Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity

R.D., Lee,; Barcel, D. Anthony; Williams, Julie C.; J.G., Wang,; Boles, Jeremiah; Manly, David A.; Key, Nigel S.; Mackman, Nigel

doi:10.1016/j.thromres.2011.06.004

Cited by 110 publications

(136 citation statements)

References 33 publications

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“…Measurement of the level of human TF protein in plasma is easier than the measurement of MP TF activity, but has a lower sensitivity, and concerns have been raised about the specificity of some of the commercial ELISAs. 6,40,41 The MP TF activity assay has greater sensitivity but more variability than an ELISA for detecting elevated levels of TF in the plasma of cancer patients. These results suggest that it would be best to measure plasma MP TF activity in addition to plasma TF protein levels to identify cancer patients who have an increased risk of thrombosis.…”

Section: Discussionmentioning

confidence: 99%

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

Wang

Geddings

Aleman

et al. 2012

Blood

179

167

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IntroductionThe link between cancer and venous thromboembolism (VTE) is referred to as Trousseau syndrome. Interestingly, different cancer types are associated with different rates of VTE, with pancreatic cancer having one of the highest rates. 1,2 A VTE risk-scoring model has been developed that stratifies ambulatory cancer patients undergoing chemotherapy into 3 VTE risk categories based on 5 parameters: (1) the site of the primary tumor, (2) prechemotherapy leukocyte count, (3) platelet count, (4) hemoglobin level, and (5) body mass index. 3 Recently, this model was expanded to include the biomarkers D-dimer and soluble P-selectin. 4 Another potential circulating biomarker of VTE risk in pancreatic cancer patients is microparticle (MP) tissue factor (TF). [5][6][7][8][9] Full-length TF (flTF) is a transmembrane protein that activates the coagulation cascade. 10 In addition, an alternatively spliced form of TF (asTF) has been identified that lacks a membrane anchor and therefore can be released as a soluble protein. 11 Increased TF expression is correlated with poor prognosis in pancreatic cancer. [12][13][14] Cultured human pancreatic tumor lines express variable levels of both flTF and asTF and release TF-positive MPs containing flTF into the culture medium. [14][15][16][17][18][19] In some patients with pancreatic cancer, high levels of TF-positive MPs are found in the circulation and, in a small pilot study, were predictive of VTE. [5][6][7]9,20 In a mouse model of human colorectal tumors, human TF protein is released into the circulation. 21 In nude mice bearing orthotopic human pancreatic tumors (L3.6pl) plasma levels of human TF protein were correlated with the levels of thrombin-antithrombin (TAT) complex, a marker of the activation of coagulation. 22 Further, plasma from these tumor-bearing mice was found to enhance thrombin generation in vitro in a human TF-dependent manner. 22 Another study found that human (SOJ-4) and mouse (PANC02) pancreatic cell lines expressed TF, and the investigators observed an accumulation of tumor-derived MPs at the site of thrombosis and increased thrombosis in a microvascular model. 18 The objective of the present study was to determine the role of tumor-derived TF in the activation of coagulation and thrombosis in a xenograft mouse model of human pancreatic tumors. We found that only TF-positive tumors activated coagulation and that this activation was abolished by inhibition of human TF. Two TF-positive pancreatic tumor cell lines activated coagulation, but only one had detectable levels of circulating TF-positive MPs, which suggested that activation of coagulation was due to TF expression by the tumor itself rather than to TF on the MPs. Mice with elevated levels of TF-positive MPs exhibited increased thrombosis in a saphenous vein model, but not in an inferior vena cava (IVC) stenosis model. Methods Cell linesHuman pancreatic (MIAPaCa-2 [CRL-1420] Abs and proteinsPE-labeled mouse IgG control (#555574), mouse anti-human TF (#550312) and FITC-conjugated anti-human MUC...

