The document discusses trichrome staining, which is used in histopathology laboratories to differentiate collagen, muscle, and other tissues. It describes the most common uses in liver, renal, dermatology, cardiac, and muscle/nerve biopsies. The Masson's, Gomori One Step, Martius Scarlet Blue, and Mallory techniques are widely used. Collagen stains blue, muscle stains red, and elastin stains pink or yellow. Trichrome staining is useful for identifying fibrosis or changes in tissues.

2. TRICHROME STAIN
• Utilized special stains in the Histo-pathology
Laboratory
• Most common uses for requesting a trichrome stain are
–
 liver biopsies
 Renal biopsies
 Dermatopathology
 Cardiac biopsies and
 Muscle and Nerve Biopsies
• Widely utilized techniques are the Massons, Gomori
One Step, Martius Scarlet Blue and Mallory.

3. TRICHROME STAIN cont’
 First account of a triple stain was by H. Gibbs in 1880 followed by B.W. Richardson in
1881.
 The triple stain became known as the Trichrome stain: tri – 3 & Chrome – Colour.
 Frank Burr Mallory developed a method suitable for studying connective tissue.
 Then tissue sections were stained in acid fuchsuine, aniline blue orange and fibrils of
collagen stained BLUE.
 Neuroglia and muscle fibers stained RED.
 Fibrils of Elastin stained PINK or YELLOW.

4. TRICHROME STAIN
This is a stained
liver biopsie
picture
Massons
Trichrome
Stained Tissue

5. TRICHROME STAINS PURPOSE
Is to demonstrate primarily between collagen and muscle in normal and
abnormal tissue.
Or to differentiate collagen and muscle tissues in tumours.
Used to identify an increase in collagenous tissue or indicate fibrotic change in
cirrhosis of the liver or in renal disease.
It is also used to distinguish tumours that have arisen from muscle cells and
fibroblasts.
Gomori’s trichrome is the trichrome stain of choice for distinguishing
changes that occur in neuromuscular diseases.
Whereas Masson’s trichrome is responsible for detection of collagen fibers in
tissues such as heart, skin etc.

6. NORMAL LIVER
CIRRHOSIS
LIVER

7. • Nuclei – nuclear stain is an iron haematoxylin (Welgert’s or
Heidenhain’s hematoxylin) It is used due to the acidity of the stain
solutions following the staining of the nuclei then the standard
aluminium-based hematoxylins are decolorized.Iron haematoxylin are
more resistant to these acid solutions.
• Collagen – is a common protein found in the body that forms Fibres.
White colour when fresh.Do not branch, wavy and present in
bundle.Fibres composed of fibril made of microfibrils. The more the
collagen in the extracellular matrix, the stronger the tissue.
TRICHROME STAIN ENTITIES

8. TRICHROME STAIN ENTITIES cont
• There are at least 27 types of collagen
• Type 1,2,3,4, and 11 are the fibrillar collagens and most abundant
• Muscle – one of the four primary types of tissue (3 types)
1. Smooth Muscle – non-striated, involuntary and found primarily in tubular
organs. Lacks striations and are elongated
2. Skeletal Muscle – striated and voluntary and have distinctive striations and
its generally attached to bones
3. Cardiac Muscle – involuntary and striated with a centrally located nucleus
composed of branching fibers connected by junctions known as “intercalated
discs”

9. Factors that affect Trichrome Staining
• Fixation – 10% neutral buffered formalin will not yield optimal trichrome staining
results. The longer those tissues remain in formalin fixatives, the less optimal the
staining results. The recommended fixatives are Bouins Zenkers, Formal-mercury
and Zinc formalin
• pH –the dyes utilized in the trichrome are prepared as low pH solutions, usually in
the range of 1.5 – 3.0
• Tissue permeability and dye molecule size – interaction between the protein chains
and the fixative usually formed a 3-D insoluble protein network.

10. GENERALLY SPEAKING
• The most common technique for trichrome staining are the Masson (multi-
step) and Gomori One Step. The more traditional Masson technique
incorporates what often is referred to as polyacids, (PTA- Phosphotungstic
or PMA-Phosmolybdic Acid)
• The Masson utilizes all of the mordanting and staining steps individually
whereas the one-step incorporates all of the staining steps in one
staining solution except the mordant (Bouin’s) and the nuclear stain.

11. • RDBs - protein will produce a dense network with small pores between
protein elements.
• Muscle cells - will form a more open structure with larger pores.
• Collagen - will demonstrate the least dense network with more pores.
• The smaller molecule dyes will penetrate muscle and collagen but will not
react with the RBCs.
• With trichrome staining usually the smaller dye molecule will penetrate and
stain a tissue element. When a larger dye molecule can penetrate the same
element then the smaller molecule will be replaced by the larger molecule.
FACTORS AFFECTING TRICHROME
STAIN cont

12. LIVER
BIOPSIE:

13. •RENAL
BIOPSIE:
DERMATO-PATHOLOGY

14. MULTI-STEP TECHNIQUE
The Masson
- differentiate between collagen and muscle
- demonstrate a change in the amount of collagen
present.
- For aniline blue staining the polyacid mixture of
PTA/PMA must be utilized which will demonstrate
small amounts ofcollagen. For light green the polyacid
PTA must be utilized.
The final staining results are:
1. Erthrocytes – yellow or red
2. Cytoplasm, fibrin and muscle – red
3. Collagen, bone – blue or green, depending upon the
collagen stain

15. ONE-STEP TECHNIQUE
• The one-step techniques such as the Gomoris One-Step and Van Giesons method
combine all of the dyes and reagents into one single solution that is applied for a
specific amount of time.
• The various tissue entities are stained differentially.
• The one step techniques are protocol dependant and will perform well if everything is
standardized.
• The final staining results are:
1. Erythrocytes – yellow or red
2. Cytoplasm, fibrin, muscle – red
3. Collagen – light green

16. CONCLUSION
• The trichrome stain is utilized as the stain of choice of distinguishing histologic
changes in tumors, connective tissue diseases, muscle and fibroblast tumors, renal
diseases and dermatology cases.
• Even the disciplines of forensics, archaeology and hematopathology incorporate
the trichrome stain for specific tissue entities and structures.
• With the utilization of immunohistochemistry expressions, the trichrome
techniques still offer a great deal of diagnostic results

17. REFERENCING
• 1. Bancroft, J; Gamble, M, Theory and Practice of Histological Techniques, 6th
edition Churchill-Livingstone, London, England, 2008.
• 2. Bricegirdle, B, A History of Microtechnique, 2nd edition, Science Heritage
Ltd, Chicago, 1986.
• 3. Carson, FL, Histotechnology A Self Instructional Text, 2nd edition, ASCP
Press, Chicago, 1997.
• 4. Sheehan, D, Hrapchak, BB, Theory and Practice of Histotechnology, 2nd
edition, Mosby, St. Louis, 1980.

18. THE END !

19. THANK
YOU !

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