Lund Kyle, Menlyadiev Marlen, Lee Kyunghoon, Kelner Michael J, Fitzgerald Robert L, Suhandynata Raymond T
Department of Pathology, UC San Diego Health, San Diego, CA, United States.
Department of Laboratory Medicine, Seoul National University, Bundang Hospital, Seongnam, South Korea.
J Mass Spectrom Adv Clin Lab. 2023 Mar 1;28:91-98. doi: 10.1016/j.jmsacl.2023.02.010. eCollection 2023 Apr.
The VALID Act is a legislative effort that, if enacted, would alter the regulatory requirements of laboratory developed tests (LDTs) used for clinical testing in the United States. Benzodiazepines, which are primarily excreted into urine as glucuronidated metabolites such as lorazepam, cross-react poorly with FDA-cleared immunoassays, leading to false-negatives. This shortfall can be addressed with LDTs created by adding glucuronidase to the immunoassay reagents producing "high sensitivity" assays that detect glucuronidated metabolites.
Precision and stability of two high-sensitivity (HS) benzodiazepine immunoassays from Roche and Thermo Scientific were evaluated using manufacturer-supplied quality control (QC) material and glucuronidated QC material. The immunoassays were directly compared to an LC-MS/MS LDT benzodiazepine assay to determine clinical sensitivity/specificity using urine specimens (n = 82 for Thermo Scientific; n = 265 for Roche). The clinical impact of the HS LDT immunoassay was determined by analyzing clinical testing results 60 days before and after its implementation.
The precision and clinical sensitivity/specificity of the HS-Thermo Scientific and HS-Roche benzodiazepine assays were acceptable. The reagent stability of the HS-Thermo Scientific immunoassay was poor, whereas the HS-Roche immunoassay was stable. After implementation of the HS-Roche benzodiazepine immunoassay as an LDT, there was a 30-fold increase -value: < 0.00001) in the percentage of lorazepam confirmations.
We demonstrate the development and validation of an immunoassay LDT with improved sensitivity for glucuronidated benzodiazepines. This LDT can detect glucuronidated benzodiazepines in clinical urine specimens and is stable for 60 days. Importantly, we were able to validate the immunoassay as an LDT by utilizing an LC-MS/MS LDT.
《VALID法案》是一项立法举措，若得以颁布，将改变美国用于临床检测的实验室自建检测方法（LDT）的监管要求。苯二氮䓬类药物主要以如劳拉西泮等葡萄糖醛酸化代谢物的形式排泄到尿液中，与美国食品药品监督管理局（FDA）批准的免疫测定法交叉反应性较差，会导致假阴性结果。通过在免疫测定试剂中添加葡萄糖醛酸酶来创建LDT，从而产生能够检测葡萄糖醛酸化代谢物的“高灵敏度”检测方法，可解决这一不足。
使用制造商提供的质量控制（QC）材料和葡萄糖醛酸化QC材料，评估了罗氏公司和赛默飞世尔科技公司的两种高灵敏度（HS）苯二氮䓬免疫测定法的精密度和稳定性。将这些免疫测定法与一种液相色谱 - 串联质谱（LC-MS/MS）LDT苯二氮䓬测定法直接进行比较，以使用尿液标本确定临床灵敏度/特异性（赛默飞世尔科技公司n = 82；罗氏公司n = 265）。通过分析HS LDT免疫测定法实施前后60天的临床检测结果，确定其临床影响。
HS - 赛默飞世尔科技公司和HS - 罗氏公司苯二氮䓬测定法的精密度和临床灵敏度/特异性是可接受的。HS - 赛默飞世尔科技公司免疫测定法的试剂稳定性较差，而HS - 罗氏公司免疫测定法是稳定的。将HS - 罗氏公司苯二氮䓬免疫测定法作为LDT实施后，劳拉西泮确认率增加了30倍（P值：< 0.00001）。
我们展示了一种对葡萄糖醛酸化苯二氮䓬具有更高灵敏度的免疫测定LDT的开发和验证。这种LDT能够检测临床尿液标本中的葡萄糖醛酸化苯二氮䓬，并且在60天内保持稳定。重要的是，我们通过利用LC-MS/MS LDT验证了该免疫测定法作为LDT的有效性。
