Hamot Gael, Ammerlaan Wim, Mathay Conny, Kofanova Olga, Betsou Fay
Integrated BioBank of Luxemburg (IBBL), Luxembourg.
Biopreserv Biobank. 2015 Jun;13(3):152-63. doi: 10.1089/bio.2014.0054. Epub 2015 Apr 1.
This article is part of a series of publications providing formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report the optimization and validation for fitness-for-purpose of automated and manual protocols for isolating peripheral blood mononuclear cells (PBMCs) from whole blood, and compare the two methods.
The manual method was optimized for whole blood centrifugation speed, gradient type (Ficoll, Leucosep, CPT), and freezing method (Mr Frosty, Controlled Rate Freezing). Various parameters of the automated protocol using a CPT gradient on a Tecan liquid handler were optimized. Optimal protocols were validated in parallel for reproducibility and robustness. Optimization and validation were assessed in terms of cell yield, viability, recovery, white blood cell (WBC) subpopulation distribution, gene expression, and lymphoblastoid cell line (LCL) transformation.
An initial centrifugation of whole blood at 2000 g was considered optimal for further processing, allowing isolation of plasma and PBMCs from a single sample. The three gradients gave similar outcomes in terms of cell yield, viability, and WBC subpopulation distribution. Ficoll showed some advantages and was selected for further evaluations. Optimization of the automated protocol script using a CPT gradient gave 61% cell recovery. No significant differences in quality, quantity, and WBC subpopulation distribution were seen between the two freezing methods, and Mr. Frosty was selected. The manual and automated protocols were reproducible in terms of quantity, recovery, viability, WBC subpopulation distribution, gene expression, and LCL transformation. Most (75%-100%) of the 13 robustness parameters were accepted for both methods with an 8 h pre-centrifugation delay versus 38%-85% after 24 h. Differences identified between the automated and manual methods were not considered consequential.
We validated the first fully automated method for isolating viable PBMCs, including RNA analysis and generation of LCLs. We recommend processing within 8 h of blood collection.
本文是一系列出版物的一部分，旨在为实验室和生物样本库认证背景下的生物样本处理提供正式的方法验证。我们报告了从全血中分离外周血单个核细胞（PBMC）的自动化和手动方案的适用性优化及验证，并比较了这两种方法。
手动方法针对全血离心速度、梯度类型（Ficoll、Leucosep、CPT）和冷冻方法（Mr Frosty、程序降温冷冻）进行了优化。对使用Tecan液体处理仪上的CPT梯度的自动化方案的各种参数进行了优化。并行验证了最佳方案的重现性和稳健性。从细胞产量、活力、回收率、白细胞（WBC）亚群分布、基因表达和淋巴母细胞系（LCL）转化方面评估优化和验证情况。
全血初始以2000 g离心被认为最适合进一步处理，可从单个样本中分离出血浆和PBMC。这三种梯度在细胞产量、活力和WBC亚群分布方面产生了相似的结果。Ficoll显示出一些优势并被选用于进一步评估。使用CPT梯度对自动化方案脚本进行优化后细胞回收率为61%。两种冷冻方法在质量、数量和WBC亚群分布方面未见显著差异，选择了Mr Frosty。手动和自动化方案在数量、回收率、活力、WBC亚群分布、基因表达和LCL转化方面具有可重复性。对于两种方法，13个稳健性参数中的大多数（75%-100%）在预离心延迟8小时的情况下被接受，而在24小时后为38%-85%。自动化和手动方法之间发现的差异不被认为具有重要意义。
我们验证了第一种用于分离有活力PBMC的全自动方法，包括RNA分析和LCL生成。我们建议在采血后8小时内进行处理。
