Newborn Screening for Cystic Fibrosis by Use of a Multiplex Immunoassay

Abstract

Background Since its beginnings newborn screening for cystic fibrosis (CF) using an assay for immunoreactive trypsinogen (IRT) has been plagued by a high number of false positive results, i.e., screen-positive / diagnosis negative, despite attempts to reduce them through use of altered cutoffs and 2nd tier DNA testing. IRT exists as two isoforms: IRT1 and IRT2, with IRT2 being more closely aligned with pancreatic disease, including CF. Standardization among programs using current IRT assays recognizing either one or both isoforms is a continuing problem. Here we report the development of a multiplexed assay for both forms of IRT simultaneously. Methods Using two different Luminex bead sets, assays for each IRT isoform were developed separately and then combined. Using the sum of IRT1 and 2 values, comparisons were made with a CF kit currently in use. Results In a sample set consisting of 16 cases confirmed positive for CF, a cut-off was established at >97 ng/mL total IRT. Seven of eight carriers with one CF mutation screen positive by the standard method also were screen positive by the IRT1-IRT2. Of 32 cases screen positive by the standard IRT, 11 were screen negative by the IRT1-IRT2. None of these cases had CF mutations as identified by the screening program. Conclusions This data indicates that the multiplex method with specificity for two isoforms of IRT has comparable performance to a standard IRT method and the advantage of improved standardization by detection of the two isoforms.

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“…In future studies, taking advantage of the experience gained in developing multiplex immunoassays, biomarkers from the best performing models could be converted into multiplex assays for use in larger studies on newborns selected as described here [14]. Additional biomarkers could also be added to the panel simply through addition of appropriate beads.…”

“…Details of the immunoassay procedure have been previously published [13,14]. The Rules-Based Medicine platform is a multiplex commercial product (kit) already validated for precision, sensitivity, specificity, accuracy and assay limitations.…”

Thirty-years of screening for cystic fibrosis in East Anglia

“…However, our data does not demonstrate a difference in the false positive rate pre and post the change to testing at day 5–8 (IRT—IRT protocol). The kits utilised at various stages may have affected the results, as there are various forms of IRT in blood that may react differently in the assays; however their relative merits are the subject of continuing debate 14 20. Between 1980 and 1990, as other centres adopted this screening strategy, poor interlaboratory performance in a quality assurance scheme highlighted problems related to the adaptation of the first generation of IRT radioimmunoassays for use with dried blood spots 21.…”

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