Validation of Accuracy-based Amino Acid Reference Materials in Dried-Blood Spots by Tandem Mass Spectrometry for Newborn Screening Assays

Abstract

Background: Advances in technology and the earlier release of newborns from hospitals have pressed the demand for accurate calibration and improved interlaboratory performance for newborn screening tests. As a first step toward standardization of newborn screening aminoacidopathy tests, we have produced six-pool sets of multianalyte dried-blood-spot amino acid reference materials (AARMs) containing predetermined quantities of five amino acids. We describe here the production of the AARMs, validation of their amino acid contents, and characterization of their homogeneity and their stability in storage. Methods: To each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0–200 mg/L), Leu (0–200 mg/L), Met (0–125 mg/L), Tyr (0–125 mg/L), and Val (0–125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs. Results: The absolute mean biases across the analytic range for five amino acids were 2.8–9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P >0.05). Regression slopes (0 ± 0.01) for amino acid concentrations vs storage times and their P values (>0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 °C, or −20 °C during the intervals tested. Conclusion: The validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.

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

“…A multi-analyte DBS amino acid reference material has also been produced by the CDC in the United States. 33 However, these materials are only available in limited quantities to kit manufacturers and EQA scheme organizers.…”

The use of mass spectrometry to analyze dried blood spots

“…In NBS for instance, it is admitted that DBS samples can stay at room temperature from sample collection to analysis for several days, without refrigeration and with only limited precautions (Therrell et al, ; Mei et al, ). Most of typical biomarkers such as acylcarnitines (Fingerhut et al, ) and amino acids (Lundsjö et al, ; Chace et al, ) are stable, respectively, for 14 and 30 days at room temperature. Nevertheless, no general rules can be drawn, and stabilities must be investigated on a case‐by‐case basis.…”

The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States

“…Blood spot samples, collected from blood obtained by heel pricks, applied to filter paper and dried, are used to screen newborns for more than 50 treatable inborn disorders [1,2]. For more than 30 years, investigators have studied the relationship between temperature and the stabilities of markers for many of these disorders in dried-blood-spot (DBS) samples [3,4,5,6,7,8,9,10,11,12]. These fragmentary retrospective and prospective studies of DBS samples illustrate the adverse effects of uncontrolled or elevated temperatures and/or humidities on marker stabilities, but differences in markers studied, study durations, DBS storage conditions, and data analysis methods make comparison of results among studies difficult.…”