the utility of immunohistochemistry in forensic pathology

1. Immunohistochemistry
in
Forensic Pathology
VKS

2. Utility of histological examination
• To confirm – macroscopic findings
• Detect/exclude – pathological findings
• Establish – cause of death
• Mechanism of death – not shown by gross
anatomic findings
• Detect cells/ biological material – further
investigation

3. • IHC – vital tool – appends findings – definitive
diagnosis
• What is IHC?
Synergy between 3 scientific disciplines

4. • Biochemical assay – chemical reaction – locate
and visualize - interaction between antigens
and antibodies
• Antigens – from the tissues
• Antibodies – manufactures – monoclonal /
polyclonal
• histological techniques – tissue processing
• Chemistry – antigen/ab reaction, visualisation

5. Immuno-
• Antigen – toxin/foreign substance – elicit an
immune response
• Epitopes – present of outer surface of antigen
- 3D structure of protein
• Antibody – globulin protein – variable region –
attach to epitope
• Antibodies – monoclonal/polyclonal –
hybridoma technique

6. Histo-
• Biopsy
• Fixation – 10% neutral buffered formalin (5%-
better for antigen retrieval)
• Dehydration and clearing
• Embedding
• Microtomy ( 5-8 micron)
• Transfer to the slide
• Pre treatment – deparaffinization, rehydration,
epitope/antigen retrieval (removal of cross links)

7. Chemistry-
• Primary ab
• Secondary ab
• Substrate
• Counter stain
• Dehydration
• Clearing
• Mount
• Ready for viewing

9. Uses
• Keratins – pancytokeratin
• In detecting amniotic fluid embolism
• c/o – intrapartum/postpartum maternal
deaths
• No gross changes- diagnosis is histological
• Difficulty – amniotic fluid particles- scanty –
missed

11. Food allergens
• Anti IgE antibodies to food allergens, bee
stings
• c/o – anaphylactic shock – post mortem
serum IgE levels
• Demonstration of positivity in the residual
food/ bee poison – diagnostic importance
• Ig E positive mast cells – lung, skin etc

12. Myocardial ischemia
• C5b-9(m) , fibronectin– early
• Myogenin, desmin, cardiac troponin I
• C5b-9(m): autolysis resistant, do not show non
specific localisation
Myogenin, desmin, cardiac troponin C5b-9 , fibronectin
•Depletion
•Early infarct/ ishemic changes – few
fibers
•Late infarct – diffuse
•Deposition
•Seen in myocytes
•Sub endothelial

13. • Expression of C5b-9, FN, MB, cTnI – Acute MI,
Acute cardiac death, Sudden coronary death,
acute traumatic death
• AMI- C5b-9, FN
• ACD – MB, cTnI
• CD- MB
• ATD – all negative

15. Myocarditis
In addition : use of
molecular
techniques – PCR
– for detection of
etiological agent

17. Drug abuse
• Anti-myoglobin : myoglobin cylinders in renal
tubules – rhabdomyolysis
• Pulmonary haemorrhage – collagen IV –
identifies breaks in alveolar wall – D/f blood
aspiration
• Myocardial injury – increased MHC II, TNF
alpha
• Renal glomerulopathies – IgM deposits
• Hepatocellular – Hepatitis B

19. • Drug toxicity – metmizole intolerance – liver
Kupffer cell hyperplasia – CD 68
• Clozapine – myocarditis
• Alcohol – liver –varying degree of steatosis –
more specific – Mallory denke bodies –
ubiquitin labelled.

