Enumeration of leukocyte infiltration in solid tumors by confocal laser scanning microscopy.

BACKGROUND: Leukocytes commonly infiltrate solid tumors, and have been implicated in the mechanism of spontaneous regression in some cancers. Conventional techniques for the quantitative estimation of leukocyte infiltrates in tumors rely on light microscopy of immunostained thin tissue sections, in which an arbitrary assessment (based on low, medium or high levels of infiltration) of antigen density is made by the pathologist. These estimates are relatively subjective and often require the opinion of a second pathologist. In addition, since thin tissue sections are cut, no data regarding the three-dimensional distribution of antigen can be obtained. RESULTS: To overcome these problems, we have designed a method to enumerate leukocyte infiltration into tumors, using confocal laser scanning microscopy of fluorescently immunostained leukocytes in thick tissue sections. Using image analysis software, a threshold was applied to eliminate unstained tissue and residual noise. The total antigen volume in the scanned tissue was calculated and divided by the mean cell volume (calculated by "seeding" ten individual cells) to obtain the cell count. Using this method, we compared the calculated leukocyte counts with those obtained manually by ten laboratory personnel. There was no significant difference (P > 0.05) between the cell counts obtained by either method. We then compared leukocyte infiltration into seven tumors and matched non-malignant tissue obtained from the periphery of the resected tissue. There was a significant increase in the infiltration of all leukocyte subsets into the tumors compared to minimal numbers in the non-malignant tissue. CONCLUSION: From these results we conclude that this method may be of considerable use for the enumeration of cells in tissues. Furthermore, since it can be performed by laboratory technical staff, less time input is required by the pathologist in assessing the degree of leukocyte infiltration into tumors.

Experimental metastasis and primary tumor growth in mice with hemophilia A.

During experimental lung metastasis, tumor cells adhere to the pulmonary microvasculature and activate coagulation via surface-expressed tissue factor (TF), leading to local fibrin deposition and platelet aggregation. While interventional studies have demonstrated great efficacy of anticoagulants and antiplatelet agents in inhibiting metastasis, no information is available on how tumor biology may be affected by congenital bleeding disorders such as hemophilia A. We therefore used a syngeneic model to study experimental metastasis and primary tumor growth in factor VIII (FVIII)-deficient mice. By conventional reverse transcription-polymerase chain reaction, flow cytometry, and one-stage clotting assays, we demonstrated constitutive expression of TF mRNA, antigen, and procoagulant activity in the murine B16F10 melanoma cell line. In hemophilic mice, B16F10 lung metastasis was significantly (P < 0.001) enhanced by a single dose of human FVIII (100 U kg(-1)), suggesting that FVIII played a critical role during the early blood-borne phase of the metastatic cascade. In contrast, lung seeding was significantly (P < 0.05) reduced by lepirudin, a direct thrombin inhibitor, suggesting that thrombin generation contributed to pulmonary metastasis even in the absence of FVIII. Consistent with this finding, intravenous injection of B16F10 cell-evoked laboratory changes of a hemolytic thrombotic microangiopathy and consumptive coagulopathy in both hemophilic and non-hemophilic mice. Subcutaneous implantation of B16F10 cells into mice with hemophilia A gave rise to primary tumors in an exponential growth pattern similar to that observed in non-hemophilic mice. Although TF expression by B16F10 cells may promote thrombin-dependent metastasis in mice with hemophilia A, amplification of coagulation by host FVIII appears to be necessary for maximum lung seeding.

Tissue procoagulant activity may be important in sustaining metastatic tumour growth.

There is strong evidence for an association between the haemostatic system and malignancy. Thus, cancer may adversely affect the host coagulation system while the haemostatic system may play a role in the development of both primary and metastatic tumours. Metastatic growth is not dependent simply on haemodynamic factors, and properties of both the tumour cell and host organ are important determinants of the site of metastatic growth. Previous studies have demonstrated that some organs are preferred sites for metastasis while others are less preferred or resistant. We have measured the procoagulant activity (PCA) of normal rat and human tissues and correlated the results with the previously reported ability of these organs to support metastatic tumour growth. In addition, we determined changes in PCA in rat tissues during oral anticoagulant therapy, and following colonic anastomosis and partial hepatectomy, procedures which are known to affect experimental metastasis. In both rat and human studies, organs which are preferred sites for metastasis had significantly higher PCA than non-preferred organs (P less than 0.001). The PCA of adrenal, lung and colon was significantly reduced by administration of warfarin (P less than 0.001). PCA was significantly (P less than 0.001) increased in both colonic anastomoses and regenerating liver and followed a time course similar to that of the enhanced tumour growth usually seen in these situations. Although the exact source of the procoagulant activity remains to be determined, the results suggest that there is a broad correlation between tissue PCA and the ability of a tissue to support metastatic tumour growth.

