Hemostatic abnormalities in malignancy, a prospective study of one hundred eight patients. Part I. Coagulation studies.

A prospective study of hemostatic abnormalities in 108 cancer patients was undertken at an oncology clinic in a university teaching hospital. Tests included Quick prothrombin time, activated partial thromboplastin time, thrombin time, platelet count, modified Ivy bleeding time, fibrinogen, fibrin degradation products (FDP), euglobulin lysis time, protamine sulfate test, and factor V, VII, VIII and X assays. Ninety-eight per cent of the patients had one or more abnormal coagulation tests. The commonest abnormalities were elevated fibrin degradation products and prolonged thrombin time. Thrombocytosis occurred in 57% of patients, hyperfibrinogenemia in 46%, thrombocytopenia in 11%, and non had hypofibrinogenmia. It is suggested that platelet count, fibrinogen concentration, and serum FDP assay are the most useful tests in assessing the hemostatic abnormalities in cancer patients, although thrombin time, factor V assay, and bleeding time may also be helpful. The peripheral blood smears of 53 patients were reviewed, and only one showed microangiopathic hemolytic anemia. The data illustrate that subclinical coagulopathy is relatively frequent in patients with malignancy.     

Measurement of plasma fibrin D-dimer levels with the use of a monoclonal antibody coupled to latex beads

Recently, monoclonal antibody (DD-3B6) to fibrin D-dimer was prepared and coupled to latex beads to provide a specific test (Dimertest) for fibrinolysis. The purpose of this study was to evaluate the Dimertest assay as a clinical laboratory test for the measurement of plasma fibrin D-dimer derivatives. The Dimer-test assay specifically detected 2 micrograms/mL of purified fibrin D-dimer or fibrin D-dimer/fragment E complex added to afibrinogenemic plasma but did not detect 500 micrograms/mL of either fibrinogen fragments X, D, E, or 160 micrograms/mL cross-linked fibrinogen. The fibrin(ogen) degradation product (FDP) assays of American Dade or Wellcome Diagnostics detected 5.0 micrograms/mL of fibrin D-dimer and from 1 to 10 micrograms/mL of the other FDPs. Twenty-eight percent of 150 random plasma samples assayed from hospitalized patients were positive for fibrin D-dimer derivatives. Plasma samples from 152 patients suspected of having disseminated intravascular coagulation (DIC) were assayed for serum FDP (Wellcome Diagnostics) and plasma fibrin D-dimer derivatives. Samples from 69% of patients with serum FDP levels less than 10 micrograms/mL, and more than 90% of those with serum FDP levels greater than 10 micrograms/mL, were positive for fibrin D-dimer derivatives. Dimertest results were not modified by heparin, streptokinase, freeze-thawing, or clotting plasma. Serum fibrinogen-related antigens were immunoadsorbed from Dimer-test positive sera by anti-fibrinogen antibody and formalin-fixed Cowan I strain Staphylococcus aureus. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein blotting with the use of monoclonal antibody DD-3B6 demonstrated a protein band with similar mobility to purified D-dimer. The measurement of plasma fibrin D-dimer derivatives by the Dimertest assay is a rapid, sensitive, and specific laboratory test for fibrinolysis. The Dimertest assay has proven to be a useful addition to the clinical laboratory and should be helpful in the diagnosis and management of patients with diseases associated with fibrinolysis.     

Comparison of a direct latex-agglutination technic with the tanned red cell hemagglutination inhibition immunoassay (TRCHII) for semiquantitation of fibrinogen/fibrin degradation products.

A new latex-agglutination kit for rapid screening for fibrinogen/fibrin degradation products (FDP/fdp) has been compared with a standard tanned red cell hemagglutination inhibition immunoassay (TRCHII). The latex-agglutination test results agreed with the TRCHII results for 489 of 588 samples (83%). FDP/fdp values were elevated by the latex-agglutination kit when normal by the TRCHII for 79 (13%) samples, and the reverse was true for 20 (3%) samples. Serial dilution of serum sample allowed reliable semiquantitation of FDP/fdp levels compared with TRCHII results (r=less than .001). The latex-agglutination test gave FDP/fdp values of larger than or equal to 10 mug. per ml. for sera from 11 of 13 patients who had acute pulmonary embolism and for only one of 24 normal control samples. The direct latex-agglutination kit for FDP/fdp appears to have appropriate sensitivity to serve as a screening test for acute pulmonary embolism in patients not receiving heparin therapy.     

