2. • The term amyloid, meaning starch-like, is a
misnomer coined by Virchow when he observed
that amyloid deposits would stain blue with the
iodine reaction, suggesting the presence of
starch or cellulose.
3. • Amyloid – is a protein that has an alternation in
its secondary structure which imparts a
particular insoluble form, called the beta-pleated
sheet conformation.
• By definition, any protein deposits staining with
Congo red and exhibiting green birefringence
when viewed with polarized light are amyloid
4. Components of amyloid:
A. Fibril protein:
• proteins make up 85-90% of the amyloid
weight
• responsible for tissue dysfunction due to
pressure effects
5. B. P Component:
• makes up about 10% of the amyloid’s weight
• found on all amyloid fibrils, except those associated
with Alzheimer’s senile plaques.
• stabilizes fibril amyloid protein and decreases their
clearance
• plays a role in some of the other special stains that can
demonstrate amyloid (TFT)
6. C. Non-amyloid components:
• Collagen, fibrin and reticulin
• Part of the amyloid OR are trapped by the amyloid
OR are part of the body’s repair process from the
cellular destruction caused by the amyloid.
• This is one of the reasons why connective tissue
stains (e.g. trichromes, PAS) stain amyloid the same
color as collagen
7. Classification of Amyloidosis
Process Type Designation
Acquired systemic Primary AL
Secondary AA
Familial AF
Organ limited Senile AS
Localized Endocrine AE
8. Important Systemic amyloidosis
Amyloid protein Precursor Syndrome or
involved tissue
AL Immunoglobulin light Primary, Myeloma
chain associated
AA Serum AA (SAA) Reactive, chronic
inflammatory condition
Aß2m ß2 microglobulin Hemodialysis
AA SAA Familial mediterranean
fever
ATTR Transthyretin Familial amyloidotic
polyneuropathies
ATTR Transthyretin Systemic senile
Amyloidosis
9. Important localized amyloidosis
Amyloid protein Precursor Syndrome or
involved tisssue
Aß Aß protein precursor Alzheimer’s disease
(AßPP)
APrP Prior protein Spongiform
encephalopathy
Acal Calcitonin Medullary carcinoma of
thyroid
AIAPP Islet amyloid Type II diabetes,
polypeptide insulinomas
AANF Atrial natriuretic factor Isolated atrial amyloid
10. • The progressive accumulation of extracellular
protein fibrils can lead to cellular atrophy,
ischemia, necrosis,and, ultimately, organ failure
due to the effects of these accumulated fibrils on
blood supply and normal cellular function
11. Demonstration of Amyloid
• H & E – homogenous pale pink color
• Van Gieson – yellow to yellow/brown
• Iodine (Gram’s or Lugol’s) – mahogany brown
turning into blue on treating with 10% sulphuric
acid
12. • Light microscopy – postive staining with congo
red together with a resultant green polorization
color
• Electron microscopy – typical fibrillar ultra
structure of amyloid
• X-ray diffraction – cross beta pleat structure
13. I. Metachromatic methods
• Staining with 1% aqueous methyl violet for 5
minutes, followed by differentiation with 1 %
acetic acid
• Sections should be mounted in aqueous media of
high sugar or salt content to prevent diffusion of
the stain
14. Lendrum’s technique
1. Bring sections to water
2. Stain with 1% aqueous methyl violet for 3 minutes
3. Differentiate in 70% formalin (controlling
microscopically)
4. Wash in running water for 1 minute
5. Flood with saturated aqueous sodium chloride for 5
minutes
6. Rinse in water and mount in corn syrup
Results:
Amyloid- Pink to red
Other elements- Violet
15.
16. II. Congo red methods
• Specificity of methods of this type are dependent
on
▫ Beta pleated sheet configuration of amyloid
▫ Linearity of the dye molecule
• Congo red is a linear dye molecule. It binds to
amyloid by hydrogen bonds
17. • The characteristic green polarization color
wasn’t present in very thin or thick section –
thickness of 5-10 micron was optimal
• Sirius red – similar properties to congo red
• Tissue sections containing amyloid AA protein,
affinity for Congo red is lost after pretreatment
with potassium permanganate
18. Bennhold’s Technique
1. Bring sections to water
2. Stain with Ehrlich’s haematoxylin for 20 mins
3. Differentiate with 1% acid alcohol
4. Wash in running water for 1 min to remove acid
5. Stain with 1% aqueous Congo red for 20-30 mins
6. Pour off stain and flood slide with a saturated aqueous solution of
lithium carbonate; leave for 15 secs
7. Differentiate in 80% alcohol until excess Congo red is removed
8. Wash in running water for 10 mins
9. Dehydrate, clear and mount in Canada balsam or synthetic resin
Results
Amyloid- Pink to red
Nuclei- Blue
19. Alkaline Congo red technique
(Puchtler, Sweat and Levine)
• Advantage of not requiring differentiation
( Congo red is in alkaline solution)
• The use of alcoholic solutions, high salt content,
and high pH, as in the Puchtler Congo red
method, greatly increase staining specificity for
amyloid
20. • A saturated salt solution in alcohol at alkaline
pH is used in both the dye solution and as a
pretreatment of the tissue sections just before
staining.
