Chemical Metrology Division, Applied Sciences Group, Health Sciences Authority, 1 Science Park Road, #01-05/06, The Capricorn, Singapore Science Park II, Singapore, 117528, Singapore.
Department of Laboratory Medicine, National University Hospital, Singapore, Singapore.
Anal Bioanal Chem. 2024 Nov;416(28):6611-6620. doi: 10.1007/s00216-024-05561-w. Epub 2024 Oct 3.
The biuret method is currently recognized as a reference measurement procedure for serum/plasma total protein by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). However, as the reaction involved in this method is highly time-dependent, to ensure identical measurement conditions for calibrator and samples for high accuracy, a fast and simple measurement procedure is critical to ensure the precision and trueness of this method. We measured serum/plasma total protein using a Cary 60 spectrophotometer coupled with a fiber optic probe, which was faster and simpler than the conventional cuvette method. The biuret method utilizing alkaline solutions of copper sulfate and potassium sodium tartrate was added to the sample and calibrator (NIST SRM 927e) incubated for 1 h before measurement. A panel of samples consisting of pooled human serum, single donor serum, and certified reference materials (CRMs) from three sources were measured for method validation. Sixteen native patient samples were measured using the newly developed biuret method and compared against clinical analyzers. Additionally, the results of three cycles of a local External Quality Assessment (EQA) Programme submitted by participating clinical laboratories were compared against the biuret method. Our biuret method using fiber optic probe demonstrated good precision with within-day relative standard deviation (RSD) of 0.04 to 0.23% and between-day RSD of 0.58%. The deviations between the obtained values and the certified values for all three CRMs ranged from -0.38 to 1.60%, indicating good method trueness. The routine methods using clinical analyzers were also found to agree well with the developed biuret method using fiber optic probe for EQA samples and native patient samples. The biuret method using a fiber optic probe represented a convenient and reliable way of measuring serum total protein. It also demonstrated excellent precision and trueness using CRMs and patient samples, which made the method a simpler candidate reference method for serum protein measurement.
双缩脲法目前被联合溯源性实验室委员会（JCTLM）认可为血清/血浆总蛋白的参考测量程序。然而，由于该方法涉及的反应高度依赖时间，为了确保校准品和样品的测量条件完全相同，以实现高精度，快速而简单的测量程序对于确保该方法的精密度和准确性至关重要。我们使用 Cary 60 分光光度计和光纤探头测量血清/血浆总蛋白，与传统比色皿法相比，这种方法更快更简单。双缩脲法利用硫酸铜和酒石酸钾钠的碱性溶液加入样品和校准品（NIST SRM 927e）中，孵育 1 小时后进行测量。使用三种来源的混合人血清、单供体血清和认证参考物质（CRM）的样品进行方法验证。使用新开发的双缩脲法测量了 16 份本地患者样本，并与临床分析仪进行了比较。此外，还比较了参加临床实验室的本地外部质量评估（EQA）计划提交的三个周期的结果与双缩脲法。我们使用光纤探头的双缩脲法显示出良好的精密度，日内相对标准偏差（RSD）为 0.04%至 0.23%，日间 RSD 为 0.58%。所有三个 CRM 的获得值与认证值之间的偏差范围为-0.38%至 1.60%，表明方法准确性良好。使用临床分析仪的常规方法也与使用光纤探头开发的双缩脲法在 EQA 样本和本地患者样本上吻合良好。使用光纤探头的双缩脲法是一种方便可靠的测量血清总蛋白的方法。它还使用 CRM 和患者样本证明了出色的精密度和准确性，这使得该方法成为血清蛋白测量更简单的候选参考方法。
