Evaluation of dried-blood spots and a hematocrit-independent procedure in lysosomal diseases screening using multiplexed tandem mass spectrometry assays.

Abstract

Dried blood spots (DBS) are widely used as a non-invasive sampling method, especially in newborn screening (NBS). Despite its numerous advantages, conventional DBS might be limited by the hematocrit effect when analyzing a punch, depending on its position in the blood spot. This effect could be avoided using hematocrit-independent sampling devices such as the hemaPEN®. This device collects blood through integrated microcapillaries, and a fixed blood volume is deposited on a pre-punched paper disc. NBS programs are increasingly poised to include lysosomal disorders, given the availability of treatments that improve clinical outcomes if detected early. In this study, the effect of hematocrit and punch position in the DBS on the assay of 6 lysosomal enzymes was evaluated on 3mm discs pre-punched in hemaPEN® devices compared to 3mm punches from the PerkinElmer 226 DBS. The enzyme activities were measured by multiplexed tandem mass spectrometry coupled to ultra-high performance liquid chromatography. Three hematocrit levels (23%, 35%, and 50%) and punching positions (center, intermediary, and border) were tested. Three replicates have been performed for each condition. A multivariate approach has been used along with a univariate method to assess the effect of the experimental design on each enzyme activity. Hematocrit, punch position, and whole blood sampling method do not affect the assessment of enzyme activity using the NeoLSD® assay. The results obtained from conventional DBS and the volumetric device HemaPEN® are comparable. These results underline the reliability of DBS for this test.