The interference of fibrinogen and heparin with the determination of circulating immune complexes in the C1q-binding assay.

When citrate plasma and serum of the same individual were tested simultaneously in the C1q-binding assay (C1qBA), binding levels in plasma were found to be 90-400% higher than in serum. The difference in 125I-Clq binding was due to the presence of fibrinogen in plasma. It was shown that complex formation between fibrinogen and 125I-Clq occurs and that this complex precipitates in the presence of polyethylene glycol, leading to the false positive results in the ClqBA. When heparin plasma was used to the assay, heparin itself also induced an increase in 125I-C1q binding that was not based on the presence of immune complexes. The effect of both fibrinogen and heparin could be inhibited by addition of protamine sulphate. Therefore, pretreatment of plasma with protamine sulphate makes it possible to use plasma samples for a reliable determination of C1q-binding levels. However, serum that is well clotted should be used preferentially.     

Elevated fibrinogen in an acute phase reaction prolongs the reptilase time but typically not the thrombin time

The effects of elevated fibrinogen on thrombin and reptilase times have not been well documented. High fibrinogen levels are common (38% of specimens submitted to our coagulation laboratory). Among 102 patients in the present study, an endogenously elevated fibrinogen level was significantly associated, as follows, with prolonged reptilase times: 1 (4%) of 28 with normal fibrinogen levels, 6 (20%) of 30 with levels in the 400 to 700 mg/dL (4.0-7.0 g/L) range, 10 (34%) of 29 with levels in the 700 to 1,000 mg/dL (7.0-10.0 g/L) range, and 7 (47%) of 15 with fibrinogen levels greater than 1,000 mg/dL (10.0 g/L). This association was independent of patient age and fibrin degradation product titer. In contrast, thrombin time was not altered notably by elevated fibrinogen levels. In 4 patients studied further, the prolonged clotting times could be corrected or nearly corrected by adding calcium chloride or albumin, whereas no such corrections were demonstrable in samples from several hereditary dysfibrinogenemia control subjects. An elevated fibrinogen level is common and is associated with reptilase time prolongations. For patients with prolonged reptilase times, a fibrinogen assay is suggested before establishing a diagnosis of dysfibrinogenemia.     

Serum fibrin/fibrinogen degradation products as a prognostic index in acute myocardial infarction.

A study of the prognostic value of serum fibrin/fibrinogen degradation products (FDP) in 137 consecutive patients with acute myocardial infarction showed a positive correlation between high FDP levels and poor prognosis. Both the frequency of complications and the mortality were related to increased levels of FDP, the highest of which were found between the fourth and eighth days after infarction.     

Differentiation of fibrinolysis and fibrinogenolysis by analysis of FDP fragments]

We previously analysed the fragments of fibrin/fibrinogen degradation products (FDP) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with immunoblotting. In this report, we studied the semi-quantitative analysis of fibrinolysis (degradation of cross-linked fibrin) and fibrinogenolysis (degradation of fibrinogen and/or unstable fibrin) of patients' samples by our method. In vitro study of FDP made it clear that an appearance of D fragment confirmed fibrinogenolysis and an appearance of DD fragment and/or high molecular weight fragments which have higher molecular weight than DY or X fragment confirmed fibrinolysis. In addition, a study with mixtures of various concentrations of fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP) demonstrated a dose dependent intensity of band by immunoblot method. These results show that our method is favorable for the semi-quantitative analysis of fibrinolysis and fibrinogenolysis. We applied the method to 6 samples from patients with disseminated intravascular coagulation (DIC). Consequently, fibrinogenolysis was observed in all of 6 samples, in which fibrinogenolysis was more enhanced than fibrinolysis in one sample, and an equivalent degree of fibrinolysis and fibrinogenolysis were observed in 3 of 6 samples. Although our method was probably devoid of the ability to distinguish FgDP from degradation products of unstable fibrin, these findings indicate that fibrinogenolysis is, at any rate, enhanced in the majority of patients with DIC, besides fibrinolysis.     

