Performance of laboratory tests used to measure blood phenylalanine for the monitoring of patients with phenylketonuria

Abstract

Analysis of blood phenylalanine is central to the monitoring of patients with phenylketonuria (PKU) and age‐related phenylalanine target treatment‐ranges (0‐12 years; 120‐360 μmol/L, and >12 years; 120‐600 μmol/L) are recommended in order to prevent adverse neurological outcomes. These target treatment‐ranges are based upon plasma phenylalanine concentrations. However, patients are routinely monitored using dried bloodspot (DBS) specimens due to the convenience of collection. Significant differences exist between phenylalanine concentrations in plasma and DBS, with phenylalanine concentrations in DBS specimens analyzed by flow‐injection analysis tandem mass spectrometry reported to be 18% to 28% lower than paired plasma concentrations analyzed using ion‐exchange chromatography. DBS specimens with phenylalanine concentrations of 360 and 600 μmol/L, at the critical upper‐target treatment‐range thresholds would be plasma equivalents of 461 and 768 μmol/L, respectively, when a reported difference of 28% is taken into account. Furthermore, analytical test imprecision and bias in conjunction with pre‐analytical factors such as volume and quality of blood applied to filter paper collection devices to produce DBS specimens affect the final test results. Reporting of inaccurate patient results when comparing DBS results to target treatment‐ranges based on plasma concentrations, together with inter‐laboratory imprecision could have a significant impact on patient management resulting in inappropriate dietary change and potentially adverse patient outcomes. This review is intended to provide perspective on the issues related to the measurement of phenylalanine in blood specimens and to provide direction for the future needs of PKU patients to ensure reliable monitoring of metabolic control using the target treatment‐ranges.

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

“…In our study, the preparation was performed in a controlled setting, with a single technician preparing the DBSV samples and with guidance from a researcher in the preparation of DBS from capillary finger prick. When DBSC is prepared by (parents of) patients at home, which is the usual situation, one can assume that the variation in DBS quality is much higher than in the current study 9,11,16 . In addition, there were only samples from adults included in this study, while especially for children or neonates more difficulties in adequate filling of the DBS can be expected, as well as effects of higher hematocrit.…”

“…Analysis of a full amino acid profile is considered as the gold standard method for Phe measurement. Recent studies have raised concerns that plasma and DBS methods do not render comparable Phe results 6‐10 …”

“…Recent studies have raised concerns that plasma and DBS methods do not render comparable Phe results. [6][7][8][9][10] Besides different analysis techniques and origin of the blood sample (capillary or venous), there are multiple causes that can contribute to the variability of Phe measurements in DBS. 11,12 The European PKU guidelines acknowledged the high variation in Phe measurements, but did not address the possible differences between analysis-and sampling methods in much detail.…”

Use of Dried Blood Spot Specimens to Monitor Patients with Inherited Metabolic Disorders

“…This is preferable to reporting patient results against different target treatment ranges as this may cause confusion for both the patient and the clinician. Particularly in conditions where both specimen types are used, e.g., plasma amino acids and DBS phenylalanine in patients with PKU [5]. It is essential that a rigorous evaluation of the bias between plasma and DBS analyte results is undertaken in laboratories to derive a calibration factor in order to report DBS results as plasma equivalents (ideally on an individual patient basis), thereby ensuring meaningful comparison of patient results to the recommended target treatment ranges [5].…”

“…The bias from the enrichment concentration values should be <±15% [52]. However, the performance of DBS assays used to monitor patients may need to be more stringent, especially when patient results are compared to consensus target treatment ranges [5].…”