A new milestone on the road to global standardization of apolipoprotein measurements

In this issue of Clin Chem Lab Med, the study by Smit et al. [1] from Leiden University Medical Center (LUMC, The Netherlands) highlights the importance of a standardized preanalytical phase for accurate mass spectrometry applications in clinical chemistry.

For medical decisions guided by in vitro diagnostic (IVD) tests, it is crucial to have accurate and standardized diagnostic assays able to attenuate errors in patient risk stratification and treatment decisions due to between-method variations. To declare a particular method as standardized, a clinical laboratory or IVD manufacturer must demonstrate adequate agreement between their measurement and a consensus reference method and/or consensus reference material to establish metrological traceability of results. But doing this once is not good enough. Creating a standardized preanalytical phase is an equally important objective to attenuate variability of measurements that may occur over time, on treatment follow-up. Preanalytical errors relate to biological variations or procedures to obtain and preserve a sample, and add up to total measurement uncertainty (MU). If the preanalytical phase of measurement of a marker is not standardized, results obtained from one laboratory using one sample matrix may not be comparable to results obtained from another laboratory using a different sample matrix to measure the same marker, even if the same analytical method by the same manufacturer is used.

The LUMC paper [1] emphasizes the importance of the measurand (that is, the quantity intended to be measured). With mass spectrometry, attention should focus on: what do we aim to measure and how can preanalytical variation disrupt the measurement? Quantitative protein mass-spectrometry (QPMS) depends on transition of the measurand by tryptic digestion of intact proteins and subsequent measurement of representing peptides – the so-called bottom-up proteomics approach [2, 3].

In QPMS with proteolytic digestion of the protein of interest, and triple quad technology as we use it, we expect the conversion of protein to proteotypic peptide to be complete and selective. After all, we make a surrogate measurand and calculate that back to protein concentration, assuming equimolarity and no disruption. To ensure accurate test results, the total testing process should be evaluated, including matrix-dependent variation. The blood sample matrix is incredibly decisive for recovery. Anything in the matrix that can interfere with the digestion step can negatively affect the test result [4, 5]. We must therefore minimize and standardize the preanalytical variables, for the sake of reproducible quantitative-“omics” applications.

Whether serum and plasma can be used interchangeably and whether in-vitro anticoagulants affect the recovery of proteolytic digestion, and ultimately the quantitation of proteins, was so far unknown. Errors may be caused by serum or plasma contamination by platelets, erythrocytes or coagulation factors, but also altered proteolytic activity, impacting the peptide production. Tryptic digestion kinetics of blood-based biomarkers may be affected by sample matrix and preparation conditions [4]. Similarly, differences between serum and plasma matrices, related to effects of the clotting process in serum or the complexing agents such as EDTA and citrate in plasma, are known to cause changes in the blood sample composition. Not only the anticoagulant, but also the standardized way of sample processing (do proteins remain intact after blood sampling or are they degraded?), the type of body material, the phlebotomy procedure, the processing time and conditions, and other variation in the matrix all influence the completeness of the digestion [5]. For example, urine proteomics is even more difficult to standardize than serum, because of the large variation between individuals in disease and health. The preanalytical phase needs to be optimized per protein [5].

The present LUMC study [1] demonstrates for the first time the influences of the varying matrices in blood collection tubes on the targeted quantification of apolipoproteins (apo) by QPMS. Serum (with and without gel) and four different plasma matrices (lithium heparin, K₂EDTA without and with protease inhibitors, and sodium citrate) were investigated for measurements of nine apos: A-I, A-II, A-IV, (a), B, C-I, C-II, C-III and E. The study revealed that apolipoprotein concentrations are interchangeable between serum tubes with and without gel, and they are highly similar to the lithium heparin plasma matrix. Contrarily, measured apolipoprotein concentrations are lower in sodium citrate plasma matrix, attributable to dilution of the blood samples by the sodium citrate buffer. Reduced recovery in EDTA-plasma is dependent on altered proteolytic digestion efficiency and also affects QPMS test results. Trypsin does not cleave at all sites with equal efficiency. Tryptic digestion kinetics revealed that especially slow forming peptides showed reduced formation in EDTA-plasma [1].

This study demonstrates that quantification of proteins from variable matrices by targeted QPMS approach using bottom-up proteomics is not necessarily interchangeable and depends on tryptic digestion kinetics in the serum and plasma matrices [1]. The fact that serum and heparin plasma, unlike citrate and EDTA plasma, show comparable QPMS results is a practical advantage, because in our clinical laboratories serum and heparin samples are the preferred matrices and are often used interchangeably for routine chemistry measurements including apolipoproteins.

The LUMC paper is yet another milestone on the road to global standardization of apolipoprotein measurements, initiated by the International Federation of Clinical Chemistry (IFCC). This is especially important for apoB. Multiple consensus groups, including the joint statements by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM), the European Atherosclerosis Society (EAS), and the European Society of Cardiology (ESC) have declared that apoB is recommended for assessment of cardiovascular risk attributable to atherogenic lipoproteins, and its measurement can be adequately standardized to meet requirements for application in clinical care [6, 7]. In 1994, the IFCC and World Health Organization (WHO) endorsed the SP3-07 reference material, value-assigned using immunonephelometry as interim reference method for apoB [8]. Common calibration with the IFCC/WHO SP3-07 reference material, although intended to be an interim solution, has been proven adequate to reduce between-laboratory variability of apoB measurements [8]. Nevertheless, given the increasing evidence base for using apoB in patient care, and promising applications of quantifying multiple apolipoproteins for precision medicine approaches of dyslipidemia management, further work on refinement of standardization and between-method comparability of apolipoproteins is justified [9, 10]. Accordingly, efforts by IFCC to develop a new Primary Reference Measurement System with a mass spectroscopy-based primary reference method and serum-based reference materials are well underway and will allow true SI-traceable standardization of apolipoproteins [3].

Given these improvements by the IFCC, measurements of apolipoproteins will meet predefined analytical performance criteria including accuracy, harmonization across laboratories, unambiguous definition of the measurand, and unequivocal test results – important prerequisites for medical use of a test which cannot be met with traditional lipid profiles based on low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and non-HDL cholesterol measurements or calculations [9]. I have focused on preanalytical and analytical standardization, which add up to total MU and should not exceed allowable MU, but it is the overall clinical performance and clinical effectiveness of an assay that are determinative to justify its medical use. The EFLM, EAS, and ESC have all concluded that apoB is the preferred accurate marker over LDL cholesterol, particularly at low concentrations, for estimating risk of atherosclerotic cardiovascular disease [6, 7].