The effect of pentoxifylline on spontaneous and experimental metastasis of the mouse Neuro2a neuroblastoma.

Pentoxifylline (PTX) has been reported to have both direct and indirect anti-tumor effects in experimental tumor models. We studied the effect of PTX on (1) the proliferation of Neuro2a mouse neuroblastoma cells in vitro and in vivo, (2) spontaneous and experimental metastasis, (3) tumor cell membrane fluidity and (4) adhesion to a fibronectin-coated surface. PTX significantly reduced the proliferation of Neuro2a cells in vitro as determined by DNA measurement (P < 0.01) and total cell count (P < 0.02). In vivo, PTX reduced the growth of subcutaneously transplanted primary tumors in syngeneic A/J mice (P < 0.01; n = 15). All seven animals (100%) receiving intravenous tumor cells developed extensive liver metastasis. In contrast, only 1/11 (9%) of animals pre-treated with oral PTX and injected with PTX-treated cells developed liver metastases. Of five mice receiving PTX-treated cells without oral pretreatment of PTX, two out of five (40%) developed liver metastases. There was a slight, but not significant (P = 0.08) increase in both experimental and spontaneous lung metastases formation in PTX-treated animals. However, tumor nodule formation on the lung surface was inefficient. PTX also increased membrane fluidity of the Neuro2a cells and significantly decreased tumor cell adhesion to fibronectin-coated microtiter wells (P < 0.01). We conclude that PTX has a cytostatic effect on the Neuro2a mouse neuroblastoma and exerts an anti-tumor effect on liver metastases following intravenous administration of neuroblastoma cells. Whether these results are directly related to the changes in membrane properties caused by pentoxifylline remains to be established.

Soluble fibrin augments platelet/tumor cell adherence in vitro and in vivo, and enhances experimental metastasis.

There is considerable evidence for a relationship between hemostasis and malignancy. Since platelet adhesion to tumor cells has been implicated in the metastatic process and plasma levels of fibrinogen (Fg) and soluble fibrin (sFn) monomer are increased in cancer, we hypothesized that these molecules might enhance tumor-platelet interaction. We therefore studied binding of sFn monomer to tumor cells in a static microplate adhesion assay and determined the effect of pre-treating tumor cells with sFn on tumor cell-induced thrombocytopenia and experimental metastasis. Soluble fibrin (produced by adding thrombin to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro-amide (GPRP-NH2) significantly increased platelet adherence to tumor cells. This effect was primarily mediated by the integrins alphaIIb beta3 on the platelet and CD 54 (ICAM-1) on the tumor cells. Platelets adhered to untreated A375 cells (28 +/- 8 platelets/tumor cell) and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or GPRP-NH2. Although thrombin treatment increased adherence, pre-incubation of the tumor cells with sFn resulted in a further increase in platelet binding to tumor cells. In contrast to untreated tumor cells, intravenous injection of sFn-treated A 375 cells reduced the platelet count in anticoagulated mice, supporting the in vitro finding that sFn enhanced tumor cell-platelet adherence. In a more aggressive model of experimental metastasis, treating tumor cells with sFn enhanced lung seeding by 65% compared to untreated cells. Extrapolation of our data to the clinical situation suggests that coagulation activation, and subsequent increase in circulating Fn monomer, may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

Antimetastatic effect of tinzaparin, a low-molecular-weight heparin.

