Amyloid - RCPAQAP

Aim of special stain:: Demonstrate the presence of amyloid deposits
Diagnostic applications:: Amyloidosis in different organs
Material supplied for surveys:: Colonic tissue cut at 6-8µm
Recommended control:: Known case of amyloidosis – veterinary samples can also be used

Congo Red Stain for Amyloid (Highman’s Technique)

Reagents – commercial source unknown, unless specified:

1. Congo Red – 0.5% Congo red in 50% ethanol
a) Congo red	0.5 g
b) 50% alcohol	100 mL

2. Differentiator – 0.01% potassium hydroxide in 80% ethanol
a) Potassium hydroxide	0.01 g
b) 80% ethanol	100 mL

Method:

Take sections to distilled water.
Stain with Congo red solution for 40 minutes.
Drain slide and then differentiate with the potassium hydroxide solution for 4-5 seconds until the background is clear.
Wash well in water.
Take sections through hematoxylin 10 seconds, wash in water, Scott’s blue 10 seconds, wash well in water, dehydrate, clear and mount.

Results:

Light Microscope:
Amyloid, elastin and eosinophils – pink to orange / red
Nuclei – blue
Background – clear

Polarizing microscopy:
Amyloid – apple green birefringence
*Tissue elements such as collagen, elastin (and others) may display birefringence of varying colours that may be misinterpreted as amyloid*

Disclaimer:

These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.

References

2022 RCPAQAP Technical General Survey 2.
Geoffrey H Vowles. Theory and practice of Histological Techniques. John D Bancroft; Marilyn Gamble. 6^th edition. New York, NY: Churchill Livingstone; 2008:270-273.
Bancroft, JD and Cook HC. Manual of Histological Techniques. Published by Churchill Livingstone. 1984:91.
Bancroft. JD and Stevens A, The Theory and Practice of Histological Techniques, 6^th edition. New York, NY: Churchill Livingstone; 2008:164.
Clement CG, Truong LD. An evaluation of Congo red fluorescence for the diagnosis of amyloidosis. Hum Pathol. 2014 Aug;45(8):1766-72. doi: 10.1016/j.humpath.2014.04.016. Epub 2014 May 8. PMID: 24935061.