SPECIAL STAINS USED IN DIAGNOSING LIVER PATHOLOGY

1. Special stains in
diagnostic liver
pathology
PRESENTER:DR.ARGHA BARUAH
MODERATOR:DR.SWAROOP RAJ

2. Brief recap:
Indications of liver biopsy
Adequacy of liver biopsy
Procedure of liver biopsy

4. Why special stain???
Staging of NASH,
Establishing a diagnosis of cirrhosis and check for regression
Assessment of fatty liver disease and its complications,
Iron and copper overload,
Identifying cholestatic disease

5. Ready for few questions?
To check normal liver architecture?
To check for fibrosis?
To check for regenerative hyperplasia?
To check for iron overload?
To check for recent phagocytosis?
To check α1 antitrypsin deficiency?
To check for copper overload?

6. Normal liver :
Shows a preponderance of type I collagen within the capsule and portal tracts (readily
demonstrated by trichrome stain)
Type III collagen within the space of Disse, serving as the structural backbone surrounding
liver-cell plates (demonstrable on reticulin stain)

7. Masson trichrome Stain
Masson's trichrome stain is among the most common special stains applied to liver specimens.
The stain imparts a blue color to collagen against a red background of hepatocytes and other
structures.
It stains type 1 collagen that is normally present in the portal tracts and vessel walls, but also
highlights the presence and distribution of reactive fibrosis as a result of liver injury.

8. Uses:
Standard method for demonstrating bridging fibrosis and cirrhosis
Helps to delineate patterns of injury
Perisinusoidal fibrosis associated with steatohepatitis
Periductal fibrosis in primary sclerosing cholangitis
Portal tracts (eg, chronic hepatitis, chronic biliary tract disease,genetic hemochromatosis)
More recently generated areas of scar tissue can be differentiated from native or old fibrous
tissue
Staging: steatohepatitis
Fibrosis vs. necrosis(pale)

9. Used in diagnosis of
steatohepatitis(along with Inflammation )

10. Thin fibrous septa with perforations • Prominent vessels
• Nodularity may persist

12. Stage 2 periportal fibrosis in chronic hepatitis C.
(B) Perisinusoidal “chicken-wire” fibrosis in a case of
steatohepatitis is seen here near a central vein (CV).
Mallory bodies in swollen hepatocytes appear dark red

13. Bright blue staining of portal tract native type I collagen fibers
from the grayer, more recently deposited periportal scar tissue
(containing relatively more ground substance and type III collagen) in
chronic hepatitis.
A ground-glass hepatocellular inclusion of hepatitis B surface
antigen appears pale pink and homogeneous in contrast to the
more darkly stained,granular and vacuolated adjacent
hepatocytes.

14. Reticulin stain:
Reticulin stain uses silver impregnation to detect reticulin fibers, which are made of type 3
collagen.
The fibers appear black against a gray to light pink background. In the liver
Aldehyde groups of reticulin reduces colourless silver complex to a dark brown oxide of silver

15. Uses:
It helps in the assessment of the architecture of the hepatic plates, such as expansion in
regenerative and neoplastic conditions, compression of plates in nodular regenerative
hyperplasia, and collapse of the reticulin framework in necrosis
The stain is also used to evaluate such fibers in other tissues, such as bone marrow and kidney.

16. Case:
• 60/F with long history of rheumatoid arthritis • Portal hypertension • Ultrasound: cirrhosis

17. Nodular regenerative hyperplasia
• Hepatocellular nodules, often <0.3cm
• Diffuse involvement
• Fibrosis absent or minimal

18. Hepatic involvement by sarcoidosis shows a cauliflower-like
cluster of portal and periportal granulomas surrounded by
fibrosis.
Hepatocellular carcinoma with a trabecular pattern shows the
diagnostic “paucireticulin” pattern, with only a few twigs of
collagen in the tumor

19. (C) Proliferating bile ductular structures (ductular reaction) is prominent within and at the edge of this portal tract in a case of
extrahepatic biliary atresia.
E) Nodular regenerative hyperplasia (NRH) shows a nodular mass of parenchyma (between long arrows) composed of thickened
plates. Resumption of single-cell thick plate architecture away from the mass, accompanied by sinusoidal dilation (short arrow) is
highlighted in the Inset.

20. Iron Stain:
The Perl’s iron stain (Prussian blue reaction) is a common and reliable stain for detecting iron.
Iron is stored in the hepatocytes as a soluble form (ferritin) and an insoluble form (hemosiderin). With
the H&E stain, the hemosiderin is seen as coarsely granular brown refractile granules in the
cytoplasm, whereas ferritin is not seen.
Perl’s stain highlights hemosiderin as coarse blue granules, while ferritin is seen as a faint blue
cytoplasmic blush .
K ferrocyanide + HCl
Hydrochloric acid releases the protein bound to ferric iron, then potassium ferrocyanide binds with
ferric iron to form ferric ferrocyanide, an insoluble blue compound

21. Uses:
In hemochromatosis, iron accumulates primarily in the cytoplasm and initially in the periportal
hepatocytes.
In secondary iron overload, the accumulation is mainly in the Kupffer cells.
When a large quantity of iron is present or if there is concurrent active hepatocellular injury,
the distribution of iron may become mixed, both in hepatocytes and Kupffer cells.
Iron stain may be supplemented with tissue iron quantitation in the appropriate
clinicopathologic setting.