The importance of coagulation activation in cancer patients is suggested by the clinical finding of hypercoagulability, experimental enhancement of metastasis and angiogenesis by coagulation factors such as tissue factor (TF) and thrombin and the possible antitumor effects of anticoagulant agents. Tinzaparin is a low-molecular-weight heparin (LMWH) with a relatively high molecular weight distribution and high sulfate to carboxylate ratio. In addition to its ability to inhibit thrombin and factor Xa, tinzaparin is particularly effective at releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of both procoagulant and non-coagulant effects of TF. The present study was undertaken to investigate the effect of tinzaparin on lung metastasis using a B16 melanoma model in experimental mice. Tinzaparin's anticoagulant effect in mice and its ability to release TFPI from human endothelial cells at various time points were demonstrated. Subcutaneous (s.c.) injection of tinzaparin (10 mg kg-1) 4 h before intravenous administration of melanoma cells (2.0 x 105) markedly (89%) reduced lung tumor formation (3 +/- 2) compared with controls (31 +/- 23; P < 0.001). In a second group of animals, tinzaparin (10 mg kg-1, s.c.) administered daily for 14 days following the initial (pretumor cell) dose, before assessment of lung seeding, reduced tumor formation by 96% (P < 0.001). No bleeding problems were observed in any of the tinzaparin-treated animals, despite a 4-fold prolongation of the whole blood clotting time after a single s.c. dose of tinzaparin (10 mg kg-1). Administration of tumor cells (2 x 106) caused a rapid and significant fall in platelet count 15 min after injection (a sensitive marker of intravascular coagulation) in controls (939 +/- 37 vs. 498 +/- 94 x 106 mL-1, P < 0.01), but this was prevented by tinzaparin treatment (921 +/- 104 x 106 mL-1). These data provide further experimental evidence to support the potential for LMWH as antimetastatic agents.

Changes in the contact system during orthotopic liver transplantation with and without aprotinin.

The main cause of nonsurgical bleeding during orthotopic liver transplantation has been attributed to be hyperfibrinolysis due to high plasma levels of tissue plasminogen activator. The aim of this study was to investigate contact activation and its possible contribution to fibrinolysis during OLT with and without aprotinin. Aprotinin or placebo was given to 20 patients undergoing OLT as part of a randomized double-blind trial. Plasma samples were collected before, during, and after OLT. There were decreased preoperative levels of prekallikrein and factor XIIa (P < 0.05), with a trend for kallikrein and factor XIIa activity to increase during OLT peaking on reperfusion (P < 0.05). Kallikrein inhibition, C1 esterase inhibitor, and alpha-2-macroglobulin levels were normal before surgery, with low normal levels of antithrombin III and alpha-2-antiplasmin; these levels decreased during OLT with no specific change on reperfusion. In the aprotinin-treated group, kallikrein inhibition levels increased (P < 0.05) from preoperative mean (+/- SD) values of 101 +/- 47% to 154 +/- 42% and antiplasmin levels increased (P < 0.05) from 72 +/- 28% to 243 +/- 53% during the anhepatic phase, reflecting the effect of aprotinin. The antifibrinolytic effect of aprotinin was demonstrated by decreased levels of D-dimer on reperfusion (P < 0.05) and at the end of OLT (P < 0.001) in the aprotinin-treated group. We have shown that contact activation during OLT is minimal and that aprotinin does not alter the pattern of contact activation, but provides an antikallikrein effect.

Fibrinolytic activity during orthotopic liver transplantation with and without aprotinin.

Hyperfibrinolysis during orthotopic liver transplantation (OLT) has been attributed to high plasma levels of tissue plasminogen activator (t-PA). This study investigated the contribution of urokinase plasminogen activator (u-PA) to hyperfibrinolysis and the effects of high-dose perioperative aprotinin (Trasylol) on fibrinolytic activation. Plasma samples were collected before, during, and after OLT in fifty five patients receiving either high dose aprotinin or placebo in a randomized double-blind trial. t-PA antigen and u-PA antigen and activity levels were increased preoperatively compared with normal controls (P < 0.05). Hyperfibrinolysis was seen during the anhepatic phase as shown by shortened euglobulin clot lysis times (ECLT) and an increase in D-dimer titers. t-PA levels peaked on reperfusion and fell at the end of the operation, and u-PA levels did not increase during OLT, but showed a decrease at the end of the operation. With aprotinin treatment, t-PA levels were lower on graft reperfusion than the placebo group (P < 0.05), but there was no difference in u-PA antigen or activity levels between groups. Fibrinolytic inhibition during OLT by aprotinin was demonstrated by prolonged ECLT (P < 0.05), reduced D-dimer levels (P < 0.05), and an increase in antiplasmin activity (P < 0.05). This study showed that the main antifibrinolytic action of aprotinin is as an antiplasmin agent with some effect on t-PA-but not u-PA-mediated fibrinolysis.

Coagulation activation by MC28 fibrosarcoma cells facilitates lung tumor formation.

