2. Immunohistochemistry
• Application of immunologic principles and
techniques to the study of cells and tissues
• The method for in situ detection of
antigens in tissues by Ag-Ab recognition,
by using specificity provided by Ab with its
Ag at a light microscopic level.
3. Principle of IHC
• The basic critical principle of IHC, as with
any other special staining method, is a
sharp visual localization of target
components in the cell and tissue, based
on a satisfactory signal-to-noise ratio.
5. Deparaffinize and hydrate the tissue
section
1. Put the slides into a rack for IHC
2. Dry slides at 58℃ overnight (recommended)
or at 65℃ for 1-2 hours(for fast experiment)
3. Dip the rack into 4 consecutive stain jars
containing xylene to remove paraffin
- 10 minutes every step
4. Dip the rack into ethanol to remove xylene
- 100% Ethanol 5min
- 95% Ethanol 5min
- 80% Ethanol 5min
- 70% Ethanol 5min
5. Rinse the rack with tap water to remove ethanol for 5 minutes
6. Quench the peroxidase
1. Dip the rack in 3% H2O2 for 13 minutes
2. Rinse the rack with tap water for 15 minutes
The blocking can be done
(1) After rehydration to water and before antigen
retrieval,
(2) After antigen retrieval and before primary antibody
incubation,
(3) After primary antibody incubation,
(4) After biotinylated secondary antibody incubation.
7. Antigen retrieval
• The retrieval techniques of unmasked
antigens :
HIER/Heat induced epitope retreival
(1) Microwave
(2) Microwave and trypsin
(3) Pressure cooker
Proteolytic enzyme digestion
8. HIER
• Buffer solutions for heat-induced epitope
retrieval
– Sodium Citrate Buffer (10mM Sodium Citrate,
0.05% Tween 20, pH 6.0)
– 1 mM EDTA, adjusted to pH 8.0
– Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA
Solution, 0.05% Tween 20, pH 9.0)
9. • Put citrate buffer into a pressure cooker or
heat it without a lid in the microwave for 5
minutes (Prewarming)
• Put the rack into the citrate buffer
• Put a lid on the cooker and heat the
cooker for 10~15 minutes
10. • Remove the cooker and cool it down at RT
for 30 minutes
• Rinse the rack with tap water for 10
minutes
• Dip the slides in PBS buffer (at RT) for 10
minutes
11. • Enzymatic method
– Pepsin
– Trypsin
– Proteinase K
– Pronase
• Cover the slide with the above working
solutions for 10-20 min at 37degC
15. Procedure for application of primary
and secondary Ab
• Primary Ab - Apply the appropriately diluted marker specific
Ab, incubate in a moist chamber for 1 hour
• Rinse slides in TBS for 5 min
• Secondary Ab – Apply 1:100 diluted biotinylated species
specific secondary Ab.
• Incubate the sections in a moist chamber for 30 min.
• Rinse the slides in TBS for 5 min.
• ABC incubation – Apply 1:1:100 diluted pre made avidin-
biotin complex
• Incubate at RT in moist chamber for 1 hr
17. ChromogensChromogens End product colourEnd product colour
Diaminobenzidine (DAB)Diaminobenzidine (DAB)
BrownBrown
Diaminobenzidine (DAB)Diaminobenzidine (DAB)
BrownBrown
3-amino-9-3-amino-9-
ethylcarbazoleethylcarbazole
RedRed
4-chloro-1-naphthol4-chloro-1-naphthol BlueBlue
Hanker-Yates raegentHanker-Yates raegent Dark blueDark blue
Alpha-naphtol pyroninAlpha-naphtol pyronin Red-purpleRed-purple
18. Fluorochromes
DAPIDAPI ColorColor
FluoresceinFluorescein BlueBlue
Hoechts 33258Hoechts 33258 GreenGreen
R-phycocyaninR-phycocyanin BlueBlue
B-phycoerythrinB-phycoerythrin RedRed
R-phycoerythrinR-phycoerythrin Orange, redOrange, red
RhodamineRhodamine Orange, redOrange, red
Texas redTexas red RedRed
19. COUNTERSTAINING AND MOUNTING
SLIDES
• Hematoxylin is used as the nuclear
counterstain for most routine IHC staining.
• Either one drop is placed on the tissue section
and a coverslip is lowered slowly onto the
slide, or the coverslip is placed on a paper
towel, one drop is placed in the center of the
coverslip, and the inverted slide is lowered
slowly onto the coverslip.
• For alcohol-insoluble stains (e.g., DAB or new
fuchsin), a permanent mounting medium,
such as Permount, may be used.
20. Reporting
• Focus on what type of cells are staining
( tumor cells, endothelial cells,
stromal cells).
• Pattern of immunoreactivity must follow
anatomic distribution of antigen before
it is called +ve.
• Note the no. of cells staining, intensity
of staining & pattern of staining
(cytoplasmic, membranous, nuclear, dot
like).