21. Asphyxia
• P selectin : increased endothelial expression –
non inflamed lung tissue – hanging, drowning,
CO intoxication
• SP –A : positive in intraaveolar - aggregates –
acute mechanical asphyxia, negative in normal
lung tissue
• HIF 1 alpha- positive in vessels – acute
mechanical asphyxia – probable role in
vasoconstriction

22. P- selectin : endothelial cells of vessels ,
c/o hanging
HIF 1 alpha: increased expression in
the vessel wall, c/o drowning
SP-A: intra alveolar aggregates , c/o drowning

23. Wound age determination

24. Use of IHC
Marker Appearance
Collagen I 4-6 days
Collagen III 2-3 days
Collagen IV 4 days
Collagen V 3 days
Collagen VI 3 days
Collagen VII 4 days
Laminin – in myofibroblasts
Basement membrane
1.5-4 days
4-8 days
HSP 1.5- 4 days
Fibronectin 10-20 min
SMA 5 days
Selectins 1hr – 17 days
Adhesion molecules 1.5 hr – 3.5 days
Keratin (CK 5/6) 13 days

25. • Asthma : CD117/ Ig E – mast cells – pulmonary
interstitium , bronchial walls
• Viral pneumonia : non specific histopatho findings –
IHC – CMV, E selectins
• Shock : vasular changes – endothelium – E selectin,
ICAM – in lungs – markers for septic shock
• Still born – positive pulmonary flotation test –
histological evidence –
– All alveoli expanded – CD68 alveolar macrophages
– Partly expanded – amniotic fluid embolism – CK in alveoli

27. • Neuronal injury dating
• Tissue and organ determination – distorted
histology – organ specific IHC
Parameter Appearance
CD15 10 min
Beta APP 2-3 hrs
Apolipoprotein E >3-4 hrs
GFAP loss 3 hrs
T cells, macrophages > 2-4 days
Tenascin 7 days

29. • Limitations:
– Artefacts : autolysis, altered staining, formalin
pigment, displaced tissue, postmortem changes
– Careful search for a preserved area
– 10% formalin – fixation- cross linking of proteins-
hampers staining in IHC
– Use 5% formalin – less cross linkages
– Unmasking/ pre treatment – heating in buffer,
enzymatic treatment

30. – Use of controls – to comment of the positivity –
positive controls – external/internal
– Controls – withdrawal studies – once each without
primary and secondary antibody (negative
controls)
– Trained pathologist

31. An interesting case!
• 45 yr old Mrs X and her 5 yr old girl – murdered in their
house – multiple blows to their head
• Crime scene – blood and brain tissue splattered all over
• Husband Mr Y –prime suspect – out of town- was
informed on phone by the police – rushed back home
• Police searched – hotel room – all belongings were
seized as evidence. A blue polo shirt – two faint blood
marks on it (examined under UV light)
• Mr Y – he used the shirt to wipe out blood from a
finger cut of Mrs X– 2 weeks back

32. • Pathologist perspective –
• Piece of shirt with the mark – histopath
examination
• Which tissue? - battery of IHC : CK, LCA, GFAP,
S100, NF
Brain tissue on shirt !!! –
unequivocal evidence – DNA
extraction – Mrs X- Mr Y was
arrested ......................................

33. Take home message
• Histopathological examination - H&E and
special stains – appropriate gross autopsy
findings – pivot role
• IHC – Inherent limitation – tissue
state/processing/antigen retrieval/ staining
patterns
• Gross + H&E+ appropriate IHC – diagnosis

34. References
• Dettmeyer R B: Forensic histopathology, Fundamentals and perspectives.
Springer
• Dettmeyer R B. The role of histopathology in forensic practise: an
overview. Forensic Sci Med Pathol. 2014; 10:401-12.
• Sinicina I, Pankartz K, Bise K. Forensic aspects of post mortem histological
detection of amniotic fluid embolism. Int J Legal Med. 2010; 124:55-62.
• Tsokos M: Forensic Pathology Reviews. Humana Press
• Cecchi R, Sestili C, Prosperini G, Cecchetto G, Vicini E et al. Markers of
mechanical asphyxia: immunohistochemical study on autopsy lung tisses.
Int J Legal Med. 2013
• Grandmaison G, Charlier P, Durien M. Usefulness of systemic histological
examination in routine forensic autopsy. J Forensic Sci. 2010;55(1): 85-88.
• Franklin R W : New diagnostics in forensic pathology
• Casse J M, Martrille L, Vignaud J M, Gauchotte G. Skin wounds vitality
markers in forensic pathology: An updated review. Medicine Science and
the Law.2016;56(2): 128-137.
• Miller R T: Immunohistochemistry in Forensic pathology. Pro Path