Tumor cells interact with the hemostatic system in various ways and may thus influence malignant growth and spread. MC28 fibrosarcoma cells possess a potent procoagulant activity (PCA) and form lung tumors following intravenous injection. The aim of this work was to study the relationship between PCA, intravascular coagulation and lung seeding in the MC28 model. MC28 cells were injected into control, warfarinized and heparinized hooded Lister rats. Coagulation changes were monitored by thromboelastography (TEG) and Sonoclot analysis (SA), lung fibrin formation by light and electron microscopy, tumor seeding by macroscopic counting and tumor cell and platelet deposition in the lungs by radiolabelling. PCA was measured by chromogenic assay. MC28 PCA was characterized as a tissue factor-factor VIIa complex that probably arose during cell culture or disaggregation of solid tumors. Injection of tumor cells caused marked coagulopathy and was rapidly (within 30 min) followed by fibrin deposition in the lungs and accumulation of radiolabelled platelets. Heparin and warfarin significantly reduced lung seeding (p < 0.001) and reduced retention of radiolabelled tumor cells in the pulmonary circulation (p < 0.01). Inhibition of cellular PCA by prior treatment with concanavalin A markedly reduced intravascular coagulation and lung seeding. We conclude that MC28 cells cause intravascular coagulation as a direct result of their procoagulant activity. The data suggest that tumor cells form complexes with platelets and fibrin which are retained in the lungs long enough for extravasation and seeding to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay.

Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean +/- SD) expression of monocyte TF in normal subjects was very low (1.1 +/- 0.95%, Mean Fluorescence [Mean FL] 0.20 +/- 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 +/- 11.2% (Mean FL 0.32 +/- 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.

Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor.

Tissue factor (TF), the membrane glycoprotein that initiates blood coagulation, is constitutively expressed by many tumor cells and is implicated in peri-tumor fibrin deposition and hypercoagulability in cancer. Upregulation of tumor TF correlates with enhanced metastatic potential. Furthermore, TF has been colocalized with VEGF in breast cancer, specially at sites of early angiogenesis. There are no data on the effect of hypoxia on tumor cell TF expression. Since hypoxia is known to stimulate VEGF production, we studied whether this also induces tumor cell TF expression. Confluent monolayers of A375 melanoma, MCF-7 breast carcinoma and A549 lung carcinoma were cultured in either 95% air, 5% CO2 (normoxic) or 95% N2, 5% CO2 (hypoxic; 25-30 mmHg) for 24 h. Procoagulant activity (PCA) was measured by amidolytic and clotting assays, surface TF antigen by flow cytometry, early apoptosis by annexin V binding and VEGF levels in culture supernatants by ELISA. Hypoxia significantly increased tumor cell PCA in all three cell lines tested and TF antigen on A375 cells was increased four-fold (P <0.05). Pentoxifylline (PTX), a methylxanthine derivative, significantly inhibited the hypoxia-induced increase in PCA as well as VEGF release in all three cell lines tested. In A375 cells, PTX significantly inhibited TF antigen expression by both normoxic and hypoxic cells. Hypoxia induced a slight (5%) but not significant, increase in early apoptosis. Intravenous injection of hypoxic A375 cells into nude rats produced more pronounced thrombocytopenia (n = 5, P <0.01) and more lung metastases (n = 3, P <0.05) compared to normoxic cells. We conclude that hypoxia increases TF expression by malignant cells which enhances tumor cell-platelet binding and hematogenous metastasis. Hypoxia-induced upregulation of TF appears to parallel that of VEGF, although the mechanism remains unclear.

A new chromogenic assay for the specific determination of prothrombin.

A new method for the specific assay of prothrombin is described which utilises Taipan snake venom and the chromogenic substrate Tos-Gly-Pro-Arg-pNA (Chromozym TH). The method is sensitive and reproducible and shows good correlation with a conventional specific assay for prothrombin. The reaction is dependent on calcium ions and phospholipid, and is therefore sensitive to the coumarin-induced prothrombin defect. The assay is rapid and well suited for use in the routine coagulation laboratory.

Acquired dysfibrinogenaemia in liver disease.

Using a new and sensitive screening method, dysfibrinogenaemia (DF) was detected in 76% of patients with cirrhosis, 78% with chronic active liver disease and 86% with acute liver failure. The incidence was much lower in obstructive jaundice (8%) and miscellaneous liver disorders (4%). It is concluded that the fibrin monomer polymerisation (FMP) ratio test is a simple and sensitive test for the detection of DF, and is useful in the differential diagnosis of hepatocellular and obstructive jaundice. Hyperfibrinogenaemia, particularly in patients with obstructive jaundice, may explain the high incidence of abnormal thrombin and Reptilase clotting times despite normal FMP ratios. Dysfibrinogenaemia dose not appear to be related to the degree of liver function impairment, but may be associated with regeneration of hepatic tissue.