Analysis of cross-linked fibrin and fibrinogen degradation products with SDS-PAGE and immunoblot

SDS-PAGE and immunoblot technique with anti-FDP-D, -FDP-E, -fibrinogen antibody or anti-FDP-DD monoclonal antibody were applied to analyze FDP fragments prepared from cross-linked fibrin and fibrinogen with plasmic digestion in vitro. FDP fragments of DY(260K), DD(187K), X(245K), Y(166K), D(77K, 97K), E1(58K), E2(46K) and E3(40K) were identified from data of molecular weight and reactivity to four antibodies referred to reports of other investigators. Serum FDP fragments from five DIC suspected patients were analyzed by the same methods. In two patients' sera, DD fragment was a main component, and in the other three patients' sera, D fragment was a main fraction. Proportions of high molecular weight fragments of FDP were considerably different in patients' sera. Appearance of D fragment in our cases was considered to be derived from unstable fibrin (fibrin monomer and dimer) rather than from fibrinogen. Molecular weight of DD fragments from patients' sera had heterogeneity (160 approximately 180K), and the values were different from that (187K) prepared from cross-linked fibrin. In conclusion, SDS-PAGE and immunoblot analysis of serum fragments of FDP will be an useful technique to investigate the clinical and pathological condition of DIC.     

Plasminogen activators in ovarian tumours.

Ovarian tumours obtained at laparotomy were histochemically examined for their local fibrinolytic activity, and simultaneous fibrin/fibrinogen degradation products (FDP) were determined in the serum. The fibrinolytic activity was confined mainly to vessels of both malignant and benign tumours. A very close correlation was demonstrated between the fibrinolytic activity and the vascularity of the sections. FDP were found in the serum in 13 of 14 patients with malignant tumours, but in none with benign tumours. The difference in occurrence of FDP in patients with malignant and benign tumours might be due to the invasive growth of the former with the entrance of thromboplastic substances, fibrinolytic activators or locally formed FDP into the bloodstream.     

Optimization of heparinization in clinical double filtration plasmapheresis

Heparin has generally been used as an anticoagulant during plasmapheresis. In this study plasma heparin levels were studied in nine patients before double filtration plasmapheresis (DFPP), 30, 60 and 120 minutes after the start of DFPP and at the end of DFPP. Prothrombin time (PT), activated partial thromboplastin time (APTT), bleeding time (BT), plasma fibrinogen levels, FDP and general blood cell examination (CBC) were measured pre and post DFPP. Heparin levels in plasma were lower than 1 (IU/ml) under the dosage of heparin nearly 40 IU/kg/h of heparin administered during DFPP. APTT before DFPP (36.5 +/- 8.2 sec) was nearly double the post-DFPP value (61.4 +/- 12.2 sec). In two patients who were given 30 IU/kg/h of heparin during DFPP, clotting occurred in the DFPP circuit. In conclusion, the optimized dosage of heparin was 40 IU per kg of body weight per hour during DFPP.     

Fibrinolysis in idiopathic menorrhagia.

Serum fibrin/fibrinogen degradation product (FDP) was studied in 64 patients of menorrhagia without any organic cause in addition to 24 healthy women by Thrombo - Wellcotest HA - 13 Kit (Wellcome, England). Serum FDP levels were found to be less than 10 micrograms/ml in healthy subjects, whereas in idiopathic menorrhagia it was more than 10 micrograms/ml in 59.34% patients. Semi-quantitative estimation of FDP in 14 patients of idiopathic menorrhagia indicated a positive correlation between duration of bleeding and FDP levels. Bleeding appears to be due to increased fibrinolytic activity in uterus secondary to plasminogen activator. Such patients are likely to be benefitted with anti-fibrinolytic agents.     

Detection of fibrin monomer. Comparison of the immune precipitate method with the serial-dilution protamine sulfate test and the ethanol gel test.

