Benzene Derivatives from Ink Lead to False Positive Results in Neonatal Hyperphenylalaninemia Screening with Ninhydrin Fluorometric Method.

Shuren Feng,Xiaonan Wang,Li Xin,Yuan Wang,Lei Pan,Joanne Mei,Ping Luo,Jingyi Yao,Lu Yang,Lan Cui,Zefang Wang

doi:10.3390/ijns6010014

Abstract

Ninhydrin-based fluorometric quantification of phenylalanine is one of the most widely used methods for hyperphenylalaninemia (HPA) screening in neonates due to its high sensitivity, high accuracy, and low cost. Here we report an increase of false positive cases in neonatal HPA screening with this method, caused by contamination of blood specimen collection devices during the printing process. Through multiple steps of verification, the contaminants were identified from ink circles printed on the collection devices to indicate the positions and sizes of blood drops. Blood specimens from HPA-negative persons collected on these contaminated collection devices showed positive results in the fluorometric tests, but negative results in tandem mass spectroscopy (MS/MS) experiments. Contaminants on the collection devices could be extracted by 80% ethanol and showed an absorption peak around 245 nm, suggesting that these contaminants may contain benzene derivatives with similar structure to phenylalanine. High-performance liquid chromatography (HPLC) analysis of the ethanol extracts from contaminated collection devices identified two prominent peaks specifically from the devices. Methyl-2-benzoylbenzoate (MBB, CAS#606-28-0) was found as one of the major chemicals from contaminated collection devices. This report aims to remind colleagues in the field of this potential contamination and call for tighter regulation and quality control of specimen collection devices.

Highlights

Hyperphenylalaninemia (HPA) or more commonly known as phenylketonuria (PKU) is characterized by elevation of blood phenylalanine (Phe) levels [1]
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Considering that the MS/MS method quantifies phenylalanine in blood samples by testing the position and intensities of phenylalanine on the mass/charge spectrum and their ratio to that of isotopically labelled phenylalanine of known concentrations, this result clearly showed that the contaminants were unlikely to be phenylalanine

Summary

Introduction

Hyperphenylalaninemia (HPA) or more commonly known as phenylketonuria (PKU) is characterized by elevation of blood phenylalanine (Phe) levels [1]. The most deleterious effect of this disease is its irreversible impairment on the developing brains of patients. Due to its harmful effects on the developing brain of newborns, early diagnosis and treatment is crucial to the normal development of the patients [4,5]. The methodologies adopted for HPA screening may vary among screening laboratories, but specimen collection devices, consisting of filter paper with preprinted circles used to collect blood and attached to a demographic section, are employed for all these screening methods to obtain dried blood samples (DBS) for new born screening (NBS). Chemical and physical properties of filter paper used to collect DBS have been shown to be important for maintaining stability and shelf life of blood samples [6].

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