Standard Operating Procedure (SOP) for Methenamine Silver-Periodic Acid Schiff Stain of Tissue Sections
1.0 Purpose:
To establish a standardized procedure for the silver methenamine stain of tissue sections.
2.0 Scope:
This procedure is applicable to all tissue sections received in the surgical pathology section intended for silver methenamine staining.
3.0 Introduction:
The purpose is to visualize fungus within the tissue and basement membranes in kidney samples.
4.0 Principle:
Carbohydrate moieties within the periodic acid oxidize basement membrane/fungal components to aldehydes. Aldehyde groups then reduce the methenamine silver solution to elemental silver within the basement membrane/fungus. Excess silver is eliminated by sodium thiosulphate.
5.0 Analytical Measuring Range and Limit of Detection:
Not applicable.
6.0 Responsibility:
Technical staff and pathologists are responsible for executing this procedure.
7.0 Abbreviations:
PAS - Periodic Acid Schiff.
8.0 Requirements:
Kit Reagents:
a) 0.5% Periodic acid
b) 0.25% Gold Chloride
c) 0.5% Borax
d) 0.3% Hexamine
e) 0.5% Silver Nitrate
f) 0.1% Light green or 1% eosin
g) 3% Sodium Thiosulphate
Instrumentation and Software:
None required.
Disposables:
None required.
9.0 Precautions:
All specimens should be handled with gloves and masks.
Care should be taken with instruments to prevent injury.
10.0 Limitations & Interferences:
Not applicable.
11.0 Documentation:
All documents are maintained in:
a) Daily routine register
b) Special stains log/register.
12.0 Instructions:
12.1 Pre-Analytical Steps:
Includes sample preparation, reagent preparation, etc.
12.1.1 Making Working Solution:
Combine 97 ml of 0.3% Hexamine with 3 ml of 0.5% Silver Nitrate. Take 20 ml of the resulting solution, add 3 ml of 0.5% Borax, and dilute with distilled water to 60 ml.
12.2 Analytical Steps:
12.2.1
Sections are deparaffinized by dipping in two changes of
xylene and two changes of alcohol.
12.2.2 Pour 0.5% periodic acid solution for 15 minutes.
12.2.3 Preheat the working solution in a brown bottle in a
water bath set at 60°C.
12.2.4 Wash the slides in running water, then immerse them
in the preheated working solution and check under a microscope after 10
minutes.
12.2.5 Wash the slides in running water.
12.2.6 Treat with 0.25% gold chloride for 1 minute or as
required (verify under microscope).
12.2.7 Wash the slides in running water.
12.2.8 Immerse the slides in 3% sodium thiosulphate for 5
minutes.
12.2.9 Counterstain with 0.1% eosin for fungus or light
green for kidney biopsies.
12.2.10 Dehydrate, clear, and mount in DPX.
12.3 Quality Control and Assurance:
Includes the use of positive and negative controls.
12.3.1 Known positive controls are run with each batch.
12.4 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:
Basement membranes/Fungi: black
Nuclei: blue
Background: green
16.0 Contingency Plan:
In case of reagent shortages, manpower issues, or instrument breakdowns, 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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