In a previous publication, a method for measuring fibrin monomer in plasma with the use of an immune precipitate of fibrinogen was described. The method was found to be more sensitive to unstabilized fibrin monomer than the serial-dilution protamine sulfate test, or the ethanol gel test, and detected fibrin in a mixture of the plasmin digestion products of fibrin. In the present study the sensitivity of the immune precipitate method for detecting specific plasmin digestion products of fibrin X, Y, D, and E, its sensitivity for stabilized fibrin monomer complexes, and its sensitivity for fibrin retaining B-peptide were measured and compared with the corresponding sensitivities of the serial-dilution protamine sulfate test and the ethanol gel test for detecting these same products. The immune precipitate method was found to be highly sensitive to stabilized fibrin monomer complexes and to fibrin retaining B-peptide; to be significantly less sensitive to the plasmin digestion fragments X, Y, and E; and to be insensitive to fragment D. The serial-dilution protamine sulfate test and the ethanol gel test were found to be insensitive to all of the plasmin digestion products of fibrin and less sensitive to the other fibrin complexes.     

Heparin reduction with the use of cardiotomy suction is associated with hyperfibrinolysis during distal aortic perfusion with a heparin-coated semi-closed cardiopulmonary bypass system

We sought to elucidate the effects of different anticoagulation levels and the use of cardiotomy suction on the postoperative coagulatory and fibrinolytic systems in patients undergoing distal aortic perfusion using a fully heparin-coated (semi-)closed cardiopulmonary bypass (CPB) system incorporating a soft reservoir bag. Thirty-two patients were divided into two groups: those who underwent cardiotomy suction (S group, 18 patients) and those who did not (N group, 14 patients). We administered 1–2 mg/kg heparin in the S group, which achieved an activated clotting time (ACT) of 345 ± 71 s. In the N group, we administered 0.7–1 mg/kg heparin, which achieved an ACT of 297 ± 52 s. Data on platelet counts and serum levels of fibrinogen, antithrombin III, D-dimer, and fibrin degradation products (FDP) were collected, and factors influencing these variables were analyzed by multiple regression analysis. Both the patient group and the initial ACT level were independent factors influencing postoperative levels of FDP and D-dimer, whereas peak ACT level and the use of selective visceral/renal shunt/perfusion, but not the patient group, were independent factors influencing the postoperative platelet counts. In the S group, a significant inverse correlation was found between the ACT and levels of FDP or D-dimer, whereas no correlation was found in the N group. The use of cardiotomy suction was associated with elevated FDP and D-dimer levels even when a fully heparin-coated semi-closed CPB system was used. Lower ACT levels with the use of cardiotomy suction were associated with higher FDP and D-dimer levels, whereas such a relationship did not exist when cardiotomy suction was not used.     

Clinical significance of a new fibrinogen/fibrin degradation products(FDP) test using plasma samples for the diagnosis of fibrinogenolysis and fibrinolysis

Recently, a new fibrinogen/fibrin degradation products(FDP) test using monoclonal antibodies against FDP(LPIA FDP-P: FDP-P) has been developed, which is able to measure FDP directly in plasma. The objective of this study is to clarify clinical significance of the test in the diagnosis of fibrinogenolysis and fibrinolysis in comparison with a conventional FDP test using polyclonal antibodies against fibrinogen(FDP-S) and D-dimer test using monoclonal antibodies against D-dimer(D-D). The monoclonal antibodies used in FDP-P test was shown to recognize fragment X, Y and D1 derived from fibrinogen digested by urokinase, and was also to recognize XDP fragments, D-dimer and D derived from cross-linked fibrin digested by tissue plasminogen activator using SDS-PAGE and immunoblotting analysis. There was a good correlation of FDP levels between FDP-P test and FDP-S test. However, levels of FDP in both tests were discrepant in several samples. There was a tendency that the levels of FDP were higher in FDP-S test than in FDP-P test. Such discrepancy was suggesting that soluble fibrin monomer complex(FM) was recognized by the antibodies used in FDP-S test, but not recognized by the antibodies used in FDP-P test. There was also a good correlation of FDP levels between FDP-P test and D-D test. However, the levels of FDP in both tests were discrepant in several samples. The levels of FDP were higher in FDP-P test than in D-D test. These discrepant samples had lower levels of antiplasmin and higher levels of plasmin antiplasmin complex(PIC), and also showed XDP fragments, D-dimer, X, Y, and D1 by using SDS-PAGE. These observations suggest that D-D test measures only fibrinolytic fragments, while FDP-P test measures fibrinogenolytic fragments as well as fibrinolysis. In results, the FDP-P test was confirmed to be a useful tool to examine fibrinogenolysis as well as fibrinolysis more specifically than the conventional FDP test.     

