Inter-laboratory analytical improvement of succinylacetone and nitisinone quantification from dried blood spot samples

Background: Nitisinone is used to treat hereditary tyrosinemia type 1 (HT-1) by preventing accumulation of toxic metabolites, including succinylacetone (SA). Accurate quantification of SA during newborn screening is essential, as is quantification of both SA and nitisinone for disease monitoring and optimization of treatment. Analysis of dried blood spots (DBS) rather than plasma samples is a convenient method, but interlaboratory differences and comparability of DBS to serum/plasma may be issues to consider. Methods: Eight laboratories with experience in newborn screening and/or monitoring of patients with HT-1 across Europe participated in this study to assess variability and improve SA and nitisinone concentration measurements from DBS by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantification of nitisinone from both DBS and plasma was performed to assess sample comparability. In addition, efforts to harmonize laboratory procedures of SA and nitisinone quantifications during 5 rounds of analysis are described. Results: Nitisinone levels measured from DBS and plasma strongly correlated (R2 = 0.93). Due to partitioning of nitisinone to the plasma, levels were higher in plasma by a factor of 2.34. In the initial assessment of laboratory performance, all had linear calibrations of SA and nitisinone although there was large inter-laboratory variability in actual concentration measurements. Subsequent analytical rounds demonstrated markedly improved spread and precision over previous rounds, an outcome confirmed in a final re-test round. Conclusion: The study provides guidance for the determination of nitisinone and SA from DBS and the interpretation of results in the clinic. Inter-laboratory analytical harmonization was demonstrated through calibration improvements