How to handle liver biopsy specimens
Prof B Portmann 11.20 - Virtual Pathology at the University of Leeds
Prof B Portmann 11.20 - Virtual Pathology at the University of Leeds
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Liver Biopsy in the Assessment of Medical Liver Disease<br />
Wednesday 9 Dec 2009<br />
<strong>How</strong> <strong>to</strong> <strong>handle</strong> <strong>liver</strong> <strong>biopsy</strong> <strong>specimens</strong><br />
Fixation, processing and staining methods<br />
Bernard Portmann<br />
King’s College Hospital<br />
London
Percutaneous <strong>liver</strong> <strong>biopsy</strong><br />
• Invasive technique<br />
• Risk of morbidity<br />
• Risk of mortality<br />
exceedingly low, but<br />
existent<br />
Essential not <strong>to</strong> loose<br />
information during<br />
handling / processing<br />
of these small <strong>specimens</strong>
Liver Biopsy – Processing / Interpretation<br />
Potential problems<br />
• Small specimen (1:50’000th of<br />
the <strong>liver</strong>)<br />
⇒ US guided specimen taken<br />
by radiologists = thinner / smaller<br />
• Fragmentation<br />
? artefact or fibrotic <strong>liver</strong><br />
• Sampling variation<br />
• Limited numbers of tissue responses<br />
<strong>to</strong> a broad range of injuries of different aetiologies
Liver Biopsy – Processing / Interpretation<br />
Importance of<br />
• High standard processing<br />
• Sensible use of available techniques<br />
• His<strong>to</strong>pathologist’s ‘expertise’<br />
• Clinical information<br />
• Interactive discussion between pathologist<br />
and clinician
Liver <strong>biopsy</strong><br />
Specimen collection / fixation<br />
• For conventional his<strong>to</strong>logy<br />
Prompt immersion in buffered formalin or<br />
formol saline<br />
• Special fixative for EM (Limited diagnostic<br />
value for <strong>liver</strong> disease)<br />
• Culture (PUO)<br />
• Fresh tissue on wet blotting paper mainly Cu<br />
[Fe] (use water for injection)<br />
• Snap frozen tissue : enzymology - metabolic<br />
disorder (specialized centres)
Handling the <strong>biopsy</strong> specimen<br />
Liver <strong>biopsy</strong> <strong>specimens</strong> are best<br />
left floating in the fixative solution<br />
No blotting paper<br />
Avoid rough packing<br />
between foam sponge<br />
Use fine mesh cassettes or<br />
lens-paper wrapping
Handling the <strong>biopsy</strong> specimen<br />
Essential <strong>to</strong> embed specimen flat, especially<br />
when dis<strong>to</strong>rted, in order <strong>to</strong> get most of the<br />
core(s) cut by each micro<strong>to</strong>me stroke<br />
Paraffin wax blocks<br />
Glass slides
Routine staining in our Labora<strong>to</strong>ry<br />
• 16 sequential sections are pair-mounted on 8<br />
slides numbered 1 <strong>to</strong> 8<br />
1, 8 = H & E (x 2)<br />
<br />
<br />
<br />
<br />
3 = Silver for reticulin<br />
5 = Perls’ for iron<br />
6 = Periodic acid Schiff after diastase<br />
7 = Orcein (modified Shikata’s)<br />
2, 4 = Held unstained<br />
Xxxxxxxxxxx<br />
Xxxxxxx<br />
xxxxxxxx
Routine slides x 8 (2 held unstained)<br />
3285/09<br />
H&E-1<br />
3285/09<br />
Perls’<br />
3285/09<br />
Reticulin<br />
3285/09<br />
Orcein<br />
3285/09<br />
dPAS<br />
3285/09<br />
H&E-2
Gordon-Sweets’ silver method for reticulin<br />
Normal<br />
Collagenisation<br />
Un<strong>to</strong>ned<br />
Un<strong>to</strong>ned<br />
Collapsed<br />
HV<br />
Un<strong>to</strong>ned<br />
Toned
NRH
Mild portal fibrosis<br />
Early septa<br />
Bridging septa<br />
Cirrhosis
Active<br />
Inactive
Periodic acid Schiff (PAS)<br />
• Cell loss / entrapped hepa<strong>to</strong>cytes<br />
in fibrous tissue<br />
• Negatively stained s<strong>to</strong>rage cells<br />
dPAS<br />
• Negative in glycogen s<strong>to</strong>rage disorders<br />
(highly soluble glycogen)<br />
No significant additional information<br />
Not performed routinely
PAS after diastase<br />
Performed routinely<br />
Bile duct basement membranes<br />
