Clinical relevance of the discrepancy in phenylalanine concentrations analyzed using tandem mass spectrometry compared with ion-exchange chromatography in phenylketonuria

Bridget M Stroup, Patrice K Held, Phillip Williams, Murray K Clayton, Sangita G Murali, Gregory M Rice, Denise M Ney, Bridget M Stroup, Patrice K Held, Phillip Williams, Murray K Clayton, Sangita G Murali, Gregory M Rice, Denise M Ney

Abstract

Introduction: Metabolic control of phenylketonuria (PKU) and compliance with the low-phenylalanine (phe) diet are frequently assessed by measuring blood phe concentrations in dried blood spots (DBS) collected by patients instead of plasma phe concentrations.

Objective: Our objective was to investigate the difference in blood phe concentrations in DBS collected by subjects and analyzed using either a validated newborn screening tandem mass spectrometry (MS/MS) protocol or ion-exchange chromatography (IEC) compared to plasma phe concentrations obtained simultaneously and analyzed using IEC.

Design: Three to four fasting blood samples were obtained from 29 subjects with PKU, ages 15-49 years. Capillary blood was spotted on filter paper by each subject and the DBS analyzed using both MS/MS and IEC. Plasma was isolated from venous blood and analyzed using IEC.

Results: Blood phe concentrations in DBS analyzed using MS/MS are 28% ± 1% (n = 110, p < 0.0001) lower than plasma phe concentrations analyzed using IEC resulting in a blood phe concentration of 514 ± 23 μmol/L and a plasma phe concentration of 731 ± 32 μmol/L (mean ± SEM). This discrepancy is larger when plasma phe is > 600 μmol/L. Due to the large variability across subjects of 13.2%, a calibration factor to adjust blood phe concentrations is not recommended. Analysis of DBS using IEC reduced the discrepancy to 15 ± 2% lower phe concentrations compared to plasma analyzed using IEC (n = 38, p = 0.0001). This suggests that a major contributor to the discrepancy in phe concentrations is the analytical method.

Conclusion: Use of DBS analyzed using MS/MS to monitor blood phe concentrations in individuals with PKU yields significantly lower phe levels compared to plasma phe levels analyzed using IEC. Optimization of current testing methodologies for measuring phe in DBS, along with patient education regarding the appropriate technique for spotting blood on filter paper is needed to improve the accuracy of using DBS to measure phe concentrations in PKU management.

Keywords: AAA, amino acid analyzer; Amino acid analyzer; Bland–Altman; DBS, dried blood spot; IEC, ion-exchange chromatography; MS/MS, tandem mass spectrometry; Newborn screening; PKU, phenylketonuria; Phenylalanine analytical methods; pah, phenylalanine hydroxylase; phe, phenylalanine; tyr, tyrosine.

Figures

Fig. 1
Fig. 1
The discrepancy between blood phe concentrations extracted from DBS and analyzed using MS/MS and plasma phe concentrations obtained by venipuncture and analyzed using IEC. Percent difference in Figures A & C is defined as ((plasma phe-blood) / plasma phe) x 100. (A) Bland–Altman analysis shows a 28 ± 1% (mean ± SE, p  600 μmol/L (n = 69). (D) When plasma phe concentrations ≤ 600 μmol/L (n = 41), blood phe concentration is 303 ± 115 μmol/L (mean ± SD) and plasma phe concentration is 401 ± 154 μmol/L (mean ± SD) for 41 sample pairs. When plasma phe concentrations > 600 μmol/L, blood phe concentration is 640 ± 206 μmol/L (mean ± SD) and plasma phe concentration is 927 ± 241 μmol/L (mean ± SD) for 41 sample pairs.
Fig. 2
Fig. 2
Heterogeneity of variability across subjects for phe measurement. Each line represents the range of percent differences from maximum to minimum within 3–4 sample pairs for each subject. Percent difference is defined as (plasma phe-blood phe)/plasma phe). x 100 The circle in the middle of each line represents the mean percent difference by subject. Total variability is high (SD = 13.2%) for 110 samples pairs from 29 subjects.
Fig. 3
Fig. 3
Estimated phe concentration measurements discrepancies using 3 different methodologies. The three methods include blood phe concentrations extracted from DBS and analyzed using MS/MS (DBS-MS/MS), blood phe concentrations extracted from DBS and analyzed using IEC (DBS-IEC), and plasma phe concentrations obtained by venipuncture and analyzed using IEC (plasma-IEC). Each method included 38 samples from 16 subjects. Blood phe concentration, extracted from DBS and analyzed using MS/MS, is 407 ± 216 μmol/L (mean ± SD). Blood phe concentration, extracted from DBS and analyzed using IEC, is 479 ± 283 μmol/L (mean ± SD). Plasma phe concentration, obtained by venipuncture and analyzed using IEC, is 573 ± 334 μmol/L (mean ± SD). DBS-MS/MS is 26 ± 2% (mean ± SE) lower compared to plasma-IEC for these 16 subjects (p 

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