Ammerlaan Wim, Trezzi Jean-Pierre, Lescuyer Pierre, Mathay Conny, Hiller Karsten, Betsou Fay
1 Integrated BioBank of Luxembourg (IBBL) , Luxembourg .
Biopreserv Biobank. 2014 Aug;12(4):269-80. doi: 10.1089/bio.2014.0003. Epub 2014 Jul 30.
Formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. Serum and plasma processing protocols were validated for fitness-for-purpose in terms of key downstream endpoints, and this article demonstrates methodology for biospecimen processing method validation.
Serum and plasma preparation from human blood was optimized for centrifugation conditions with respect to microparticle counts. Optimal protocols were validated for methodology and reproducibility in terms of acceptance criteria based on microparticle counts, DNA and hemoglobin concentration, and metabolomic and proteomic profiles. These parameters were also used to evaluate robustness for centrifugation temperature (4°C versus room temperature [RT]), deceleration (low, medium, high) and blood stability (after a 2-hour delay).
Optimal protocols were 10-min centrifugation for serum and 20-min for plasma at 2000 g, medium brake, RT. Methodology and reproducibility acceptance criteria were met for both protocols except for reproducibility of plasma metabolomics. Overall, neither protocol was robust for centrifugation at 4°C versus RT. RT gave higher microparticles and free DNA yields in serum, and fewer microparticles with less hemolysis in plasma. Overall, both protocols were robust for fast, medium, and low deceleration, with a medium brake considered optimal. Pre-centrifugation stability after a 2-hour delay was seen at both temperatures for hemoglobin concentration and proteomics, but not for microparticle counts.
We validated serum and plasma collection methods suitable for downstream protein, metabolite, or free nucleic acid-based applications. Temperature and pre-centrifugation delay can influence analytic results, and laboratories and biobanks should systematically record these conditions in the scope of accreditation.
在实验室和生物样本库认证的背景下，缺乏针对生物样本处理的正式方法验证。血清和血浆处理方案已根据关键下游终点进行了适用性验证，本文展示了生物样本处理方法验证的方法。
针对微粒计数，对人血的血清和血浆制备的离心条件进行了优化。基于微粒计数、DNA和血红蛋白浓度以及代谢组学和蛋白质组学谱，根据验收标准对最佳方案的方法和可重复性进行了验证。这些参数还用于评估离心温度（4°C与室温[RT]）、减速（低、中、高）和血液稳定性（延迟2小时后）的稳健性。
最佳方案是在2000g、中等制动、室温下血清离心10分钟，血浆离心20分钟。除血浆代谢组学的可重复性外，两种方案均符合方法和可重复性验收标准。总体而言，两种方案在4°C与室温下离心时均不稳健。室温下血清中的微粒和游离DNA产量更高，血浆中的微粒更少且溶血更少。总体而言，两种方案在快速、中等和低速减速时均稳健，中等制动被认为是最佳的。对于血红蛋白浓度和蛋白质组学，在两个温度下延迟2小时后的离心前稳定性均可见，但微粒计数则不然。
我们验证了适用于下游基于蛋白质、代谢物或游离核酸的应用的血清和血浆采集方法。温度和离心前延迟会影响分析结果，实验室和生物样本库应在认证范围内系统记录这些条件。
