ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, United States.
Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84112, United States.
Clin Chim Acta. 2018 Aug;483:142-144. doi: 10.1016/j.cca.2018.04.037. Epub 2018 Apr 27.
Individuals with inherited deficiencies of the pyruvate dehydrogenase complex or the respiratory chain complex can have increased concentrations of cerebrospinal fluid (CSF) lactate. Such measurements are clinical useful when measured in conjunction with pyruvate in order to calculate the lactate:pyruvate (L:P) ratio, a useful surrogate of cytosolic redox status. CSF pyruvate is measured in a protein-free supernatant prepared by the addition of CSF to perchloric acid while lactate is measured in untreated CSF. Utilizing the same sample for both lactate and pyruvate measurements is desirable.
To develop a method to measure lactate in perchloric-acid precipitated CSF and validate the L:P ratio as calculated from the analysis of both analytes in the same sample.
Samples were prepared by the addition of 1 mL CSF to 2 mL 8% (w/v) cold perchloric acid, incubated on ice for 10 min, then centrifuged to obtain a protein-free supernatant. Lactate was measured by its oxidation to pyruvate and hydrogen peroxide using lactate oxidase and the absorbance of the resulting chromogen determined at 540 nm on a Roche cobas c501 chemistry analyzer. Method accuracy, linearity, imprecision and sensitivity were determined and a reference interval was verified.
To assess accuracy, this method was compared to lactate determined in unaltered CSF at another laboratory using 41 specimens with lactate concentrations from 0.6-11.9 mmol/L. Linear regression produced a slope of 1.09 and y-intercept of 0.26 (R = 1.00). Recovery was performed by ad-mixes of a high lactate standard and a CSF pool in different ratios to create a set of 19 samples prior to preparing protein-free supernatants. Recovery was 94.6-100% (mean ± SD was 97.4 ± 1.4%) at lactate concentrations of 2.68 to 12.63 mmol/L. Linearity was determined by combining two supernatants with low and high lactate concentrations in different ratios to create a set of six samples (0.15-12.70 mmol/L) that were tested in duplicate. Linear regression generated a slope of 1.01, y-intercept of -0.04 (R = 1.00). Precision was verified by analyzing quality control materials (acid-treated lactate standard) in 3 replicates each day for 5 days. Within-laboratory imprecision was 2.3% at 1.5 mmol/L and 1.5% at 10.5 mmol/L. The limit of blank was 0.05 mmol/L as determined by the mean added to three standard deviations determined from 10 replicates of perchloric-acid treated saline pool. The limit of detection was determined to be 0.12 mmol/L calculated from 10 replicates of a patient sample treated with perchloric-acid. The manufacturer's reference interval of 1.1-2.4 mmol/L was verified using 20 residual patient CSF samples.
CSF lactate can be measured with accuracy and precision using the same perchloric-acid treated sample that is used for pyruvate.
丙酮酸脱氢酶复合物或呼吸链复合物遗传缺陷的个体，其脑脊液（CSF）中乳酸浓度会升高。当与丙酮酸一起测量以计算乳酸：丙酮酸（L：P）比值时，这种测量具有临床意义，L：P 比值是细胞溶质氧化还原状态的有用替代物。CSF 丙酮酸是通过在加入高氯酸的 CSF 中添加 CSF 来测量的，而乳酸则是在未处理的 CSF 中测量的。理想情况下，同时利用同一样本测量乳酸和丙酮酸。
开发一种测量高氯酸沉淀 CSF 中乳酸的方法，并验证从同一样本中分析两种分析物计算得出的 L：P 比值。
通过向 2mL 8%（w/v）冷高氯酸中加入 1mL CSF，将样品制备成沉淀，在冰上孵育 10min，然后离心获得无蛋白上清液。通过使用乳酸氧化酶将乳酸氧化为丙酮酸和过氧化氢，然后在罗氏 cobas c501 化学分析仪上测定生成的显色剂的吸光度，在 540nm 处测定乳酸。测定了方法的准确性、线性、精密度和灵敏度，并验证了参考区间。
为了评估准确性，将此方法与另一个实验室使用未经处理的 CSF 测定的乳酸进行了比较，使用了 41 份乳酸浓度为 0.6-11.9mmol/L 的标本。线性回归得到的斜率为 1.09，y 截距为 0.26（R=1.00）。回收率通过在不同比例下混合高乳酸标准品和 CSF 池中的 ad-mixes 来完成，在制备无蛋白上清液之前，创建了一组 19 个样本。在 2.68 至 12.63mmol/L 的乳酸浓度下，回收率为 94.6-100%（平均值±SD 为 97.4±1.4%）。通过将两种低浓度和高浓度的上清液以不同比例混合，创建一组 6 个样本（0.15-12.70mmol/L），在重复分析中进行测试，以确定线性。线性回归得到的斜率为 1.01，y 截距为-0.04（R=1.00）。通过每天在 3 个重复中分析 5 天的质控材料（处理过的乳酸标准品）来验证精密度。在 1.5mmol/L 时，实验室内部精密度为 2.3%，在 10.5mmol/L 时为 1.5%。空白限通过从 10 个重复的高氯酸处理盐水池的三个标准差中添加平均值确定为 0.05mmol/L。检测限通过从处理过的高氯酸的患者样本的 10 个重复中计算得出，为 0.12mmol/L。使用 20 份剩余的患者 CSF 样本验证了制造商的 1.1-2.4mmol/L 参考区间。