A study of two methods for estimating plasma fibrinogen and the effect of epsilon aminocaproic acid and protamine

Fibrinogen concentrations were determined in normal plasma and in plasma from patients with high and low levels. There was a good correlation between the results of a rapid semi-quantitative fibrinogen titre technique and those of a quantitative assay of coagulable fibrinogen. In normal subjects fibrinogen levels were not significantly influenced by taking blood into epsilon aminocaproic acid (EACA) or by the addition of protamine to plasma. In patients with the defibrination syndrome in whom increased plasma fibrinolysis was not detected, fibrinogen levels were not affected by taking blood into EACA but considerably increased levels were observed after the addition of protamine to plasma. In patients undergoing thrombolytic therapy the fibrinogen levels measured were increased both in blood taken into EACA and in plasma containing protamine. It is suggested that EACA acted by preventing lysis in vitro whilst protamine counteracted abnormal fibrin polymerization. The pattern of results may be of diagnostic importance.     

Anwendung von niedermolekularem Heparin bei Hämodialysepatienten

Summary Low-molecular-weight (LMW) heparin has been compared to standard unfractionated (UF) heparin in a total of 49 patients on hemodialysis and hemofiltration in order to determine the necessary therapeutic dose and its effect on the coagulation system. A LMW heparin dose corresponding to 50% of the normal UF heparin dose was found to produce similar plasma heparin levels (anti-FXa-U/ml) in particular on minimal heparinization. At higher doses, UF heparin produced a more marked increase in plasma-heparin than did LMW heparin. Highly significant differences were found between UF and LMW heparin in their effects on PTT and thrombin time. Partial thromboplastin time (PTT) increased under UF heparin by an average of 120 s whereas LMW heparin only produced an increase of 5–7 s. Thrombin time was increased by 250–280 s under UF heparin and by 5–8 s under LMW heparin. With this LMW heparin dose of 50% of the UF heparin dose, no thrombosis of the extracorporal system occurred and no macroscopic detectable thrombotic material was found in the dialyzers or filters. No significant differences were observed between the effects of UF and LMW heparin on Factor VIII activity and fibrin monomers, so that a difference in coagulation activation between the two heparins can be excluded. Furthermore, there were no changes in thromboplastin time according to Quick, fibrinogen, antithrombin III, plasminogen, and a₂-antiplasmin. Thus effective Anti-FXa levels and by similar antithrombotic activity, LMW heparin will probably present less of a bleeding risk because of its reduced effect on PTT and thrombin time. LMW heparin therefore appears to be a good alternative to UF heparin for patients with renal insufficiency requiring dialysis. LMW heparin is indicated in particular in patients at bleeding risk, with diabetic retinopathy, on therapy with oral anticoagulants or platelet aggregation inhibitors, and with thrombocytopenia.

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Abkürzungsverzeichnis F Faktor - FFA Free Fatty Acids=freie Fettsäuren - LMW-Heparin Low Molecular Weight Heparin=niedermolekulares Heparin - LPL Lipoprotein-Lipase - PTT Partial Thromboplastin Time=Partielle Thromboplastinzeit - UF-Heparin unfraktioniertes Heparin     

A case of SLE with positive antiphospholipid antibody and various coagulopathy]

