Development and characterization of dried blood spot materials for the measurement of immunoreactive trypsinogen

Abstract

Objectives In response to increasing numbers of states in the US that test newborn babies for cystic fibrosis (CF), the Newborn Screening Quality Assurance Programme initiated a pilot proficiency testing programme for immunoreactive trypsinogen (IRT), the biomarker for CF. Dried blood spot specimens (DBS) were used to evaluate the performance of laboratories that screen babies for CF. Methods DBS were prepared from human whole blood enriched with physiologically relevant levels of IRT. Various methods of making IRT-enriched DBS were used to optimize the recovery and stability of the biomarker, including preparation of DBS from either intact or lysed red blood cells, varying the timing of IRT addition to blood before dispensing onto filter paper, adding a protease inhibitor cocktail, and treating serum with charcoal before IRT enrichment. The recovery and stability of IRT in DBS were assessed. Newborn screening laboratories were offered the opportunity to test blind-coded DBS in the pilot PT programme. Results IRT was stable in the filter paper matrix when stored for one year at either À201C or 41C. Fifty percent more IRT was recovered from DBS prepared with lysed red blood cells where the IRT was added to blood just before dispensing; however, protease inhibitors did not improve IRT recovery. Conclusions IRT in the DBS matrix is stable and can be shipped worldwide under ambient conditions. Optimal IRT recovery was achieved by adjustment of DBS production practices. Laboratories receiving specimens accurately measured IRT by a variety of commercial and in-house methods.

European best practice guidelines for cystic fibrosis neonatal screening

“…Despite such variability, all the current commercial IRT assays seem to produce satisfactory results. Production of quality control material for IRT assays is extremely difficult as commercially available trypsin preparations differ between companies and batches in immunogenicity, protein concentration, or tryptic activity [59]. Traditional EQA schemes are of limited use for calibrating IRTassays used in screening since there is too much volumetric variability in the control spots themselves and in the calibrant spots used to construct the standard curve.…”

Newborn Screening for Cystic Fibrosis by Use of a Multiplex Immunoassay

“…The heterogeneous nature of IRT and differing specificity of antibodies to the various components have raised issues with the standardization and external QC of the assay. As noted by Li et al (8), the lack of a universally acceptable IRT standard has made the comparison of absolute IRT values among commercial immunoassays difficult. As reported by Lafont (11) trypsin exists in many forms in the serum but is not recognized equally among immunoassays, thus contributing to the discordant results when comparing one assay with another.…”

Variants in Solute Carrier SLC26A9 Modify Prenatal Exocrine Pancreatic Damage in Cystic Fibrosis

“…Moreover, different commercial IRT kits use different antibodies 23 , which have different specificity for trypsinogen or bind to different forms of trypsinogen present in serum 24 . Further, there is no certified IRT reference material, so the IRT provided as a reference can vary in level in different kits 40 . Finally, the stability of IRT measured from blood spots may be affected by transport time from the hospital to the testing facility and the climate of the area 41 .…”