ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT.
J Appl Lab Med. 2020 Jan 1;5(1):54-61. doi: 10.1373/jalm.2019.029579.
Pyruvate kinase (PK) deficiency is the most common cause of nonspherocytic hemolytic anemia owing to defective glycolysis. This study developed and validated an automated method to measure PK activity in red blood cells (RBCs).
PK catalyzes the reaction of phosphoenolpyruvate with ADP to form pyruvate and ATP. The pyruvate is reduced in the presence of lactate dehydrogenase and NADH to produce lactate and NAD+. The rate of absorbance decrease at 340 nm is proportional to PK activity. PK and hemoglobin (Hb) measurements were performed on a Roche cobas c501 analyzer. After establishing a k-factor, accuracy, linearity, imprecision, sensitivity, and stability were validated and the reference interval was verified.
The k-factor was -9477. Accuracy was evaluated by method comparison (n = 56). Linear regression yielded y = 1.0x - 0.57, and R2 of 0.93. Linearity was determined by combining a high sample with hemolyzing solution in 6 different ratios. Linear regression analysis yielded y = 1.02x - 2.68, and R2 of 1.0. The assay was linear to 87 U/dL. Precision was evaluated by testing hemolysates in 3 replicates/day for 10 days. Within-run imprecision was 1.9% and 2.5% and total imprecision was 4.0% and 5.6% at 14.0 and 8.1 U/g Hb, respectively. The limit of blank was 0.0, and the limit of detection was 1.0 U/dL. Stability was determined in 4 sample types at 3 different temperatures; the changes were all <10% when compared with t0. The current PK reference interval of 4.6 to 11.2 U/g Hb was verified.
This automated assay for quantifying PK in RBCs has acceptable performance characteristics and is fit for intended use.
丙酮酸激酶（PK）缺乏是由于糖酵解缺陷导致非球形红细胞溶血性贫血的最常见原因。本研究开发并验证了一种自动测定红细胞（RBC）中 PK 活性的方法。
PK 催化磷酸烯醇丙酮酸与 ADP 的反应，形成丙酮酸和 ATP。在存在乳酸脱氢酶和 NADH 的情况下，丙酮酸被还原为乳酸和 NAD+。340nm 处吸光度的下降速率与 PK 活性成正比。PK 和血红蛋白（Hb）的测量在罗氏 cobas c501 分析仪上进行。建立 k 因子后，验证了准确性、线性、精密度、灵敏度和稳定性，并验证了参考区间。
k 因子为-9477。通过方法比较（n=56）评估准确性。线性回归得到 y=1.0x-0.57，R2 为 0.93。通过将高浓度样本与溶血溶液以 6 种不同比例混合来确定线性范围。线性回归分析得到 y=1.02x-2.68，R2 为 1.0。该测定法的线性范围为 87U/dL。通过每天在 3 个复孔中测试溶血物，连续 10 天评估精密度。批内精密度分别为 1.9%和 2.5%，总精密度分别为 4.0%和 5.6%，Hb 为 14.0 和 8.1U/g。空白限为 0.0，检测限为 1.0U/dL。在 4 种样本类型和 3 种不同温度下确定稳定性；与 t0 相比，所有变化均<10%。当前的 PK 参考区间为 4.6 至 11.2U/g Hb，得到验证。
这种自动测定 RBC 中 PK 的方法具有可接受的性能特征，适合预期用途。
