Introduction of a Simple Second Tier Screening Test for C5 Isobars in Dried Blood Spots: Reducing the False Positive Rate for Isovaleric Acidaemia in Expanded Newborn Screening

Abstract

In 2015 the English Newborn Screening programme expanded to include Isovaleric Acidaemia (IVA). Screening is performed by flow injection analysis tandem mass spectrometry of isovalerylcarnitine. Isovalerylcarnitine is isobaric with pivaloylcarnitine which can be present in blood due to the use of pivalic ester pro-drugs or pivalic acid derivatives used as emollients in some nipple creams; the potential for false positives (FP) is well documented. A pilot study in England screened 438,164 babies, 18 had presumptive positive results but only 4 were confirmed as true positives (TP). We developed a simple test to separate the isobaric compounds and investigate these samples further.

We studied newborn screening blood spots from 122 randomised controls and 34 infants with an initial raised C5 result. Dried blood spots were eluted with 30% acetonitrile (150 μL) and injected into a Waters Acquity UPLC coupled to a Waters Premier XE tandem mass spectrometer operating in positive ion mode. Isocratic separation of isovalerylcarnitine, pivaloylcarnitine, valerylcarnitine and 2-methylbutyrylcarnitine was achieved within 8 min. Assay performance characteristics were acceptable and non-parametric reference ranges (n = 122) were determined for each analyte.

If this method had been used as a second tier test for the 34 presumptive positive samples, the number of FP’s would have reduced from 24 to 8 and the positive predictive value of the screening test would have increased from 29 to 56%. Introduction of this test into the screening protocol has the potential to significantly reduce FP results for IVA and prevent unnecessary anxiety.

Appendices

Synopsis

Introduction of C5 isobars as a second tier screening test for Isovaleric Acidaemia has the potential to significantly reduce the FP rate.

General Rules

The name of the corresponding author: Rachel Carling.

A competing interest statement: No competing interests.

Details of funding N/A.

Conflict of Interest

Rachel Carling, Deborah Burden, Ian Hutton, Rachel Randle, Kate John and Jim Bonham declare no conflict of interest.

This article does not contain any studies with human or animal subjects performed by any of the authors.

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.

Details of the contributions of individual authors:

Rachel Carling performed the data analysis, researched the literature and wrote the first draft of the manuscript. All authors contributed to the data interpretation, review and approval of the manuscript.