• High salt content and alkaline pH are believed to
depress dye ionization and electrostatic binding
to nonamyloid structures. Saturation of the salt
and dye solutions is very important
21. • Fixation:
▫ Best results are obtained after alcohol or Carnoy
fixed tissues
▫ Formalin or Zenker –fixed tissues were found to
stain better than with other techniques
22. • Reagents
▫ Alkaline salt solution. To 50 ml of 80% alcohol
saturated with sodium chloride add 0.5 ml of 1%
aqueous sodium hydroxide. Filter and use within
15 min.
▫ Stock stain solution. Use 80% alcohol saturated
with Congo red and sodium chloride
▫ Staining solution. Add 0.5 ml of 1% aqueous
sodium hydroxide to 50 ml of stock stain, filter
and use within 15 mins
23. Method :
1. Bring sections to water.
2. Stain in haematoxylin for 5 minutes.
3. Rinse well in distilled water.
4. Pretreat in alkaline alcohol-salt solution for 20
min.
5. Stain in alkaline Congo red solution for 20 min.
6. Dehydrate rapidly in three changes of absolute
alcohol.
7. Clear and mount in synthetic resin.
Results:
Amyloid- deep pink to red
Nuclei -blue
Elastic -pale pink
24. This bone marrow section stained with the Puchtler Congo red
procedure reveals intense amyloid deposits in the bone marrow
sample
25. This figure viewed with polarizing microscopy;
amyloid deposits exhibit characteristic apple-
green birefringence.
27. a) PROBLEM: Weakly Stained Tissues
• APPEARANCE: Faint uptake of Congo red dye
throughout the section
• CAUSES:
▫ Tissue was fixed for a prolonged period in a
formaldehyde-containing fixative.
▫ Cut sections were stored for a prolonged period.
▫ Solutions of Congo red are not stable in the presence
of salt and alkali.
28. • SOLUTIONS:
• Avoid storing tissues in formaldehyde-based
fixatives for prolonged periods of time.
• Cut the sections just before staining, or cut
only as many control sections as can be used in a
short period of time.
• Seal the cut paraffin blocks to help preserve
control tissue reactivity.
• Prepare fresh working solutions just before use
29. Congo red staining in this section appears
indistinct, suggesting that the stain was
excessively differentiated
30. Examination of the section with polarized
light reveals numerous amyloid deposits
31. b) PROBLEM: Nonspecific Staining
• APPEARANCE: Structures other than amyloid
bind the Congo red; high background staining may
make it difficult to distinguish true amyloid deposits
if present in the tissue
• CAUSES:
Collagen, elastic fibers, and keratin may stain
nonspecificallywith aqueous Congo red solution.
The pH is not sufficiently alkaline.
32. SOLUTIONS:
• Use polarizing microscopy to help distinguish connective
tissue components (grey or silver )from amyloid deposits
(green).
• Rotating the slide on the stage during polarizing microscopy
can help distinguish true amyloid deposits from connective
tissue. When the slide is rotated, connective tissue fibers will
lose the dichroism,while amyloid will not
• Use the Puchtler Congo red method, because the high salt
content of the prestain rinse and staining solutions tends to
diminish nonspecific staining.
• Avoid the use of Canada balsam mounting medium because it
will fluoresce.
33. Tissue components in this Congo red stained
kidney section appear to be nonspecifically
stained
34. Polarization of the Congo red-stained
section reveals that most of the red
staining is not amyloid. Some apple-green
birefringence is noted in the glomerulus at
the bottom of the image
35. C. PROBLEM: Incorrect Color of Birefringence
APPEARANCE: Structures may exhibit yellow, red, or white
dichroism
CAUSE:
• Section thickness may be incorrect; this artifact is especially
likely in thin sections
SOLUTION:
• Ensure that sections are cut at 5 to 10 μm.
COMMENT:
Old (large) amyloid deposits will often display diminished
birefringence.
Smaller deposits in blood vessel walls, for example, may be more
likely to demonstrate the characteristic apple green color.
