Abstract：
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.
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.
摘要：
目的：组织病理学是诊断肺外结核的重要方法，然而,在使用耐酸染色剂(AFS)后,组织切片通常对分枝杆菌呈阴性。这项研究调查了AFS使用的机制和组织学处理的有害影响-特别是，二甲苯脱蜡-在AFS和分枝杆菌检测上。
方法：使用DNA和RNA特异性染料的三重染色研究了荧光金胺O（AuO）AFS的靶标。使用AuO荧光作为定量标记研究了二甲苯脱蜡对培养物或组织切片中分枝杆菌耐酸性的影响。对二甲苯法进行了比较,无溶剂投射热空气脱蜡（PHAD）。
结果：AuO与DNA/RNA染色的共定位表明细胞内核酸是AFS的真正靶标，产生高度特定的图案。二甲苯显著降低分枝杆菌荧光(P<0.0001；中等效应大小，r=0.33）。PHAD过程在组织中产生的荧光明显高于二甲苯脱蜡（P<0.0001；大效应大小，r=0.85）。
结论：金胺O可用于产生典型珠状图案的组织中分枝杆菌的核酸染色。耐酸染色在很大程度上取决于分枝杆菌细胞壁的完整性，二甲苯似乎会损坏。无溶剂组织脱蜡方法具有显着提高分枝杆菌检测的潜力。
方法：使用DNA和RNA特异性染料的三重染色研究了荧光金胺O（AuO）AFS的靶标。使用AuO荧光作为定量标记研究了二甲苯脱蜡对培养物或组织切片中分枝杆菌耐酸性的影响。对二甲苯法进行了比较,无溶剂投射热空气脱蜡（PHAD）。
结果：AuO与DNA/RNA染色的共定位表明细胞内核酸是AFS的真正靶标，产生高度特定的图案。二甲苯显著降低分枝杆菌荧光(P<0.0001；中等效应大小，r=0.33）。PHAD过程在组织中产生的荧光明显高于二甲苯脱蜡（P<0.0001；大效应大小，r=0.85）。
结论：金胺O可用于产生典型珠状图案的组织中分枝杆菌的核酸染色。耐酸染色在很大程度上取决于分枝杆菌细胞壁的完整性，二甲苯似乎会损坏。无溶剂组织脱蜡方法具有显着提高分枝杆菌检测的潜力。
