Effects of sample matrix in the measurement of antithrombin by LC-MS: A role for immunocapture

M Kruijt; N P M Smit; J J van Ham; C M Cobbaert; L R Ruhaak

doi:10.1016/j.jmsacl.2023.01.002

Effects of sample matrix in the measurement of antithrombin by LC-MS: A role for immunocapture

J Mass Spectrom Adv Clin Lab. 2023 Jan 7:27:61-65. doi: 10.1016/j.jmsacl.2023.01.002. eCollection 2023 Jan.

Authors

M Kruijt¹, N P M Smit¹, J J van Ham², C M Cobbaert¹, L R Ruhaak¹

Affiliations

¹ Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands.
² Biobank Facility, Leiden University Medical Center, Leiden, The Netherlands.

Abstract

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 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.

Keywords: AT, antithrombin; Anticoagulants; Antithrombin; BD, Becton, Dickinson and Company; CV, coefficient of variation; FA, formic acid; Immunocapture; LC, liquid chromatography; LUVDS, Leiden University Medical Center Voluntary Donor Service; MRM, multiple reaction monitoring; PIC, protease inhibitor cocktail; QC, quality control; Quantitative clinical chemistry proteomics; SST, system suitability test; Sample matrix; VHH, single variable domain on a heavy chain.