Improving Harmonization and Standardization of Expanded Newborn Screening Results by Optimization of the Legacy Flow Injection Analysis Tandem Mass Spectrometry Methods and Application of a Standardized Calibration Approach

Abstract

Background Newborn screening (NBS) laboratories in the United Kingdom adhere to common protocols based on single analyte cutoff values (COVs); therefore, interlaboratory harmonization is of paramount importance. Interlaboratory variation for screening analytes in UK NBS laboratories ranges from 17% to 59%. While using common stable isotope internal standards has been shown to significantly reduce interlaboratory variation, instrument set-up, sample extraction, and calibration approach are also key factors. Methods Dried blood spot (DBS) extraction processes, instrument set-up, mobile-phase composition, sample introduction technique, and calibration approach of flow injection analysis–tandem mass spectrometry (FIA-MS/MS) methods were optimized. Inter- and intralaboratory variation of methionine, leucine, phenylalanine, tyrosine, isovaleryl-carnitine, glutaryl-carnitine, octanoyl-carnitine, and decanoyl-carnitine were determined pre- and postoptimization, using 3 different calibration approaches. Results Optimal recovery of analytes from DBS was achieved with a 35-min extraction time and 80% methanol (150 μL). Optimized methodology decreased the mean intralaboratory percentage relative SD (%RSD) for the 8 analytes from 20.7% (range 4.1–46.0) to 5.4% (range 3.0–8.5). The alternative calibration approach reduced the mean interlaboratory %RSD for all analytes from 16.8% (range 4.1–25.0) to 7.1% (range 4.1–11.0). Nuclear magnetic resonance analysis of the calibration material highlighted the need for standardization. The purities of isovaleryl-carnitine and glutaryl-carnitine were 85.13% and 69.94% respectively, below the manufacturer’s stated values of ≥98%. Conclusions For NBS programs provided by multiple laboratories using single analyte COVs, harmonization and standardization of results can be achieved by optimizing legacy FIA-MS/MS methods, adopting a common analytical protocol, and using standardized calibration material rather than internal calibration.

Retrospective Review of Positive Newborn Screening Results for Isovaleric Acidemia and Development of a Strategy to Improve the Efficacy of Newborn Screening in the UK

“…Whilst the geographical variation in FP results has been linked to the prescribing patterns of pivmecillinam, it is important to consider whether inter-laboratory variation is also a factor. Whilst all UK screening laboratories adhere to a defined algorithm with common COVs, equipment and methodology differ and inter-instrument variation of C5 has previously been reported to be 46% [ 10 ]. However, in addition to standard proficiency testing, population data has been monitored centrally on a quarterly basis since 2015 and confirms that the variation is not related to inter-laboratory bias (See Supplementary Material Figure S1 ).…”

Current State and Innovations in Newborn Screening: Continuing to Do Good and Avoid Harm

“…However, the practicalities of accurately preparing multiple DBS calibrators in sufficient quantity pose significant challenges, and this is before the traceability and standardization of the calibrators are considered. The alternative, more practical approach, is to rely on SIIC; however, the efficacy of this is limited by the purity of the stable isotope internal standards (IS), the accuracy with which they are prepared, and the limitations of what is, effectively, a single-point calibration [ 27 ]. With the introduction of proteomics and metabolomics profiling in screening and the rapid increase in the number of new biomarkers, the appropriate calibration of individual markers may prove to be challenging.…”

“…A major limitation of the utility of DBS specimens for biomarker analysis is the lack of commercially available, matrix-matched certified reference materials (CRMs) for the various analytes in DBS specimens with which to standardise laboratory tests [ 27 , 28 ]. As a result, DBS calibrators tend to be produced in-house by collecting blood from a healthy donor or using residual pooled patient samples and adding an aqueous enrichment prior to application onto the filter paper.…”

Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria

“…9 Another method of isotope dilution is constrained by the purity of internal standards and a single-point calibration. 10 European guidelines indicate that PHE levels in the blood of PKU patients under 12 years of age should range between 120 and 360 μmol/L, with a suggested range of 120-600 μmol/L for children older than 12. 1 A typical PHE:TYR ratio for neonatal babies has been reported as 3.3 (between 0.8 and 8.25) in the literature.…”

“…9 Another method of isotope dilution is constrained by the purity of internal standards and a single-point calibration. 10…”