Bloom Kaitlyn, Ditewig Meyers Gail, Bennett Michael J
Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA.
Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA.
J Appl Lab Med. 2016 Nov 1;1(3):271-279. doi: 10.1373/jalm.2016.020289.
Measurement of amino acids in dried blood spots has been extensively used for the detection of newborns with various inborn errors of amino acid metabolism including phenylketonuria (PKU) and maple syrup urine disease (MSUD). Whereas blood spot amino acid measurement has been invaluable for initial diagnosis, the relative insensitivity of blood spot measurement has found limited use in lifelong monitoring of patients with these disorders. The work described here outlines the evaluation of blood spot amino acid analysis using ultra-performance liquid chromatography (UPLC©) for use in follow-up testing.
Dried blood spot amino acids were derivatized with a proprietary AccQTag® reagent and separated using UPLC. Plasma amino acids from dried bloods spots were obtained from 318 patient samples and compared to corresponding plasma samples measured using the same UPLC method.
Dried blood spot amino acid concentrations were highly correlated but negatively biased vs plasma concentrations. Interassay imprecision studies using UPLC demonstrated a %CV for phenylalanine of 4.81%-16.07%, tyrosine 5.62%-20.16%, valine 4.23%-15.46%, leucine 8.3%-15.3%, and isoleucine 4.25%-16.80%. Intraassay imprecision studies using UPLC demonstrated a %CV for phenylalanine of 0.42%-3.4%, tyrosine 1.6%-7.85%, valine 0.14%-1.84%, leucine 0.28%-2.01%, and isoleucine 0.6%-2.65%. Blood spot amino acid concentrations were stable for at least 3 days at temperatures up to 65 °C.
This UPLC-based method can reliably measure clinically significant amino acids in dried blood spots.
干血斑中氨基酸的测定已广泛用于检测患有各种氨基酸代谢先天性疾病的新生儿，包括苯丙酮尿症（PKU）和枫糖尿症（MSUD）。虽然血斑氨基酸测定对于初始诊断非常重要，但血斑测定的相对不敏感性在这些疾病患者的终身监测中用途有限。本文所述工作概述了使用超高效液相色谱法（UPLC©）进行血斑氨基酸分析用于后续检测的评估。
干血斑氨基酸用专有的AccQTag®试剂进行衍生化，并用UPLC进行分离。从318份患者样本中获取干血斑中的血浆氨基酸，并与使用相同UPLC方法测量的相应血浆样本进行比较。
干血斑氨基酸浓度与血浆浓度高度相关，但存在负偏差。使用UPLC进行的批间精密度研究表明，苯丙氨酸的变异系数（%CV）为4.81%-16.07%，酪氨酸为5.62%-20.16%，缬氨酸为4.23%-15.46%，亮氨酸为8.3%-15.3%，异亮氨酸为4.25%-16.80%。使用UPLC进行的批内精密度研究表明，苯丙氨酸的变异系数（%CV）为0.42%-3.4%，酪氨酸为1.6%-7.85%，缬氨酸为0.14%-1.84%，亮氨酸为0.28%-2.01%，异亮氨酸为0.6%-2.65%。血斑氨基酸浓度在高达65°C的温度下至少3天保持稳定。
这种基于UPLC的方法能够可靠地测量干血斑中具有临床意义的氨基酸。
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