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

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

doi:10.3390/ijns6010014

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

Int J Neonatal Screen. 2020 Feb 29;6(1):14. doi: 10.3390/ijns6010014. eCollection 2020 Mar.

Authors

Shuren Feng¹, Joanne Mei², Lu Yang³, Ping Luo⁴, Xiaonan Wang⁵, Yuan Wang⁶, Jingyi Yao¹, Lan Cui¹, Lei Pan¹, Zefang Wang³, Li Xin¹

Affiliations

¹ Tianjin Women's and Children's Health Centre, Tianjin 300070, China; yaojingyi82@126.com (J.Y.); cuilan68@126.com (L.C.); panlei2121@126.com (L.P.); xinli66@126.com (L.X.).
² Newborn Screening and Molecular Biology Branch, Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA; jvm0@cdc.gov.
³ Department of Biology, College of Life Sciences, Tianjin University, Tianjin 300072, China; yanglululu@tju.edu.cn (L.Y.); zefangwang@tju.edu.cn (Z.W.).
⁴ Department of Basic Medicine, Tianjin Medical College, Tianjin 300222, China; luoping62@163.com.
⁵ Tianjin Medical Laboratory, BGI-Tianjin, BGI-Shenzhen, Tianjin 300308, China; douding.wxn@gmail.com.
⁶ Binhai Genomics Institute, BGI-Tianjin, BGI-Shenzhen, Tianjin 300308, China; yuanwang@genomics.cn.

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.

Keywords: Neonatal Screening; dried blood spots; filter paper; hyperphenylalaninemia (HPA), false-positive; newborns; ninhydrin fluorometric test; specimen collection devices.