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

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

Abstract Objectives 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 pa...

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Published in:American journal of clinical pathology Vol. 160; no. 1; pp. 81 - 88
Main Authors: Marinho, Pedro F, Vieira, Soraia L, Carvalho, Tânia G, Peleteiro, Maria C, Hanscheid, Thomas
Format: Journal Article
Language:English
Published: US Oxford University Press 05-07-2023
Subjects:
Acids
BCG
BCG vaccines
Benzophenoneidum
Cell walls
Coloring Agents
Diagnosis
Fluorescence
Histochemistry
Hot Temperature
Humans
Localization
Methods
Methylene blue
Mycobacterium
RNA
Solvents
Staining and Labeling
Stains & staining
Tuberculosis
Xylene
Xylenes
Portugal
Acid-fast stain
Histology
Novel method
Xylene deparaffinization
Mycobacteria
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Summary:Abstract Objectives 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. 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 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). Results 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). Conclusions 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.
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ISSN:0002-9173
1943-7722
DOI:10.1093/ajcp/aqad016