Immunohistochemistry

methology

1. IMMUNOHISTOCHEMISTRY
Dr. Jayalakshmi V.
Dept of Pathology
BARCH

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.

4. Steps of IHC
Slide 3 of 23
Tissue sections
Antigen retrieval
Blocking endogenous enzymes
Secondary antibody
Primary antibody
Microscopy Observation
Blocking background staining
Chromogen Substrate
Counterstain
Mounting

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

12. Antigen Recognition
Direct labeling of antibody
Secondary antibody
labeling
The methods of Identifying Tissue Antigens

13. Immunoperoxidase procedures
•Put the two photographs from
Ackermann

14. Biotin-avidin immunoenzymatic
method

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

16. Chromogen / subsrate
•Enzyme labels
–Horseradish peroxidase
–Alkaline phosphatase
–Bacterial-derived beta-D-galactosidase
•Colloidal metal labels
•Fluorescent labels
•Radiolabels

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

22. S100-Both nuclear & cytoplasmic staining
Chondrosarcoma Malignant melanoma

23. Chromogranin positivity (cytoplasmic)

24. CD45 staining in a lymph node

25. B-cells in a
lymph node
CD99

26. -Section from normal lung tissue –
TTF1

27. Immunoreactivity for CD117
in GIST
CD117- in seminoma

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

31. CD45 staining in a lymph node

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”

35. CD3 staining in a section from the
parotid gland

36. Pigmented melanophages

37. Section from the liver – non specific
b/g staining

38. Folding of the tissue – variable and
dense staining

39. Case of malignant melanoma with S-
100 positivity

40. Section from an adenocarcinoma colon
stained for CEA

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.

48. CD19 +ve
CD20 +ve
Mantle cell
Lymphoma
Follicular cell
lymphoma
B-CLL/SLL
CD5+ve
Cyclin D1
+ve
CD23 -ve
CD5- ve
Bcl2 +ve
Bcl6 +ve
CD5 +ve
Cyclin D1
-ve
CD23 + ve
Non- Hodgkins Lymphoma

49. ALK +ve in ALCL
(Anaplastic lymphoma kinase gene)
Cyclin D1- 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

52. LCA -ve
CD15+ve
CD30 +ve
CD 20
Classical HL
CD15 -ve/+ve
CD30-ve
CD 43/CD 2/CD 3
Keratin/ S100
ALCL
Embryonal Ca
Pancreatic Ca
Malignant Melanoma
CD15 -ve
CD30 +ve
+ve
+ve
-ve
+ve-ve
+ve
-ve
Keratin
Carcinoma/ Sarcoma
+ve
-ve

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
CK10/11/13/14/15/16/19Most squamous lesions and many Ca
CK8/14/15/16/18/19 Most Ca

55. EMA
Epithelial
membrane
antigen
Glycoprotei
n
Apical surface
of most
glandular
epithelial cells.
Adeno Ca of breast,
lung, bile ducts,
pancreas, endocervix,
thyroid, salivary
glands
Skin & adneaxal
tumors
Synovial sarcoma,
epithelioid sarcoma,
plasmacytoma.

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

57. Chromogranin
Loc.-secretory
granules of
neuroendocrinal
cells
Neuroendocrinal tumors
Chromo. A-Gastric &
appendiceal carcinoid
Chrom. B-
Rectal carcinoid, prolactinoma
To diff b/w adrenal cortical Ca
& pheochromocytoma
Carcinoid tumour stomach
Paraganglioma

58. S100
Intracellular Ca
binding protein,
Soluble in 100%
amm.sulphate sol.
Localised in cytopl
asm & nucleus
Glial cell
Schwann cell
Melanocytes
Chondrocytes
Adipocytes
Myoepithelial
cell
Histiocyte
Melanoma,MPNS
T-variable,
Clear cell
sarcoma,
Schwannoma,
Malignant glioma
Rare-
Leiomyosarcoma,
synovial sarcoma,
chondosarcoma
Melanocytic markers

59. S100- Both nuclear & cytoplasmic staining
Chondrosarcoma Malignant melanoma

60. HMB-45
Immature
melanosomes,
and
melanocytes
Melanoma, Spitz nevi,
cellular nevi, pigmented
nerve sheath tumors,
angiomyolipoma, tuberous
sclerosis complex
components,
clear cell tumor of lung,
lymphangiomyomatosis.
Melan A/
MART 1
Sensitive & specific
-Melanoma
Sentinel lymph nodes
Adrenal cortical tumors
Sex cord stromal
tumors

61. HMB 45 staining
Isolated melanoma cells in sentinel
lymph node- HMB 45

62. Desmin
Intermediate
filament
Smooth muscle-
cytoplasmic dense
bodies
Striated muscle-
sarcomeric Z disc
Myoblast,
myofibroblast-
variable
fetal mesothelial
cells
Endometrial stromal
cells
Leiomyoma
Rhabdomyoma,
Rhabdomyosarcom
a
Desmoplastic small
round cell tumor
Endometrial stromal
sarcoma
PNET
Muscle specific markers

63. Actin
2 imp Isomers-
•Muscle specific
•Smooth muscle
actin
Myoepithelial
cells
Vascular
smooth
muscle
Pericytes
Myofibroblasts
RMS, LMS,
Fibromatosis,
myoepithelioma
Angiomyolipoma
Adenoid cystic
carcinoma
Inflammatory
myofibroblastic
tumor
Myogenin
Skeletal muscle
(expressed only
in early
differentiation).
RMS(sensitive
& specific),
regenerative
skeletal
muscle

64. Muscle markers
Actin in myoepithelial cells
Myoglobin in MMMT

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

68. ER in an invasive ca of the breast

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 patternScoreHer2/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

71. Her2 positivity in a case of invasive ca
breast

72. Application in Infectious diseases
•Viral infection-HBV, HCV, HHV8, HSV,
Adenovirus, CMV,HPV,Rabies, Parvovirus
B19
•Bacterial infection-H pylori, Rickettsia,
Bartonella, Leptospira.
•Fungi & parasite-aspergillus, Pneumocystis
carinii, toxoplasma, cryptosporidium.

73. HbSAg localized in variable quantity
in cytoplasm & cell membrane of
hepatocytes.
Positive cytoplasmic granules in every
periportal hepatocytes.

74. H-pylori in superficial
mucus in chr gastritis