Monitoring phenylalanine concentrations in the follow‐up of phenylketonuria patients: An inventory of pre‐analytical and analytical variation

Abstract

Background Reliable measurement of phenylalanine (Phe) is a prerequisite for adequate follow‐up of phenylketonuria (PKU) patients. However, previous studies have raised concerns on the intercomparability of plasma and dried blood spot (DBS) Phe results. In this study, we made an inventory of differences in (pre‐)analytical methodology used for Phe determination across Dutch laboratories, and compared DBS and plasma results. Methods Through an online questionnaire, we assessed (pre‐)analytical Phe measurement procedures of seven Dutch metabolic laboratories. To investigate the difference between plasma and DBS Phe, participating laboratories received simultaneously collected plasma‐DBS sets from 23 PKU patients. In parallel, 40 sample sets of DBS spotted from either venous blood or capillary fingerprick were analyzed. Results Our data show that there is no consistency on standard operating procedures for Phe measurement. The association of DBS to plasma Phe concentration exhibits substantial inter‐laboratory variation, ranging from a mean difference of −15.5% to +30.6% between plasma and DBS Phe concentrations. In addition, we found a mean difference of +5.8% in Phe concentration between capillary DBS and DBS prepared from venous blood. Conclusions The results of our study point to substantial (pre‐)analytical variation in Phe measurements, implicating that bloodspot Phe results should be interpreted with caution, especially when no correction factor is applied. To minimize variation, we advocate pre‐analytical standardization and analytical harmonization of Phe measurements, including consensus on application of a correction factor to adjust DBS Phe to plasma concentrations.

Urine Phenylacetylglutamine Determination in Patients with Hyperphenylalaninemia

“…Monitoring and adjustments of individual treatments for PKU patients are still based on reliable Phe concentration measurements. Regarding the type of sample for Phe monitoring, DBS sampling is preferred for regular Phe analysis because it allows patients to obtain the sample at home and send it to the laboratory, whereas analysis of a full amino acid profile is considered the gold standard [24]. Despite the concerns about variability of DBS and plasma Phe results shown in previous studies [24][25][26][27][28][29], in this investigation, we have found a strong correlation (R = 0.98, p < 0.001) between Phe levels from DBS samples prepared from capillary finger-prick and Phe levels from venous blood without using a correction factor.…”

“…Regarding the type of sample for Phe monitoring, DBS sampling is preferred for regular Phe analysis because it allows patients to obtain the sample at home and send it to the laboratory, whereas analysis of a full amino acid profile is considered the gold standard [24]. Despite the concerns about variability of DBS and plasma Phe results shown in previous studies [24][25][26][27][28][29], in this investigation, we have found a strong correlation (R = 0.98, p < 0.001) between Phe levels from DBS samples prepared from capillary finger-prick and Phe levels from venous blood without using a correction factor. Although the analytical methods to analyze Phe levels from DBS (tandem mass spectrometry) and plasma (ion-exchange chromatography) were obviously different, pre-analytical factors that can influence Phe level variability were minimized, as all DBS and venous samples were obtained at the same time at Cruces University Hospital by an experienced nurse using the same methodology (spot size, punch location, etc.)…”

“…Therefore, the aim of this cross-sectional study was to determine (a) whether PKU patients present higher urine PAG levels than sexand age-matched healthy control participants, and (b) to determine whether there is a correlation between urine PAG levels, and Phe levels and dietary protein intake in PKU patients. As there is controversy over the inter-comparability of plasma and DBS Phe results [24][25][26][27][28][29], a secondary aim of this study was to assess the correlation of Phe levels from DBS (capillary finger-prick) and plasma samples obtained at the same time in participants with hyperphenylalaninemia. The inclusion criteria for the study were: (a) established diagnosis of mHPA or PKU, (b) regular attendance to their scheduled clinical follow-up visits at the Metabolic Unit, and (c) willingness to participate in the study.…”

Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria

“…10 European guidelines indicate that PHE levels in the blood of PKU patients under 12 years of age should range between 120 and 360 μmol/L, with a suggested range of 120-600 μmol/L for children older than 12. 1 A typical PHE:TYR ratio for neonatal babies has been reported as 3.3 (between 0.8 and 8.25) in the literature. 11,12 For the early monitoring of newborn babies with PKU, it is necessary and very important to determine 1 Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan 2 the concentrations of PHE and TYR.…”

“…[14][15][16] In the Netherlands, other methods are used to screen whole amino acid spectra in plasma, coupled with ion exchange chromatography. 1 However, previous studies have described how PHE levels cannot be compared between plasma and DBS samples. 1,[15][16][17] Therefore, we developed a quantitative method for determining the PKU disease biomarkers PHE and TYR.…”