23. (A) Iron stain in classical hemochromatosis shows marked
parenchymal hemosiderin deposits and portal-to-portal
bridging fibrosis of portal tracts (P) with the characteristic
“holly leaf” stellate pattern of fibrosis.
(B) Hepatocellular and bile canalicular cholestasis due to sepsis
(long arrows)
Minimal hepatocellular hemosiderosis is also present (short
arrows).

24. Iron stain showing marked iron deposition with a mixed distribution in hepatocytes and Kupffer cells
(magnification ×100).

25. Periodic Acid-Schiff Stain
The periodic acid-Schiff (PAS) stain is useful for identifying glycogen, but removing glycogen with
diastase digestion DPAS (diastase pretreated PAS) stain finds its major uses in liver
histopathology in identifying phagocytic material in activated macrophages, hepatocellular
globules of alpha-1-antitrypsin (AAT) in the deficiency state, and basement membrane
surrounding bile ducts and ductules.

26. USES:
1. Bile duct damage seen in primary biliary cirrhosis where rupture or interruption of the
basement membrane surrounding the epithelium by infiltrating immune cells is often
present
2. Active necroinflammation within the lobules is often followed by uptake of phagocytic debris
by Kupffer cells which stain tan-to-green on H&E (ie,ceroid-laden Kupffer cells) and positively
with DPAS

27. A) Ischemia/reperfusion injury of a transplanted cadaveric liver allograft has resulted in glycogen depletion and pale staining of centrilobular
hepatocytes with PAS. Compare with the glycogen-replete hepatocyte staining near the portal tract (PT).
(B) DPAS-positive globules of varying sizes are present in periportal hepatocytes in this case of alpha-1-antitrypsin deficiency.
(C) DPAS stain of a florid bile duct lesion in primary biliary cirrhosis shows a bile duct with disordered epithelium partly surrounded at right by its
basement membrane (arrows) and basement membrane interruption by inflammatory cells to the left.
(D) Chronic hepatitis with portal lymphoplasmacytic infiltrate and DPAS-positive macrophages containing phagocytic debris.

28. Rhodanine stain
It is used to detect copper-binding protein and is thus used to evaluate for Wilson disease.
Copper is excreted in bile and accumulates in the liver in chronic biliary diseases..

30. Victoria blue:
Identification of copper-associated protein with this stain is helpful supporting evidence of a
chronic cholestatic liver disease such as primary biliary cirrhosis and primary sclerosing
cholangitis

31. Victoria blue:
(C) A postmortem liver section shows numerous cytoplasmic
inclusions of hepatitis B surface antigen in a
case of chronic hepatitis B with cirrhosis.
(D) Demonstrates focal copper-binding protein in a periportal
hepatocyte (arrow)

32. Congo red stain
It is used to assess amyloid deposition and is combined with polarization microscopy to
demonstrate the characteristic apple-green birefringence

33. Oil Red O stain
It is used to highlight the presence of fat globules, with its most common application being
evaluation of pretransplantation donor liver biopsies . The stain is performed on frozen sections
of the liver tissue.
Shows the presence of large (macrovesicular) and small (microvesicular) globules

34. Other stains that are currently not in widespread use include :
Aldehyde fuchsin (copper-associated protein, elastic fibers, hepatitis B surface antigen),
Aniline blue (collagen).
Chromotrope aniline blue (CAB) (helpful also for recognizing giant mitochondria as well as new
collagen in conditions such as steatohepatitis)
Picrosirius red (which is suitable for morphometric analysis of fibrosis in chronic hepatitis,
Bile pigment using the Hall’s stain
Stains commonly used for microorganisms include the Ziehl-Neelsen stain for acid fast bacteria,
Gomori methenamine silver (or PAS) stain for fungus, and Gram stain for bacteria

35. Immunohistochemical stains:
They are much less commonly used in the diagnostic evaluation of nonneoplastic liver diseases
compared with the histochemical stains described above.
The most commonly used stains are those for diagnosing or confirming viral infections involving
the liver, including hepatitis B virus ,cytomegalovirus virus, and herpes simplex virus, although
the latter can also be detected via in situ hybridization.
Assessment of possible metabolic disorders and in tumor histogenesis

36. (D) Hep Par 1 (“hepatocyte”) shows positive staining of tumor
cells in a trabecular hepatocellular carcinoma.
(E) Polyclonal carcinoembryonic antigen (pCEA) immunostain
shows apical, canaliculus-like staining of malignant
hepatocytes in hepatocellular carcinoma

37. A)Cytoplasmic hepatitis B surface antigen in hepatocytes.
(B) Hepatocyte nuclei with positive staining for hepatitis B core antigen, indicative of active viral
replication.

38. Cytomegalovirus hepatitis. H&E stain shows a typical microabscess containing a cell with nuclear and
cytoplasmic inclusions.

39. Case 1:
50/F with cirrhosis, obese, serum A1AT normal

40. Globules specific for diagnosis??????
Vascular etiologies, Acute hepatitis

41. Case 2:
40/M with renal transplant • Persistent elevation of ALT, AST 5-6x • No history of viral hepatitis

42. Ground glass appearance
• Hepatitis B
• Drugs: Barbiturates, cyanamide
• Metabolic diseases Glycogen storage IV Lafora disease Hypo(a)fibrinogenemia
Glycogen inclusions (‘pseudo ground glass’)
• Often on multiple immunosuppressive medications

43. Quiz :