1.0 Purpose
To establish a standardized procedure for the reticulin staining of tissue sections.
2.0 Scope
This procedure applies to all tissue sections received in the Surgical Pathology section for reticulin staining.
3.0 Introduction
The procedure is designed to demonstrate reticulin fibers in tissue.
4.0 Principle
This technique is categorized under the oxidation-reduction silver techniques. Potassium permanganate (KMNO4) oxidizes reticulin fibers, which contain a carbohydrate component, to aldehyde groups. This oxidation step also controls the extent of oxidation. Excess permanganate is removed using oxalic acid or potassium metabisulphate. Ferric ammonium sulphate (mordant) is then used to sensitize the tissue for reaction with the ammoniacal silver solution. The silver solution, at a basic pH, is reduced to elemental silver by interacting with aldehyde groups. Elemental gold replaces this elemental silver, and both silver and gold are removed with sodium thiosulphate.
5.0 Analytical Measuring Range and Limit of Detection
Not Applicable
6.0 Responsibility
Technical staff and pathologists are responsible for following this SOP.
7.0 Abbreviations
KMNO4: Potassium permanganate
8.0 Requirements
Kit Reagents:
0.2% Potassium permanganate
0.2% Oxalic acid
0.2% Ferric alum
Silver nitrate solution
10% Formalin
5% Sodium thiosulphate
9.0 Precautions
All specimens should be handled with gloves and masks.
All instruments should be handled carefully to prevent
injury.
10.0 Limitations & Interferences
Not Applicable
11.0 Documentation
All documents should be maintained in:
Dolly routine register
Special stains log/register
12.0 Instructions
12.1 Pre-Analytical Steps:
Sample preparation
Reagent preparation
12.2 Analytical Steps:
Bring section to water, washing with distilled water at each
step.
Treat with 0.2% potassium permanganate for 2 minutes.
Wash with running tap water for 2 minutes.
Treat with 0.2% oxalic acid for 2 minutes, wash in water for
2 minutes, and rinse with distilled water.
Treat with 0.2% ferric alum for 2 minutes, wash in water for
2 minutes, and rinse with distilled water.
Treat with silver nitrate staining reagent for 1 minute.
Reduce with 10% formalin for 1 minute, wash thoroughly with
water, and rinse with distilled water.
Tone with gold chloride for 10 minutes.
Treat with oxalic acid for 2 minutes.
Fix with 5% sodium thiosulphate for 2 minutes.
Wash, dehydrate, clear, and mount.
12.3 Quality Control and Assurance:
Positive and negative controls should be used for
validation.
Known positive controls are run with each batch.
12.4 Calibration and Calibration Verification
Not Applicable
12.5 Storage of Samples
Slides are stored for 10 years.
13.0 Reference Range
Not Applicable
14.0 Critical Value
Not Applicable
15.0 Reporting of Results and Interpretation
Reticulin fibers stain black.
16.0 Contingency Plan
In case of reagent shortage, manpower issues, or instrument breakdown, suitably trained backup staff are available.
17.0 References
Bancroft Theory and Practice of Histological Techniques by John D. Bancroft and Marilyn Gamble, 2007 (6th ed.)
END OF THE DOCUMENT
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