF) was obtained upon cooling (+4°C) of heparinized plasma from dermatological inpatients, mainly affected with psoriasis arthropathica. HPF, isolated by centrifugation (+4°C) and redissolved in 0.15 M NaCl at 37°C, consisted of about 60% thrombin-clottable protein. This portion could also be removed by heating at 56°C for 10 min. Polycrylamide gel electrophoresis demonstrated the larger part of HPF to be fibrinogen. In addition varying amounts of albumin and an unidentified protein (X-component) and traces of other plasma proteins were observed. SDS gel electrophoresis demonstrated the fibrinogen-related protein to have the same molecular weight as purified human fibrinogen. Upon reduction with beta-mercaptoethanol only intact Aα, Bβ and γ-chains were observed. Quantitative N-terminal amino acid analysis showed insignificant amounts of N-terminal glycine indicating the absence of soluble fibrin. Eighteen out of 21 patients with HPF had a negative ethanol gelation test in citrated plasma, and 17 out of 21 patients demonstrated no detectable serum FDP. Possibly, HPF exists in plasma as a complex between native fibrinogen, the X-component and heparin.

Bleeding in Uremic Patients after Carbenicillin

Hemorrhagic diathesis was observed in patients with renal insufficiency after carbenicillin at serum levels greater than 300 mug/ml. Normal coagulation factors (F. I, II, V, VII, VIII, X), normal PTT, normal platelet counts, negative ethanol gelation test (fibrin monomers) were found as well as a prolongation of thromboplastin time (Quick), thrombin time, reptilase time and thrombin coagulase time. Platelet function was disturbed. In addition, the plasmatic system was involved: inhibition of fibrinogen-fibrin conversion (Belitser assay) and enhanced antithrombin III activity; in vivo the latter was ascribed to a heparin-like activity. In vitro, abnormal III was seen: however an enhanced antithrombin III activity in vitro was not found with carbenicillin and various penicillin derivatives. This study demonstrates that carbenicillin, in addition to its known effect on platelet function, also disturbs the plasmatic coagulation system. This additional effect of carbenicillin is clinically important since protamin chloride effectively blocks bleeding without interfering with antibacterial activity. Both penicillin and penicillin derivatives have been shown to interfere with hemostasis and to cause clinically manifest hemorrhagic diathesis (Fleming and Fish 1947, Lurie et al. 1970a, b, McClure et al. 1970, Yudis et al. 1972, Demos 1971, Waisbren et al. 1971). Carbenicillin interferes with ADP-, collagen- or thrombin-induced platelet aggregation and with the release reaction both in vivo (McClure et al. 1970, Cazenae et al. 1973) and in vitro (McClure et al. 1970, Cazenave et al. 1973). In addition Lurie and colleagues (1970b) concluded that an inhibition of the conversion of fibrinogen to fibrin is involved although no experimental details were given. Later Brown and colleagues (1974) concluded that carbenicillin at usual dose levels "only affects the platelet component of hemostasis and has little effect on fibrin formation or other phases of coagulation in patients with normal renal function".

The influence of large doses of hydrocortisone on the generalized Shwartzman reaction in rabbit

The influence of large doses (50 mg/kg) of hydrocortisone, given after both or only after second endotoxin dose, on the generalized Shwartzman reaction was studied. The effect of hydrocortisone alone was also investigated. The hydrocortisone injection after the first endotoxin dose lessened the decrease in platelet count but this effect was not observed after the second endotoxin dose. The addition of hydrocortisone to endotoxin did not change the pattern of fibrinogen concentration in plasma but did increase the frequency of positive ethanol gelation test. The Fibrinogen/Fibrin Related Antigen content in serum rose gradually throughout the experiments, especially in animals given hydrocortisone after both endotoxin doses. The injections of hydrocortisone alone inhibited fibrinolytic activity in euglobulins but did not change the content of plasminogen activator in the outer medullary zone of rabbit kidney. In comparison with results of previous studies on generalized Shwartzman reaction (Thrombos. Res.: 6 , 399, 1975), the addition of hydrocortisone to endotoxine increased the inhibition of fibrinolytic activity in euglobulins. The decrease of plasminogen activator content in the outer medullary zone of kidneys was less pronounced when first hydrocortisone injection followed the provocative endotoxin dose than after both injections of endotoxin and hydrocortisone. Injections of hydrocortisone following both doses of endotoxin increased fibrin deposition within kidney glomeruli. It is supposed that this effect is due to the inhibited release of tissue plasminogen activator in rabbit kidney after large doses of hydrocortisone.

Various Prothrombin Complex Concentrates and their Effect on Coagulation and Fibrinolysis In Vivo

Thromboembolic complications occurring in patients treated with factor IX concentrates have been reported. To study the thrombogenicity various types of factor IX concentrates (50 or 100 units F. IX/kg bodyweight) have been infused in dogs. As control albumin was given. The various components of the coagulation and fibrinolytic system have been assayed before the infusion and at various intervals after the end of infusion (0, 1, 4 and 24 hrs). Konyne resulted in marked activation of the coagulation process with decrease of platelets, fibrinogen, F. VIII and appearance of FDP and positive ethanol gelation test. Prothromplex and Preconativ gave no significant changes. Preconativ is prepared without addition of heparin during the procedure.