21. Interpreting positivity
• Localisation of the Ag
– Nuclear – ER/PR, S-100, TTF1
– Cytoplasm – S-100, synaptophysin
– Membrane - CD20, CD45, Her-2, CD99
– ECM, basal laminae
29. Interpretation …. cont’d
• Absence of staining of both the test tissue and positive
control
• Absence of staining of the test tissue with appropriate
positive staining of the positive control
• Weak staining of the test tissue with appropriate staining of
the positive control
• The presence of background staining on the test tissue, the
positive control, or both
• The presence of artifactual staining on the test tissue, the
positive control, or both
30. False negative results
• Ab is inappropriate, denatured or used at
wrong conc.
• Loss of Ag – through autolysis or diffusion.
• Presence of Ag at a low density, below the
level of detection with the reagents
32. Bcl-2 staining in a lymph node –
wrong results due to fixation artefact
33. False positive results
• Cross reactivity of the Ab
• Non specific binding of the Ab to the tissue
• Presence of endogenous peroxidases in –
or avidity for avidin-biotin complex by –
some cellular elements
34. • Entrapment of normal tissue by tumor
cells
• Release of proteins from the cytoplasm of
the normal cells invaded by the tumor,
with subsequent permeation of the
interstitium and non specific absorption by
the tumor cells.
• “Chromogen freckles”
41. Some examples of utility of IHC in the
diagnosis and management of malignant
tumors
• Categorization of malignant tumors
• Categorization of leukemia/lymphoma
• Determination of origin of metastatic tumors
• Detection of molecules that have prognostic or
therapeutic importance
42. Common Panels of IHC stains
Epithelial
LMW-k
(CAM5.2),
AE1-AE3 CK cocktail,
CK7, CK20, CEA,
EMA
Mesenchymal
Vimentin
S100
Endothelial
CD34,CD31,
Factor VIII
ulex europaeus
Muscle
Desmin,
Myoglobin
Actin
Melanocytic
HMB45
S100
Melan-A,
MART-1
Neuroendocrine
NSE,
chromogranin,
Synaptophysin
Fibrohistio-
cytic
CD68,
lysozyme,
HAM 56,
CD1a(Lang-
erhans).
Lymphoid
Bcell-LCA/CD45,
CD20
T cell-LCA,CD3
Hodgkins- Ki1, CD15
L26,BLA36, CD30
Neuronal
NF,GFAP,
S100
Leu7
43. Other markers-
Ewings, PNET- MIC-2(o-13/CD99)
Hormone receptors-ER/PR/AR
Germ cell-AFP,HCG, PLAP
Cell Proliferation-Ki67, PCNA
Oncogenes/tumor supressor-Her 2neu,
p53,RAS,bcl-2,Rb.WT1
Metastatic potential-Laminin, collagen, cathepsin D
Tissue specific epithelial markers
Breast-GCDFP-15
Prostate-PAP,PSA
Liver- AFP, Hep Par1
Thyroid-TG, Calcitonin
Mesothelium-Keratin
Ovary- CA 125
44. Markers in Lymphoma
CD43- +ve in Most T cell malignancies,
group of small lymphocyte B cell
CLL/SLL, Mantle cell lymphoma
-ve in Follicular lymphoma
CD45- Pan cell marker Found on all
leucocytes.
RA RB RC RO
Bcells and a subpopulation of T cells B cells & most T cells Major T cells susbet, some macrophages
and granulocytes
LCA - Ab mixture to CD 45 +ve in all
lymphomas except ALCL, HL.
45. B cell markers
CD19- Earlier marker of lineage- not useful.
CD 20- +nt in cell throughout differentiation
+ve in all mature B cell (exc. Plasma
cells, RS-cells in 25% positive)
CD21- Follicular dendritic cells & some B
lymphocytes +ve in Follicular
lymphoma, Angio-immunoblastic T cell
lymphoma (Dendr- itic cells).
46. CD 23- +ve in B cell CLL/SLL
-ve in Mantle cell lymphoma
CD 79a- + ve Precursor Bcell LL &
in Mature B cell LL
CD38 – Plasma cell neoplasms
Ig Light chain-
Lambda in plasmacytoma
B cell markers
47. T cells
CD2, CD3- +ve in T cell lymphoma
CD5- Present on most thymocytes &
immature peripheral T cells.
+ve in B- CLL /SLL, Mantle
cell lymphoma.
-ve in Follicular & marginal cell
lymphoma.
+ve in Thymic carcinoma.
49. ALK +ve in ALCL
(Anaplastic lymphoma kinase gene)
CyclinD1- Cell cycle regulatory nuclear protein
+ve in mantle cell lymphoma, hairy
cell leukemia, plasma cytoma.
-ve in B-CLL/SLL.
Bcl-2- Antiapoptotic gene-
normally in follicular mantle B
lymphocytes, occ.germinal cemtres.
+ve in follicular lymphoma
Other markers
50. ALK +ve in anaplastic large cell
lymphoma
CD30 in Anaplastic large cell
lymphoma
51. Bcl 6- Nucleus of lymphocyte in germinal centre
+ve in most B cell lymphoma
-ve in follicular lymphoma progression.