We described an 11 year-old boy with systemic lupus erythematosus (SLE) and various coagulopathy. He had purpura on the legs, pancytopenia, positive anti-DNA antibodies and hypocomplementia. Hematological examination also showed that platelet counts were 80 x 10(3)/microliter, lupus anticoagulant and anticardiolipin antibodies were positive. The aPTT was remarkably prolonged. Those laboratory findings fulfilled the criteria of antiphospholipid syndrome. Following treatment with predonisolone and heparin, thrombocytopenia improved. When heparin discontinued and renal biopsy was performed, severe thrombocytopenia recureded. FDP and FDP-DD became high, but the aPTT was not prolonged. Thrombocytopenia didn't improved by the therapy with heparin, high dose of methylpredonisolone, FOY and gamma-globulin. However by the therapy with both warfarin and cyclophosphamide, remarkable improvement of coagulopathy was absorbed. Probably anticardiolipin antibodies and disseminated intravascular coagulation (DIC) participate in the various coagulopathy in this case.     

Inhibition of in vitro platelet aggregation and release and fibrinolysis

Inhibition of in vitro platelet aggregation and release of contents of platelet granules is necessary in order to assess accurately platelet activation in vivo. This can be accomplished by using a variety of inhibitors added to blood collection containers. An additive mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD) provides a practical alternative to a mixture of acid citrate dextrose (ACD), acetylsalicylic acid (aspirin), and prostaglandin E1 (PGE1) because of the stability problems associated with PGE1. Inhibition of in vitro fibrinolysis is essential for the accurate measurement of fibrin degradation products (FDP). This can be accomplished by using a mixture of thrombin, soybean trypsin, or aprotinin into which blood is collected. However, in patients receiving heparin, the fibrinolysis inhibitor mixture is ineffective unless it is supplemented with reptilase. With increasing use of recombinant tissue-type plasminogen activator therapy (rt-PA), an inhibitor such as D-phenylalanine-proline-arginine-chloromethylketone (PPACK) used as a blood collection additive is superior to a conventional protease inhibitor, such as aprotinin.     

Clinical evaluation of a test for plasma fibrin/fibrinogen degradation products (FDP) based on monoclonal anti-FDP antibody technology: an application for the scoring system of the disseminated intravascular coagulation (DIC) diagnostic criteria

Fibrin/fibrinogen degradation products(FDP) have been measured using serum samples which were specially prepared for the FDP test because of the usage of anti-human fibrinogen antibody for the assay. Since diagnostic criteria for DIC were established by the study group on thrombosis and hemostasis which is supported by the Japanese Ministry of Health and Welfare(JMHW), serum FDP assay have been used as standard methods to diagnose DIC in Japan. Recently, a reagent using an anti-human FDP monoclonal antibody was developed and this has enabled the use of plasma samples for FDP measurement. The comparability, especially of the DIC score, of a new assay, Latex test BL-2 P-FDP, using plasma samples with a conventional assay for serum was investigated. Two sets of DIC scores based on data from the two tests were compared and the correlation was high with 97.5% of the patients being diagnosed with the same DIC status. In four disease groups--DIC, thrombosis, leukemia and solid cancer--high comparability between the two tests was also shown and no significant difference was observed in the correlation coefficient and the slope coefficient between serum and plasma samples. To conclude, it is suggested that "Latex test BL-2 P-FDP" is applicable to the diagnostic criteria for DIC from JMHW without any difficulty.     

Fibrinogen and fibrinogen/fibrin degradation products in the urine of rabbits infected with Trypanosoma (Trypanozoon) brucei

Summary Studies have been carried out on the urine of rabbits infected with Trypanosoma (Trypanozoon) brucei to determine whether fibrinogen or fibrinogen/fibrin degradation products (FDP) could be detected. No fibrinogen was found but during the last two weeks of this 7-week infection low levels of FDP were present in the urine which did not exceed 5 g/ml. Rabbit urine was shown to contain a potent proteolytic enzyme capable of breaking down rabbit fibrinogen and both early and late FDP were present in the cleavage products. No deposits of fibrin were detected in the kidney, but casts were present in the urine suggesting renal damage. The most likely explanation of the urinary FDP is that either an increase in the glomerular permeability occurs allowing filtration of plasma FDP or a local fibrinogenolysis in the kidney tubules.