Cors Julien F, Kashyap Aditya, Fomitcheva Khartchenko Anna, Schraml Peter, Kaigala Govind V
IBM Research - Zurich, Rüschlikon, Switzerland.
Institute of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland.
PLoS One. 2017 May 11;12(5):e0176691. doi: 10.1371/journal.pone.0176691. eCollection 2017.
We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and β-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol-dewaxing, rehydration and staining-with the same microfluidic probe head. Tissue lithography brings a new level of versatility and flexibility in sample processing and budgeting in biobanks, which may alleviate current sample limitations for retrospective studies in biomarker discovery and drug screening.
我们提出了一种名为组织光刻（TL）的新概念及其实现方法，该方法能够对福尔马林固定石蜡包埋的组织切片进行回顾性研究。组织光刻使用微流控探针去除福尔马林固定石蜡包埋活检样本上石蜡层的微观区域。目前在研究和诊断中样本利用的做法要求在进行分子检测之前对样本进行完全脱蜡。这在检测次数以及对单个样本进行检测的时间方面都带来了很大限制。微观脱蜡通过允许对一部分组织进行脱保护以进行检测，同时保持样本的其余部分完整以便未来分析，从而消除了这些限制。检测后，样本可以送回储存而不是像标准工作流程那样被丢弃。我们通过用探头在样本顶部以流体动力学方式限制纳升级二甲苯来实现这种微观脱蜡。我们在扁桃体和乳腺组织切片及微阵列上展示了针对多种生物标志物（p53、CD45、HER2和β - 肌动蛋白）的微米级、基于显色和荧光的免疫组织化学。我们实现了小至100μm×50μm的染色模式，以及在单个组织微阵列核心内的多重免疫染色，与标准方案相比，局部脱蜡时间减少了20倍。我们还展示了复水时间减少了10倍，从而缩短了不同染色之间的处理时间。我们进一步通过在连续四天内对同一组织微阵列中的四个单独核心依次进行脱蜡和染色，展示了组织光刻在回顾性研究中的潜力。通过将组织光刻与微免疫组织化学概念相结合，我们用同一个微流控探头实现了免疫组织化学方案的每个步骤——脱蜡、复水和染色。组织光刻在生物样本库中的样本处理和预算方面带来了新的通用性和灵活性水平，这可能缓解目前生物标志物发现和药物筛选回顾性研究中的样本限制。