α1-antitrypsin globules<br />
Scavenger macrophages
Orcein staining: requirements<br />
• Optimal fixation and processing<br />
(Folds, scratches dark brown)<br />
• Adequate pre-staining oxidation<br />
(renew KMNO4 weekly)<br />
= same as that used for reticulin)<br />
• pH of orcein solution
Orcein<br />
• Hepatitis B surface antigen<br />
Homogeneous cy<strong>to</strong>plasmic inclusions<br />
• Low sensititvity ( ↓ in buffer formalin)<br />
• Does not detect membranous deposition<br />
• Elastic tissue<br />
• Age of areas of collapse → not very useful)<br />
• Vascular thrombi
Orcein<br />
Copper associated protein Dark brown granules<br />
• Chronic cholestasis (periportal / periseptal)<br />
• Wilson’s disease<br />
random ± Kupffer cells<br />
• Indian childhood cirrhosis / Cu associated<br />
neonatal disorders / Cu <strong>to</strong>xicosis<br />
• Physiological (infant <strong>liver</strong> up <strong>to</strong> 2 mos of age)<br />
• Cirrhosis of any aetiology (patchy)
Copper handling by hepa<strong>to</strong>cytes<br />
Caeruloplasmin<br />
A<strong>to</strong>x 1<br />
Trans-Golgi<br />
Membranes<br />
(WDP – ATP7B)<br />
CCS<br />
SOD (cy<strong>to</strong>sol)<br />
Cu(I)<br />
Ctr1<br />
Cox 17<br />
Cyt C oxydase<br />
Bile<br />
canaliculus<br />
Mi<strong>to</strong>chondrion<br />
Cu(II)-albumin<br />
in portal blood<br />
Cu-GSH<br />
Cu-Metallothionein<br />
Lysosome<br />
(Excess Cu /<br />
Orcein++)
Copper and copper associated protein<br />
Thionein<br />
s<br />
s<br />
s<br />
s<br />
Cu ++<br />
Cu ++ Rhodanine +<br />
Oxidation (KMnO 4 )<br />
Thionein<br />
SO 2<br />
H<br />
SO 2<br />
H<br />
SO 2<br />
H<br />
SO 2<br />
H<br />
Orcein +
Copper / Copper-associated protein<br />
Orcein<br />
Rhodanine<br />
Immediate processing<br />
After 3 wks in formol saline
Copper and copper associated protein<br />
• Copper easily removed by formal saline<br />
(especially if acidic)<br />
•Copper-binding protein unaffected by fixation<br />
• Early Wilson :<br />
- Cy<strong>to</strong>solic Cu highly soluble + <strong>to</strong>o small<br />
<strong>to</strong> be seen his<strong>to</strong>logically<br />
- Lysosomal Cu binding protein may not<br />
be significant yet<br />
⇒ negative staining does not exclude WD
Perls’ for iron<br />
Ferritin + haemosiderin<br />
are ferric compounds stained<br />
by the Perls’ technique<br />
• Ferritin: diffuse blue blush<br />
(DD artefact)<br />
• Haemosiderin: intense blue<br />
granules<br />
Parenchymal iron graded on a<br />
0 - 4+ scale<br />
1 = mild periportal/periseptal deposition<br />
4 = massive deposits (no acinar gradient)<br />
2 and 3 = intermediate<br />
Grade 1-2<br />
Grade 4
Iron overload<br />
(Liver siderosis / haemosiderosis)<br />
• Genetic haemochroma<strong>to</strong>sis<br />
HFE / non-HFE associated (juvenile, adult)<br />
Mutational analysis (C282Y +/+)<br />
Iron tissue estimation now rarely requested<br />
• Chronic anaemia (genetic, acquired)<br />
• Blood transfusions<br />
(Kupffer cells / macrophagic siderosis ++)
Immunohis<strong>to</strong>chemistry<br />
• Numerous commercial antibodies <strong>to</strong><br />
choose from<br />
against structural components,<br />
viral or secre<strong>to</strong>ry material<br />
• Variably useful for diagnosis or research<br />
• Balance between diagnostic need,<br />
interest and cost effectiveness<br />
• To be used sensibly given the small<br />
amount of tissue
Viral proteins<br />
• Hepatitis B (HBs – HBc)<br />
HDV (delta)<br />
• [Hepatitis C]<br />
• Cy<strong>to</strong>megalovirus (CMV)<br />
• Adenovirus<br />
• Herpes virus<br />
• EBV (ISH / EBER)<br />
• Cryp<strong>to</strong>sporidium
HBV<br />
Vertical transmission<br />
HBc tissue expression ++<br />
Minimal inflammation<br />
HBc<br />
HBs carrier<br />
HBV DNA -ve, ↑↑ HBs in serum and <strong>liver</strong><br />
HBc<br />
Immunocompromized host<br />
Active viral replication<br />
HBs
HCV anti-core region antibody<br />
Not useful for diagnosis<br />
or screening<br />
• Adequate results only in<br />
