A novel and simple heat-based method eliminates the highly detrimental effect of xylene deparaffinization on acid-fast stains

Objectives Histopathology is an important method for the diagnosis of extrapulmonary tuberculosis, yet tissue sections are often negative for mycobacteria after acid-fast-staining (AFS). This study investigated the mechanism of AFS and the detrimental effect of histological processing, in particular xylene deparaffinization, on AFS staining and mycobacterial detection. Methods 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 acid-fastness of mycobacteria in cultures or tissue sections was studied using AuO fluorescence as a quantitative marker. The xylene method was compared to a novel, solvent-free projected-hot-air-deparaffinization (PHAD). Results Colocalization 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<0,0001, moderate effect size: r = 0.33). PHAD yielded significantly higher fluorescence than xylene deparaffinization in tissues (p<0.0001, large effect size: r = 0.85). Conclusions AuO can be applied for nucleic-acid-staining of mycobacteria in tissues producing typical beaded patterns. AFS depends crucially on the integrity of the mycobacterial cell-wall, which seems to be damaged by xylene. A solvent-free tissue deparaffinization method has the potential to increase mycobacterial detection significantly. Key Points Acid-fast-stains for mycobacterial detection have a notoriously poor yield, due to misconceptions of the underlying staining principle, but mainly because of the possible detrimental effect of xylene. The yield of acid-fast-stains for mycobacterial detection in histology, including fluorescent stains, can be significantly increased by eliminating solvents (xylene) in the deparaffinization step. Acid-fast-stains depend on bacterial integrity because they target nucleic acids. A simple hot air deparaffinization is significantly superior to xylene, avoiding damage to the lipid-rich cell wall.