Faculdade de Medicina, Instituto de Microbiologia, Universidade de Lisboa, Lisbon, Portugal.
Instituto de Medicina Molecular João Lobo Antunes, Lisbon, Portugal.
Am J Clin Pathol. 2023 Jul 5;160(1):81-88. doi: 10.1093/ajcp/aqad016.
Histopathology is an important method for diagnosing extrapulmonary tuberculosis, yet tissue sections are often negative for mycobacteria after use of acid-fast stain (AFS). This study investigated the mechanism of AFS use and the detrimental effect of histologic processing-in particular, xylene deparaffinization-on AFS and mycobacterial detection.
The target of the fluorescent Auramine O (AuO) AFS was investigated using triple staining with DNA- and RNA-specific dyes. The effect of xylene deparaffinization on the acid fastness of mycobacteria in cultures or tissue sections was studied using AuO fluorescence as a quantitative marker. The xylene method was compared with a novel, solvent-free projected-hot-air deparaffinization (PHAD).
Co-localization of AuO with DNA/RNA stains suggests that intracellular nucleic acids are the true target of AFS, producing highly specific patterns. Xylene reduces mycobacterial fluorescence significantly (P < .0001; moderate effect size, r = 0.33). The PHAD process yielded significantly higher fluorescence than xylene deparaffinization in tissues (P < .0001; large effect size, r = 0.85).
Auramine O can be applied for nucleic acid staining of mycobacteria in tissues producing typical beaded patterns. Acid-fast staining depends heavily on the integrity of the mycobacterial cell wall, which xylene appears to damage. A solvent-free tissue deparaffinization method has the potential to increase mycobacterial detection significantly.
组织病理学是诊断肺外结核的重要方法，但在使用抗酸染色（AFS）后，组织切片中经常找不到分枝杆菌。本研究探讨了 AFS 使用的机制以及组织学处理（尤其是二甲苯脱蜡）对 AFS 和分枝杆菌检测的有害影响。
使用 DNA 和 RNA 特异性染料的三重染色来研究荧光金胺 O（AuO）AFS 的靶标。使用 AuO 荧光作为定量标记物，研究二甲苯脱蜡对培养物或组织切片中分枝杆菌抗酸性的影响。将二甲苯法与新型无溶剂投射热空气去蜡法（PHAD）进行比较。
AuO 与 DNA/RNA 染色剂的共定位表明，细胞内核酸是 AFS 的真正靶标，产生高度特异性的模式。二甲苯显著降低分枝杆菌的荧光强度（P <.0001；中等效应大小，r = 0.33）。与二甲苯脱蜡相比，PHAD 过程在组织中产生的荧光明显更高（P <.0001；大效应大小，r = 0.85）。
金胺 O 可用于组织中分枝杆菌核酸的染色，产生典型的珠状图案。抗酸染色严重依赖于分枝杆菌细胞壁的完整性，而二甲苯似乎会破坏细胞壁。无溶剂组织脱蜡方法有可能显著提高分枝杆菌的检测率。
