Supplementary file 3_Polymer embedding of membrane lungs for histological investigations of intra-device clot formation.pdf

Extracorporeal membrane oxygenation (ECMO) is an invasive but potentially lifesaving treatment option for severe cardiac or respiratory failure. Despite its beneficial effect, coagulation-related complications, mainly due to clot formation, excessive bleeding and the accumulation of deposits in the membrane lung (ML) remain common, causing higher mortality. In this context, the formation of clots and other deposits in the ML is of particular interest. Previous histological examinations of the polymethylpentene fiber mats inside the ML could only be performed in a top view, prohibiting valid quantification and examination of the multi-layered deposits or fiber mat spanning structures. Our objective was the establishment of a polymer embedding to increase the mechanical stability of the deposits and thus enable cross-sectional microtome cutting through the ML hollow-fibers. Clinically used MLs (PLS, Getinge, Rastatt, Germany) were stabilized with a polymer resin (HistoCURE 8100). Specimens were cut out of the embedded MLs and microtome sections with a thickness of 10 µm were performed. In addition to standard histological staining with hematoxylin-eosin (HE) and Pappenheim (May-Grunwald-Giemsa), fluorescence DNA staining for nucleated cells with 4′,6-diamidino-2-phenylindole (DAPI) and SYTOX™ Green as well as immunohistochemical and immunofluorescence staining for the lysosomal enzyme myeloperoxidase (MPO) and von Willebrand factor (vWF) were established. The protocol provides a method for large volume embedding (400 mL). The cellular and extracellular deposits were securely fixed by the polymer scaffold allowing the examination of clots in MLs in native position which was not possible with conventional paraffin embedding. Multi-layered deposits and fiber mat spanning structures are no longer disrupted during specimen extraction and can now be quantified. Staining with HE, Pappenheim, DAPI, SYTOX™ Green, MPO, and vWF was successfully tested with this protocol. This method may be the foundation for new insights into the complex clotting phenomena observed in MLs.

体外膜肺氧合（Extracorporeal membrane oxygenation, ECMO）是针对重症心功能或呼吸功能衰竭的有创治疗手段，有望挽救患者生命。尽管其具有临床获益，由血栓形成、过度出血以及膜肺（membrane lung, ML）内沉积物蓄积引发的凝血相关并发症仍较为常见，进而导致患者死亡率升高。在此背景下，膜肺内血栓及其他沉积物的形成成为研究热点。此前针对膜肺内部聚甲基戊烯纤维毡的组织学检查仅能通过俯视视角开展，无法对多层沉积物或跨纤维毡结构进行有效定量与分析。本研究旨在建立一种聚合物包埋方法，以提升沉积物的机械稳定性，从而实现对膜肺中空纤维的横断面切片机切片。本研究采用聚合物树脂（HistoCURE 8100）对临床使用的膜肺（型号PLS，厂家为德国拉施塔特的Getinge公司）进行固定稳定。从包埋完成的膜肺标本中切取样本，并制作厚度为10 μm的切片标本。除采用苏木精-伊红（HE）及潘普海姆（May-Grunwald-Giemsa）染色这两种标准组织学染色方法外，本研究还建立了针对有核细胞的荧光DNA染色方案，包括4',6-二脒基-2-苯基吲哚（DAPI）与SYTOX™ Green染色，同时建立了针对溶酶体酶髓过氧化物酶（MPO）及血管性血友病因子（vWF）的免疫组织化学与免疫荧光染色方法。本方案可实现大容量标本包埋（容量可达400 mL）。聚合物支架可牢固固定细胞及细胞外沉积物，从而能够在原位状态下对膜肺内的血栓进行观察，这是传统石蜡包埋方法无法实现的。多层沉积物及跨纤维毡结构在标本取材过程中不再被破坏，如今可实现定量分析。采用本方案对HE、潘普海姆、DAPI、SYTOX™ Green、MPO及vWF染色均成功完成验证。该方法可为深入理解膜肺内复杂的凝血现象提供研究基础。