some immunocompromized<br />
hosts with high virus load<br />
in tissue<br />
HCV recurrence in <strong>liver</strong><br />
allograft with rapid<br />
progression <strong>to</strong> cirrhosis
Liver allograft<br />
CMV (microabscess)<br />
CMV early phase Ab<br />
Necrosis due <strong>to</strong><br />
adenovirus<br />
Adenovirus group Ab
Structural antigens<br />
• Cy<strong>to</strong>keratins 7,19 [AE1/AE3] = biliary epithelium<br />
• Cy<strong>to</strong>keratins 8, 18 / ‘Hepa<strong>to</strong>cyte-specific<br />
antigen’(HePar1) = hepa<strong>to</strong>cytes<br />
• CD68, HLA-DR = Kupffer cells<br />
• FVIII-associated Ag, CD34 = negative on normal<br />
sinusoidal endothelium<br />
• CD10, CD13, pCEA = canalicular regions<br />
Enzymes<br />
• Gamma-glutamyl-transpeptidase (γ GT)<br />
• Bile salt export protein (BSEP)<br />
• Multidrug resistance 3 (MDR3)
CK7<br />
Chronic cholestasis / CK7<br />
Chronic rejection<br />
Duc<strong>to</strong>paenia
Progressive familial intrahepatic cholestasis (PFIC2 – BSEP deficiency)<br />
γ-GT<br />
BSEP<br />
PFIC2<br />
Normal
Progressive familial intrahepatic cholestasis<br />
(PFIC3 – MDR3 deficiency)<br />
Morphology<br />
- Cholestasis<br />
- Progressive periportal fibrosis<br />
- Ductular reaction - Duc<strong>to</strong>paenia<br />
⇒ Biliary cirrhosis<br />
(adolescents / young adults)<br />
Imunostaining for MDR3<br />
• Infants / young children<br />
No staining in MDR3 deficiency<br />
• Adolescents/adults (late onset)<br />
Normal staining<br />
(?functional deficiency)<br />
MDR3 immuno (4 yr)<br />
MDR3 control
Biopsy taken <strong>to</strong> assess focal lesion<br />
• Best <strong>to</strong> evaluate firstly H&E sections<br />
• Spare sections on PLL coated slides<br />
held for immunohis<strong>to</strong>chemistry<br />
• Panel of antibodies <strong>to</strong> assess neoplasms does<br />
not differ from that used in any other organ<br />
• Due <strong>to</strong> scarcity of tissue more judicious<br />
selection recommended based on his<strong>to</strong>logical<br />
appearances + clinical guidance<br />
• Remember neuroendocrine neoplasms
Electron microscopy<br />
• Requires 1mm 3 pieces in glutaraldehyde<br />
• May contribute <strong>to</strong> diagnosis in<br />
some metabolic disorders<br />
- FIC1 disease (Byler)<br />
- Mi<strong>to</strong>chondriopathy, Wilson<br />
- S<strong>to</strong>rage disorders<br />
• Technique generally <strong>to</strong>o demanding for<br />
the diagnostic yield<br />
• Risk of removing information for his<strong>to</strong>logy<br />
assessment
Minute <strong>biopsy</strong> with diagnostic features<br />
Importance of careful processing + clinical information<br />
• Clinical information:<br />
? Chronic <strong>liver</strong> disease<br />
- Small nodular fragment<br />
lined by fibrosis<br />
- Dilated cholangioles<br />
with bile plugs<br />
• Differential diagnosis<br />
Cirrhotic nodule ? Biliary<br />
? Sepsis<br />
von Meyenburg complex /<br />
Congenital hepatic fibrosis
Additional clinical information<br />
– Portal hypertension / Haematemesis<br />
– No jaundice - Normal <strong>liver</strong> enzymes<br />
Diagnosis : Congenital hepatic fibrosis<br />
Subsequent confirma<strong>to</strong>ry<br />
<strong>biopsy</strong> specimen
<strong>How</strong> <strong>to</strong> <strong>handle</strong> needle <strong>liver</strong> <strong>biopsy</strong><br />
Invading technique - small specimen<br />
High standard processing <strong>to</strong> avoid lost of<br />
information<br />
Sensible use of available techniques<br />
especially immunohis<strong>to</strong>chemistry<br />
Seek second opinion for challenging cases<br />
(but limited assistance if poorly processed<br />
+ paraffin block empty)<br />
Importance of<br />
• Clinical awareness on the part of the pathologist<br />
• Clinical information given by clinicians<br />
• Interactive discussion between pathologist<br />
and clinician
King’s College Hospital<br />
Institute of<br />
Liver Studies
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