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Section: Discussionmentioning

confidence: 99%

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

Wang

Geddings

Aleman

et al. 2012

Blood

179

167

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“…On fresh samples, a second centrifugation at 13,000g has been reported to decrease endothelial and annexin V1 microvesicles by about 50% in some studies (22,23), but not others (14,20,24). If plasma samples are going to be frozen, a single centrifugation step is inadequate to remove all platelets from the sample, double centrifugation is required to prevent 100% to 1,500% increases in platelet and annexin V1 microvesicle counts due to fragmentation of residual platelets in the sample (15,20,(25)(26)(27).…”

Section: Centrifugationmentioning

confidence: 99%

Measurement of microvesicle levels in human blood using flow cytometry

Chandler

2016

Cytometry Part B Clinical

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Microvesicles are fragments of cells released when the cells are activated, injured, or apoptotic. Analysis of microvesicle levels in blood has the potential to shed new light on the pathophysiology of many diseases. Flow cytometry is currently the only method that can simultaneously separate true lipid microvesicles from other microparticles in blood, determine the cell of origin and other microvesicle characteristics, and handle large numbers of clinical samples with a reasonable effort, but expanded use of flow cytometric measurement of microvesicle levels as a clinical and research tool requires improved, standardized assays. The goal of this review is to aid investigators in applying current best practices to microvesicle measurements. First pre-analytical factors are evaluated and data summarized for anticoagulant effects, sample transport and centrifugation. Next flow cytometer optimization is reviewed including interference from background in buffers and reagents, accurate microvesicle counting, swarm interference, and other types of coincidence errors, size calibration, and detection limits using light scattering, impedance and fluorescence. Finally current progress on method standardization is discussed and a summary of current best practices provided. V C 2016 Clinical Cytometry Society

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“…56 Several functional assays have been developed that measure TF activity in isolated MPs, using either a single-stage clotting assay or a 2-stage factor Xa generation assay in the presence or absence of an inhibitory anti-TF antibody. [55][56][57][58][59] Thaler and colleagues measured levels of MP TF activity in patients with cancer, and found a good correlation between 2 assays. 60 We observed comparable levels of TF activity in MPs isolated from lipopolysaccharide-stimulated human whole blood, using 2 centrifugation speeds (either 20 000 3 g for 20 minutes [primarily MPs] or 100 000 3 g for 1 hour [MPs and exosomes]), suggesting that the majority of vesicle TF is on MPs.…”

Section: Development Of a Venous Thrombusmentioning

confidence: 99%

“…60 We observed comparable levels of TF activity in MPs isolated from lipopolysaccharide-stimulated human whole blood, using 2 centrifugation speeds (either 20 000 3 g for 20 minutes [primarily MPs] or 100 000 3 g for 1 hour [MPs and exosomes]), suggesting that the majority of vesicle TF is on MPs. 59 Light-scatter flow cytometry is the most commonly used method for the quantification of MPs in clinical samples. 61 Several studies have used flow cytometry to detect TF-positive MPs in patients with cancer.…”

Section: Development Of a Venous Thrombusmentioning

confidence: 99%

Tumor-derived tissue factor–positive microparticles and venous thrombosis in cancer patients

Geddings

Mackman

2013

Blood

284

238

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Patients with cancer have an increased risk for venous thrombosis. Interestingly, different cancer types have different rates of thrombosis, with pancreatic cancer having one of the highest rates. However, the mechanisms responsible for the increase in venous thrombosis in patients with cancer are not understood. Tissue factor (TF) is a transmembrane receptor and primary initiator of blood coagulation. Tumor cells express TF and spontaneously release TF-positive microparticles (MPs) into the blood. MPs are small membrane vesicles that are highly procoagulant. It has been proposed that these circulating tumor-derived, TF-positive MPs may explain the increased rates of venous thrombosis seen in patients with cancer. In animal models, increased levels of tumor-derived, TF-positive MPs are associated with activation of coagulation. Moreover, these MPs bind to sites of vascular injury and enhance thrombosis. We and others have found that patients with cancer have elevated levels of circulating TF-positive MPs. These MPs are derived from tumors because they express tumor markers and are decreased by tumor resection. Importantly, several studies have shown that increased levels of TF-positive MPs correlate with venous thrombosis in patients with cancer. Taken together, these results suggest that TF-positive MPs may be a useful biomarker to identify patients with cancer who are at high risk for thrombosis.

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Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity

Cited by 110 publications

References 33 publications

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

Measurement of microvesicle levels in human blood using flow cytometry

Tumor-derived tissue factor–positive microparticles and venous thrombosis in cancer patients

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