Development and validation of an assay for urinary tissue factor activity.

BACKGROUND: Activation of blood coagulation is a common complication of cancer and inflammation in both humans and experimental animals. Increased production of tissue factor--the principal initiator of the coagulation process--by endothelial cells, monocytes, and macrophages has been implicated in these conditions. AIM: To investigate whether urinary tissue factor (uTF) might reflect the state of monocyte/macrophage activation and be a useful diagnostic test. METHODS: Urine was centrifuged at 51,000 g to sediment tissue factor containing membrane vesicles. The tissue factor was then solubilised in beta-octyl-glucopyranoside and assayed in a specific chromogenic assay adapted for use in microtitre plates. RESULTS: The assay proved to be sensitive, specific, and reproducible. The normal range of uTF was relatively narrow and unaffected by age, sex, or cigarette smoking. Levels were not significantly influenced by storage of urine samples before assay or by the presence of fresh blood in the urine sample. CONCLUSIONS: This method may have diagnostic application in the study of haemostatic activation in patients with cancer and other disease states.

Whole blood procoagulant activity in breast and colorectal cancer.

Whole blood procoagulant activity was determined by measuring the recalcification time of citrated blood, with and without the addition of bacterial endotoxin, in patients with breast cancer (n = 39), colorectal cancer (n = 20), benign breast disease (n = 15), benign colorectal disease (n = 11), normal volunteers (n = 15) and inpatients with non-malignant disease (n = 22). The median clotting times of those samples incubated with endotoxin were significantly shorter in the patients with breast and colorectal cancer compared with normal controls. Furthermore, significant differences between the median clotting times of stimulated and unstimulated samples within each subject group were observed only in the two cancer groups. There was no correlation between whole blood procoagulant activity and absolute monocyte counts, with histological staging or with plasma concentrations of plasma fibrinopeptide A. The results suggest that blood from patients with cancer is more sensitive to endotoxin stimulation than that from normal or benign controls, but that in its present form the technique cannot be used to distinguish between malignant and non-malignant disease.

Urinary tissue factor in glomerulonephritis: a potential marker of glomerular injury?

AIM: To investigate the significance of urinary tissue factor (uTF) concentrations in patients with glomerulonephritis. METHODS: Urine samples were collected from normal subjects (n = 57), patients with uncomplicated renal stones (n = 30), and patients with glomerulonephritis (n = 150). Samples were then centrifuged and the pellets solubilised in n-octyl-beta-glucopyranoside. uTF concentrations were determined using a one stage kinetic chromogenic assay. RESULTS: The uTF concentration was higher in patients with glomerulonephritis than in normal controls (p < 0.01) or in patients with renal stones (p < 0.05). uTF activity correlated with the protein creatinine index (PCI, r = 0.41, p < 0.001) and seven patients with glomerulonephritis and a PCI < or = 0.1 g/mmol had raised uTF. Glomerulonephritis patients were subdivided into two groups depending on the PCI: < 0.2 g/mmol creatinine (mild to moderate proteinuria, group I) and > or = 0.2 g/mmol creatinine (heavy proteinuria, group II). In group I, uTF concentrations were higher in patients with either immune complex (IC) glomerulonephritis (p < 0.01) or non-IC (p < 0.05) glomerulonephritis than in normal controls. In group II, the IC glomerulonephritis group had higher uTF concentrations than normal controls (p < 0.001) or patients with renal stones (p < 0.01); and non-IC glomerulonephritis patients had higher uTF than normal controls (p < 0.01). When the glomerulonephritis groups were divided into broad WHO subtypes, the significance level varied with the type of glomerulonephritis. CONCLUSIONS: uTF is increased in patients with glomerulonephritis, and its concentration may reflect the aetiopathogenesis of glomerulonephritis.

Effect of kidney function and disease status on urinary tissue factor measurements.