36. Congo red staining in this section of kidney appears
specific and well differentiated
37. When viewed with polarized light, amyloid
deposits appear red to yellow, not the
expected apple-green color; incorrect
section thickness is the most likely cause
38. Collagen, appearing white to silver in this
polarized, Congo red stained section,
surrounds a characteristic apple-green
amyloid deposit.
39. D. PROBLEM: Precipitate on Tissue
APPEARANCE: A red precipitate is randomly
present throughout the tissue
CAUSE:
Salt solutions were prepared with isopropyl alcohol.
SOLUTION:
Ensure that the salt solutions are prepared with ethyl
alcohol because the salt does not dissolve well in
isopropyl alcohol and will deposit crystals on the
section.
40. Although the amyloid is well demonstrated in
this section, close inspection reveals a red
granular precipitate caused by using isopropyl
alcohol instead of ethyl alcohol as the solvent for
the salt solutions
41. III. Toluidine blue method
Standard toluidine blue
1. Bring sections to water.
2. Stain in 1% toluidine blue in 50% isopropanol for
30 min at 37o C.
3. Blot and place in absolute isopropanol for 1 min.
4. Clear in xylene and mount.
Results:
Amyloid is distinguished by its dark polarization color
42. IV. Fluorescence techniques
• Thioflavine T technique
• Extremely sensitive technique although not
specific because
▫ stained sections are not permanent and
▫ tissue components other than amyloid, including
fibrinoid, keratin, intestinal muciphages, Paneth
cells, zymogen granules, and juxtaglomerular
apparatus, all stain with thioflavine T
43. • Attempts made to increase specificity
▫ Using acid solutions
▫ Including Magnesium chloride
• Thioflavine S may be substituted for Thioflavine T
44. Method:
1. Bring sections to water.
2. Stain in alum-haematoxylin for 2 min to quench nuclear
fluorescence. The haematoxylin does not need to be
differentiated, or blued.
3. Wash in water for a few minutes.
4. Stain in 1% aqueous thioflavine T for 3 min.
5. Rinse in water.
6. Differentiate in 1% acetic acid for 20 min.
7. Wash in water.
8. Mount in Apathy’s medium.
Results:
Amyloid and mast cells fluoresce bright yellow when examined
using a BG12 exciter filter and an OG4 or OG5 barrier filter.
The finest deposits can be seen using a UG1 or UG2 exciter filter
with a colourless ultraviolet barrier filter
45. Amyloid stained with TFT in Kidney.
Hit with 490 (blue) excitor wavelength. Amyloid
fluorescing yellow
46. • Congo Red Fluorescence
Congo red stained amyloid, even if it does not birefringe
with polarization, will often fluoresce an orange color
when viewed with the auramine-rhodamine
fluorescence microscope wavelengths (540 nm excitor
wavelength) and lenses.
At the same time, connective tissue and/or other tissue
components that like to autofluoresce with green light
will usually exhibit a white to yellow color
47. Congo Red stained amyloid in kidney, .
Hit with 490 (blue) excitor wavelength.
Amyloid fluorescing orange
48. V. Alcian blue method
• Introduced by Lendrum, Slidders and Fraser
• Fixation in formal-saline is usually adequate
49. • The sodium sulphate-Alcian blue (SAB) method
• Reagents
1. Acetic alcohol
a) 95% ethanol – 45ml
b) Distilled water – 45 ml
c) Glacial acetic acid - 10 ml
Prepare fresh for use
2. SAB solution
a) 1% alcian blue in 95 % ethanol – 45ml
b) 1% aqueous sodium sulphate decahydrate – 45ml
c) Glacial acetic acid – 10ml
Prepare from stock solutions and stand for 30 mins before
use
50. Method
1. Bring sections to water.
2. Immerse in acetic alcohol for 1-2 min.
3. Stain in SAB working solution for 2 hours.
4. Transfer to acetic alcohol for 1-2 min.
5. Wash in water.
6. Alkalinize in 80% ethanol saturated with borax for 30
min.
7. Wash in water
8. Stain nuclei with Celestine blue-haemalum sequence
and counterstain with Van Gieson.
9. Dehydrate, clear and mount.
Results:
Amyloid, mast cells and some colloids- Green
51. VI. IMMUNOHISTOCHEMISTRY
• There are limitations as to the use of
immunohistochemistry for the demonstration of amyloid
• Each has its own sequence of amino acids (proteins).
Also, there are variations of amino acid sequences from
person to person, even if they have the same type of
amyloid.
• It is usually necessary to use more than one IHC for
amyloid concurrent with the Congo red stain, as well as
other amyloid histology stains, if needed.
