Buffered Romanowsky-Giemsa method for formalin fixed, paraffin embedded sections: taming a traditional stain

Area of Science:

• Histopathology
• Staining Techniques
• Cytology

Background:

• Romanowsky-Giemsa (RG) stains are historically significant for parasite identification and have become standard in hematology and cytology.
• Previous attempts to adapt RG staining for formalin-fixed paraffin-embedded tissues yielded inconsistent results due to overstaining and acid differentiation.
• Optimizing staining conditions eliminates the need for differentiation, improving reproducibility.

Purpose of the Study:

• To develop a simplified and effective Romanowsky-Giemsa staining protocol for formalin-fixed paraffin-embedded tissue sections.
• To achieve staining results comparable to routine Hematoxylin and Eosin (H&E) staining.
• To enhance the visualization of specific cellular components and microorganisms often missed by H&E.

Main Methods:

• Utilized RG stain with a 0.002 M buffer at pH 42, all at room temperature.
• Sections were stained, air-dried, washed in ethanol (96-100%) to remove excess stain, followed by xylene treatment.
• Mounted with DPX for microscopic examination.

Main Results:

• Achieved staining results comparable to H&E, with nuclei appearing blue and RNA-rich sites purple.
• Successfully visualized collagen fibers, keratin, muscle cells, erythrocytes, and CNS white matter in pinkish to reddish hues.
• Demonstrated distinct staining of cartilage matrix, mast cell granules, myxomatous degeneration (purple), sulfate-rich mucins (pale blue), hemosiderin, lipofuscin, melanin (greenish), calcium deposits (blue), and Helicobacter pylori (violet to purple).

Conclusions:

• The optimized RG staining method is technically simple and produces results similar to H&E staining.
• This method enhances the visualization of structures like hemosiderin, H. pylori, mast cell granules, and melanin, which are poorly seen with H&E.
• Advantages include shorter staining times and the elimination of acetone, making it a practical alternative for tissue staining.