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Abstract BACKGROUND
Within the working group standardization of apolipoproteins by mass spectrometry of the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC WG APO-MS), a higher order Reference Measurement Procedure (RMP) was developed and described for the quantitation of apolipoprotein(a) in multiplex with six other apolipoproteins (Ruhaak et al., Clin Chem 2023). So far the sample preparation was manual and laborious, therefore semi-automation of the sample preparation was considered not only to reduce the burden on manual labor in the lab, but also to hold the promise of increased precision. Here, we describe the semi-automation of the sample preparation and the evaluation of the analytical precision at a wide range of apo(a) concentrations.
METHODS
A protocol for semi-automation of the sample preparation of our RMP was programmed in VWorks software for Agilent BRAVO liquid handler. The sample preparation strategy consists of sample dilution, protein reduction in the presence of internal standard-TCEP mix, cysteine alkylation with iodoacetamide, proteolytic predigestion using LysC and proteolytic digestion with trypsin, followed by quenching of the digestion. Peptide enrichment is subsequently performed offline through solid phase extraction. The procedure was subdivided in three liquid handling protocols to allow for sufficient deck position flexibility. The first protocol contains the start to protein reduction, the second protocol alkylation to digestion and the last protocol the quench. For quantitation of apo(a) by LC-MRM-MS, three peptides are used: LFLEPTQADIALLK (LFLEP), GISSTVTGR (GISST) and TPENYPNAGLTR (TPENY). Calibration is performed through 5 native human serum calibrators that are value assigned with traceability to the former reference material SRM2B. The precision of the method with semi-automated sample preparation for apo(a) quantitation was evaluated by a CLSI EP-05 protocol using 20 native human serum samples spanning concentrations of 5 to 300 nmol/L.
RESULTS
Implementation of the sample preparation on an Agilent BRAVO liquid handling platform could be achieved through the use of three protocols. Using manual sample preparation and following a CLSI EP-05 protocol, average within laboratory imprecision of 8.9%, 11.9%, and 12.8% was reported for peptides LFLEP, GISST, and TPENY, respectively (Ruhaak et al., Clin Chem 2023). For the semi-automated RMP, results obtained from 15 duplicate measurements of each of the 20 clinical samples, yielded median within run imprecision of 5.8%, 6.2% and 10.8% for LFLEP, GISST, and TPENY, respectively. Median within-laboratory imprecisions were 6.8%, 7.5% and 11.3% for peptides LFLEP, GISST, and TPENY, respectively. Notably, the imprecision increased below concentrations of 20 nmol/L, with a maximum within-laboratory imprecision of 29.7%, 24.1% and 31.0% at a concentration of 6 nmol/L. However, given the larger intra-individual variation observed for individuals with lower apo(a) concentrations, the higher levels of imprecision obtained fulfills the analytical performance required for accurate clinical interpretation. More importantly, the average within-laboratory imprecision around the clinical cutoff value of 90 nmol/L was calculated based on four clinical samples with concentrations between 80 and 105 nmol/L was 6.6%, 6.9% and 10.9% for peptides LFLEP, GISST, and TPENY, respectively, suggesting precise measuring results in the clinically relevant range.
CONCLUSIONS
We here present the semi-automation of the reference measurement procedure for the quantitation of apolipoprotein(a). Employment of a liquid handler not only makes the sample preparation procedure more lean, but also significantly increases the precision of the RMP. The impact of the increased precision of the method is large, as it now better feasible to stay within analytical performance specifications of the RMP that are set as a rule of thumb at 1/3rd of the allowable measurement uncertainty for end users at medical laboratories. The RMP for apo(a) is now suitable for value assignments of reference materials with traceability to SRM2B. Moreover, within the Leiden Apolipoprotein Calibration Laboratory, an Lp(a)/Apo(a) certification programme for IVD-manufacturers has been established which guarantees traceability of Lp(a)/apo(a) test results to SRM2B. Participation of IVD-manufacturers in this certification programme is encouraged in order to reduce intermethod variability and to facilitate universal application of clinical decision limits in specific target populations. In this era of looming Lp(a)/apo(a)-lowering medication, global Lp(a)/apo(a) standardization is a conditio sine qua non for correct classification and monitoring of patients with ASCVD risk related to high Lp(a) levels.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
