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

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.

NeoSeq: a new method of genomic sequencing for newborn screening

“…Due to the high sensitivity of TMS, the high false-positive rate and low positive predictive value of TMS are always a problem, particularly for some special populations such as premature infants [21]. Additionally, the technique is easily affected by external interference [22]. The high false-positive rate will bring a large number of healthy infants to be excessively recalled, which will not only burden the medical service but also result in psychological burden to the parents [23].…”

NeoSeq: A New Method of Genomic Sequencing for Newborn Screening

“…Due to the high sensitivity of TMS, the high false-positive rate and low positive predictive value of TMS are always a problem, particularly for some special populations such as premature infants 20 . Additionally, the technique is easily affected by external interference 21 . The high false-positive rate will bring a large number of healthy infants to be excessively recalled, which will not only burden the medical service but also result in psychological burden to the parents 22 .…”

Predictive value of fluorometric method and tandem mass spectrometry for hyperphenylalaninemia and its subtypes in China: A systematic review and meta‑analysis

“…There were a total of 12 items, each with a score of 0-2 and a total score of 24. Studies were classified as moderate quality (9)(10)(11)(12)(13)(14)(15)(16) or high quality (17)(18)(19)(20)(21)(22)(23)(24). The Meta-analysis was performed in accordance with the relevant items in the Preferred Reporting Items for Systematic Reviews and Meta Analysis statement.…”

“…These two methods are widely employed in clinical practice; however, their diagnostic accuracy and predictive value have remained the subject of ongoing research and clinical scrutiny (9). Fluorometric assays, which are relatively simple, cost-effective and amenable to large-scale application, are nonetheless associated with debates regarding its specificity and accuracy (10). By contrast, MS/MS offers higher resolution and sensitivity but requires more complex instrumentation and specialized expertise (11).…”