AIM: To investigate factors that influence urinary tissue factor (uTF) measurements: glomerular permeability and filtration, tubular function, haematuria, and urine bacterial growth. METHODS: uTF, protein creatinine index, glomerular filtration rate, retinol binding protein, N-acetyl-beta-D-glucosaminidase (NAG) and urinary haemoglobin (uHb) were measured in patients with hypertension, diabetes mellitus and nephrotic syndrome (n = 342), tubulo-interstitial disease (n = 50), and haematuria of uncertain cause (n = 50); measurements were also made in urine samples from healthy subjects for "simulated" haematuria (n = 6) and bacterial growth (n = 4) studies. RESULTS: There was a weak correlation of uTF with glomerular permeability and filtration (protein creatinine index and glomerular filtration rate) and with markers of tubular function (retinol binding protein and NAG). uTF concentrations were not affected by the presence of blood or bacteria in the urine sample. CONCLUSION: uTF concentrations are relatively stable. This is an important finding if the assay is to be used in clinical practice.

Assessment of hypercoagulability in patients with cancer using the Sonoclot Analyzer and thromboelastography.

Patients with cancer have an increased incidence of thrombosis and abnormal haemostasis detectable by sophisticated laboratory tests. Whether abnormalities in such highly sensitive assays is clinically relevant to bleeding or thrombosis is not clear. The thromboelastograph (TEG) and Sonoclot analyzers assess the coagulation process in whole blood and may therefore be physiologically more relevant than assays of isolated haemostatic components. Blood was collected from healthy volunteers and patients with breast cancer, colorectal cancer or benign disease and tested in the TEG and Sonoclot. Results in the cancer group were compared to appropriately sex-matched controls. The TEG parameters R (P < 0.02), angle (P < 0.05) and MA (P < 0.001) were abnormal in colorectal cancer; up to 8/17 (47%) patients being assessed as hypercoagulable. R (P < 0.05), angle (P = 0.05) and MA (P < 0.001) were abnormal in breast cancer; 2/21 (9%) patients having abnormal results. In the Sonoclot Analyzer, 11/17 (64%) patients with colorectal cancer had a significant increase in clot rate (P < 0.001) while 2/17 had a decreased SonACT time (P = 0.05). 4/21 (19%) breast cancer patients had a significant increase in clot rate (P < 0.001) and the SonACT was shortened (P = 0.05). Platelets and fibrinogen levels were generally normal and only one patient had clinical evidence of thrombosis. There were no significant coagulation changes in patients with benign colon or breast disease. In conclusion, hypercoagulability was detected in a high proportion of breast and colorectal cancer patients by both techniques. The clot rates of the TEG and Sonoclot were significantly correlated but the latter was abnormal in a greater proportion of cancer patients.

Factor X-activating activity in normal and malignant colorectal tissue.

The factor X-activating activity (FXAA) of homogenates from human colorectal tumours and corresponding normal colonic mucosa from the same patients was assessed with a specific chromogenic substrate technique. FXAA was detected in all normal and tumour tissue tested, but was significantly higher in tumour tissue. The procoagulant activity was inhibited by DFP, but was unaffected by iodoacetamide and mercuric chloride. FXAA was largely abolished by prior incubation of both normal and tumour tissue homogenates with a rabbit anti-human factor VII serum, but was greatly enhanced by the addition of purified factor VII. FXAA was partially adsorbed on to aluminium hydroxide and almost completely abolished by treatment with barium citrate. It is concluded that the FXAA of both normal and malignant colorectal tissue is the result of tissue factor-factor VII interaction.

The role of sodium citrate in the dysfibrinogenaemia of liver disease.

The addition of excess sodium citrate to plasma was found to inhibit fibrin polymerisation (clot opacity) from patients with cirrhosis, hepatitis and hepatoma but not from normal controls. Abnormal clot opacity in plasma from patients with liver disease could be partly or completely abolished by removal of citrate ions by dialysis against citrate-free buffer, but not by dialysis against buffer containing citrate. Similar results were observed in plasma freed of calcium ions by treatment with EGTA. Treatment of plasma with neuraminidase largely abolished the inhibitory effect of excess citrate, and the thrombin times and clot opacity of asialofibrinogen were less affected by citrate than native fibrinogen. In addition, the effects of citrate on the clotting of purified, calcium-free fibrinogen from cirrhotic patients correlated with the sialic acid content. It is concluded that binding of citrate ions to fibrinogen renders the molecule acutely more sensitive to elevations in the sialic acid content, and that a simple plasma clot opacity test in the presence of excess citrate may be a useful aid in the differential diagnosis of liver disease. These findings may also explain why defects in fibrin polymerisation observed in plasma are not always reproduced in purified fibrinogen or fibrin monomer preparations.