CD 10 Markers of germinal centre origin
(CALLA) Precursor B cell lymphoma,
Burkitts lymphoma., Follicular lymphoma.
Tdt- DNA polymerase
Early B & T lymphoblast
Sensitive & specific for lymphoblastic
lymphoma.
Other markers
53. Most commmon keratins ad their
distribution
CK1 Epidermis of palms and soles, keratinizing squamous epidermis
CK2 Epithelia, all locations
CK3 Cornea
CK4 Non keratinizing squamous epithelia
CK5 Basal cells of squamous and glandular epithelia, myoepithelia,
mesothelium
CK6 Squamous epithelia, especially hyperproliferative
CK7 Simple epithelia
CK8 Basal cells of squamous and glandular epithelia, Simple epithelia of
stomach, intestines, Merkel cells.
54. CK Ag Positivity
Pankeratin (AE1/AE3) Ca of simple and complex epithlium
CK8 Ca of simple epithelium
CK1/10 SCC
CK7 Non GI derived ca
CK20 Most GI, mucinous ovarian, biliary, TCC, Merkel cell Ca
CK19 Most Ca, ca with squamous component, myoepithelial
cells
CK1/10/5/14 Basal cells of prostate, most duct derived Ca
CK18/19 Most Ca
CK10/11/13/14/15/16/19 Most squamous lesions and many Ca
CK8/14/15/16/18/19 Most Ca
56. Neuron specific
Enolase (NSE)
Gamma gamma
isoenzyme
Glycolytic enzyme
2PGL PEP
Neurons
Neuroendocrinal
cells
Neuroectodermal
& Neuroendocrinal
tumors, Melanoma
Synaptophysin Presynaptic
vesicles
Neuroendocrinal
tumors
Leu 7/CD57
T cell Ag
Indicative of NK
cell activity
Myelin of
CNS/PNS,
Neuroendocrine
cells
MPNST, Carcinoids,
Pheochromocytoma,
Small cell Ca of lung
Neuroendocrine markers
65. Applications in Ca breast
• Presence of ER/PR – treatment and
prognosis
• Traditionally measured using dextrn
coated charcoal and sucrose gradient
assay
• Two paramertes are evaluated in IHC
– Number of tumor cell nuclei stained
– Percentage of entire tumor cell nuclei
population
66. • Not much correlation between type of breast ca
and hormone receptors noted
• However, most medullary, intraductal and
comedoca are negative
• Mucinous ca – highest rates of positivity
• DCIS with a predominance of large cells – best
morphological predictor of ER-neg status
67. • Percentage of ER and androgen receptors
are LOWER in premenopausal women
• Presence of ER – high nuclear and low
histologic grade, absence of tumor
necrosis, marked tumor elastosis and
older patient’s age
69. Her2/neu
• Her2 /neu is an oncogene
• Tranmembrane GP with TK activity known as
p185
• Belongs to EGFR
• Detected by IHC, FISH
• Importance lies in the fact that Her2 positive
tumors are sensitive to t/t with monoclonal Ab
(Trastuzumab) to it.
70. Grading the IHC staining of Her2/neu.
Staining pattern Score Her2/neu protein
overrexpression
asssessment
No staining is observed or
membrane staining is observed in
<10% of tumor cells
0 Negative
Faint/barely perceptible membrane
staining is detected in >10% of
tumor cells. The cells are stained
only in a part of the membrane
1+ Negative
A weak to moderate complete
membrane staining is observed in
>10% of the tumor cells
2+ Weakly positive
A strong and complete membrane
staining is observed in >10% of the
tumor cells
3+ Strongly positive
Section of lymph node stained with CD45, where the tissue is adequately fixed.
Sections of lymph node stained with CD20 showing bright cytoplasmic membrane staining of the B cells in the germinal center.
Section of normal lung tissue stained with TTF-1 (thyroid transcription factor), exhibiting specific nuclear positivity of the alveolar lining cells (pneumocytes
Section of lymph node stained with CD45 showing variable fixation artifact. Well-defined cytoplasmic membrane staining is seen in the subcapsular region
Another lymph node section stained with Bcl-2 showing similar variable fixation artifact with gradual loss of staining appreciated toward the center of the node.
Section of parotid gland stained with CD3 showing scattered T-lymphocyte and non-specific cytoplasmic granular staining in the glandular epithelium.
Pigmented melanophages, not to be confused with chromogen.
Section of liver illustrating endogenous biotin resulting in non-specific background staining.
Section of lymph node stained with CD43 demonstrating processing problem with cleavage and folding of the tissue, which subsequently resulted in variable staining intensity and precipitation of the chromogen. All these artifacts can adversely affect interpretation.
Sections of malignant melanoma stained with S-100. The spindle-shaped neoplastic cells are overstained to the extent that it is difficult to appreciate true nuclear staining.
Section of adenocarcinoma stained with CEA demonstrating positive polymorphonuclear neutrophils showing strong endogenous peroxidase staining, which are also seen scattered in the stroma.
