Introduction: The sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence... Show moreIntroduction: The sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence test results. We developed an LC-MRM-MS test to molecularly characterize antithrombin in citrate plasma. The test principle differs greatly from traditional laboratory tests and the influence of varying plasma sample matrices is largely unknown. Objectives: To identify whether variations in sample matrix affect the LC-MRM-MS test for antithrombin and assess whether sample pre-processing by immunocapture reduces matrix-specific effects. Methods: Samples (n = 45) originating from four different blood collection tubes (sodium citrate, lithium heparin, K2-EDTA and K2-EDTA with protease inhibitors) were processed directly or after immunocapture. Antithrombin was digested into proteotypic peptides, which were monitored by LC-MRM-MS. Results from lithium heparin and the K2-EDTA matrices were compared to the standard sample matrix, sodium citrate, using Deming regression analysis and repeated measures one-way ANOVA.Results: Deming regression analysis of directly processed samples revealed slopes deviating >5% from the line of identity for at least six out of 22 peptides in all matrices. Significant differences between all matrices were found upon analysis by ANOVA for at least 10 peptides. Pre-processing by immunocapture led to slopes within 5% of the line of identity for nearly all peptides of the matrices. Furthermore, significant differences between matrices after immunocapture were only observed for four peptides.Conclusion: Variations in the sample matrix affect the measurement of antithrombin by LC-MRM-MS, but observed effects are greatly reduced upon pre-processing by immunocapture. Show less
Kruijt, M.; Smit, N.P.M.; Ham, J.J. van; Cobbaert, C.M.; Ruhaak, L.R. 2023
IntroductionThe sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence... Show moreIntroductionThe sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence test results. We developed an LC-MRM-MS test to molecularly characterize antithrombin in citrate plasma. The test principle differs greatly from traditional laboratory tests and the influence of varying plasma sample matrices is largely unknown.ObjectivesTo identify whether variations in sample matrix affect the LC-MRM-MS test for antithrombin and assess whether sample pre-processing by immunocapture reduces matrix-specific effects.MethodsSamples (n = 45) originating from four different blood collection tubes (sodium citrate, lithium heparin, K2-EDTA and K2-EDTA with protease inhibitors) were processed directly or after immunocapture. Antithrombin was digested into proteotypic peptides, which were monitored by LC-MRM-MS. Results from lithium heparin and the K2-EDTA matrices were compared to the standard sample matrix, sodium citrate, using Deming regression analysis and repeated measures one-way ANOVA.ResultsDeming regression analysis of directly processed samples revealed slopes deviating >5% from the line of identity for at least six out of 22 peptides in all matrices. Significant differences between all matrices were found upon analysis by ANOVA for at least 10 peptides. Pre-processing by immunocapture led to slopes within 5% of the line of identity for nearly all peptides of the matrices. Furthermore, significant differences between matrices after immunocapture were only observed for four peptides.ConclusionVariations in the sample matrix affect the measurement of antithrombin by LC-MRM-MS, but observed effects are greatly reduced upon pre-processing by immunocapture. Show less
Local coagulation activation has been shown to impact both primary tumor growth and metastasis in mice. It is well known that components of the blood clotting cascade such as tissue factor and... Show moreLocal coagulation activation has been shown to impact both primary tumor growth and metastasis in mice. It is well known that components of the blood clotting cascade such as tissue factor and thrombin play a role in tumor progression by activating cellular receptors and local formation of fibrin. However, whether venous thromboembolism (VTE) or a hypercoagulable state has a direct impact on cancer progression is unknown. Here we have combined an orthotopic murine breast cancer model, using female Nod-SCID mice, with siRNA-mediated silencing of antithrombin (siAT) leading to the induction of a systemic hypercoagulable state. We show that, compared to control siRNA-treated (not experiencing a hypercoagulable state) tumor-bearing mice, siAT treated tumor-bearing mice do not show enhanced tumor growth nor enhanced metastasis. We conclude that, in this murine model for hypercoagulability, induction of a hypercoagulable state does not contribute to breast cancer progression. Show less
Minno, M.N.D. di; Ambrosino, P.; Ageno, W.; Rosendaal, F.; Minno, G. di; Dentali, F. 2015
Thrombosis is considered to be a multifactorial disease in which both genetic and acquired risk factors are involved to cause disease. Over the past years epidemiological studies have revealed a... Show moreThrombosis is considered to be a multifactorial disease in which both genetic and acquired risk factors are involved to cause disease. Over the past years epidemiological studies have revealed a number of acquired risk factors that increase the risk of venous thrombosis. Some acquired risk factors of venous thrombosis are associated with a hypercoagulable state, which may be to a certain extent dependent on the dysregulation of gene expression in the liver, as the liver is the major organ that produces coagulation factors. Coagulation gene transcription can be modulated at different levels through hepatic transcription factors, co-regulatory or intermediate proteins, however, the exact contribution of these modulators to coagulation gene transcription is largely unknown. We aimed to study the mechanisms by which hepatic coagulation gene transcription is regulated, in order to increase our understanding of how thrombotic risks conditions coincide with hypercoagulable state. RNA interference (via synthetic small interfering RNA; siRNA) was used as a tool to study genes involved in coagulation and coagulation control in mice. Studies described in this dissertation may contribute to a better understanding of which genes are involved in coagulation (control) and how thrombotic risk factors result in a hypercoagulable state Show less
