immunohistochemistry

1. IMMUNOHISTOCHEMISTRY
IN HISTOLOGICAL
DIAGNOSIS
Dr. Sapana Bhandari
JR II Pathology
Dr. V.M.G.M.C. Solapur

2. INDEX
• DEFINITION
• PRINCIPLE
• TECHNIQUE
• CLASSIFICATION OF MARKERS
• APPLICATION IN NEOPLASTIC
CONDITIONS.
• APPLICATION IN INFECTIOUS DIS.

3. Immunohistochemistry
• Def- 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.
• The site of antibody binding is identified by
tagging the antibody with a visible label as
antibody molecules cannot be seen by light
or electron microscope ie. Enzymes –
Horseradish peroxidase

4. Dr.Sonal Agrawal
PRINCIPLE
• The basic critical principle of IHC is sharp
localization of target components in the cell &
tissue, based on satisfactory signal to noise ratio.
• Amplifying the signal while reducing nonspecific
background staining (noise) is the major strategy.

5. Dr.Sonal AgrawalDr.Sonal Agrawal
SAMPLES FOR IHCSAMPLES FOR IHC
Immunohistochemistry can be performed onImmunohistochemistry can be performed on
Formalin fixed paraffin embedded sectionsFormalin fixed paraffin embedded sections
Frozen sectionsFrozen sections
SmearsSmears
ImprintsImprints
CytospinsCytospins

6. PREPARATION
• Fixation, dehydration, embedding
• FIXATION-
• 1. Freezing- Rapidly frozen tissue
* Adv- Superior preservation of antigens
Optimal reaction
* Disadv- Not routinely available
Morphology is not so good.

7. FIXATION
Other fixatives
Crosslinking fix. Microwave Coagulant fix.
irradiation fix.
10% formalin
Act- covalent crossli-
nking between macro-
molecules
Adv- Mcly used, inex-
pensive ,Morphology
is preserved.
Ethanol
Act- Precipitation
of protein
Adv-primary structure
is unmodified.
Disadv- LMW Ag lost
during fixation,
dislocation artifacts

8. Principle of cross linking fixatives &
Antigen Retrieval
Deleterious
effects of
formaldehyde
countered by
Antigen
retrieval in
attempts to
retrieve/unmask
target Ag.

9. Dr.Sonal AgrawalDr.Sonal Agrawal
METHODOLOGYMETHODOLOGY
1.1. Signal detectionSignal detection
2.2. Signal amplificationSignal amplification
3.3. Reducing the noise : Blocking ofReducing the noise : Blocking of
background stainbackground stain
a)a) Nonspecific antibody bindingNonspecific antibody binding

10. Dr.Sonal AgrawalDr.Sonal Agrawal
1. SIGNAL DETECTION METHODS1. SIGNAL DETECTION METHODS
Labeled methodLabeled method
Direct conjugate labeled methodDirect conjugate labeled method
Indirect conjugate labeled method (sandwich method)Indirect conjugate labeled method (sandwich method)
Avidin Biotin Method (Direct & Indirect)Avidin Biotin Method (Direct & Indirect)
Polyvalent methodPolyvalent method
Protein A MethodProtein A Method
Enzyme Labeled Antigen MethodEnzyme Labeled Antigen Method
Polymeric Labeling Two Step MethodPolymeric Labeling Two Step Method
Unlabeled methodUnlabeled method
Enzyme Bridge TechniqueEnzyme Bridge Technique
Paroxidase Antiperoxidase Technique (PAP)Paroxidase Antiperoxidase Technique (PAP)
Avidin Biotin Conjugate Procedure (ABC)Avidin Biotin Conjugate Procedure (ABC)
Biotin Streptavidin SystemsBiotin Streptavidin Systems
Alkaline Phosphatase Anti Alkaline Phosphtase method (APAAP)Alkaline Phosphatase Anti Alkaline Phosphtase method (APAAP)

11. Dr.Sonal AgrawalDr.Sonal Agrawal
Direct Conjugate Labeled Antibody MethodDirect Conjugate Labeled Antibody Method
One step methodOne step method
Labeled primary antibody reactsLabeled primary antibody reacts
directly with tissue antigensdirectly with tissue antigens
AdvAdv – short & quick– short & quick
DisadvDisadv ––
Less sensitiveLess sensitive
Require large number ofRequire large number of
conjugated primary antibodiesconjugated primary antibodies

12. Dr.Sonal AgrawalDr.Sonal Agrawal
Indirect or Sandwich ProcedureIndirect or Sandwich Procedure
Secondary antibody is raisedSecondary antibody is raised
to gamma globulin of theto gamma globulin of the
species producing primaryspecies producing primary
antibodyantibody
Secondary Ab is conjugatedSecondary Ab is conjugated
Adv –Adv –
Increased versatilityIncreased versatility
primary Ab can be used at aprimary Ab can be used at a
higher working dilutionhigher working dilution

13. Dr.Sonal AgrawalDr.Sonal Agrawal
Peroxidase Antiperoxidase MethodPeroxidase Antiperoxidase Method
Further development of indirect techniqueFurther development of indirect technique
It involves third layer of PAP complexIt involves third layer of PAP complex
This complex is made up of 2 Ab molecules & 3This complex is made up of 2 Ab molecules & 3
horseradish peroxidase moleculeshorseradish peroxidase molecules
Adv –Adv –
The sensitivity is about 100 to 1000 times higher sinceThe sensitivity is about 100 to 1000 times higher since
peroxidase molecule is not chemically conjugated to antiperoxidase molecule is not chemically conjugated to anti
IgG but immunologically bound & loses none of itsIgG but immunologically bound & loses none of its
enzymatic activityenzymatic activity
Allows use of much higher dilution of primary antibodiesAllows use of much higher dilution of primary antibodies
Disadv –Disadv –
Antibody incorporated into PAP reagent should be ofAntibody incorporated into PAP reagent should be of
same species as the primary antibodysame species as the primary antibody

14. Dr.Sonal AgrawalDr.Sonal Agrawal
Peroxidase Antiperoxidase MethodPeroxidase Antiperoxidase Method

15. Dr.Sonal AgrawalDr.Sonal Agrawal
Biotin Avidin ProcedureBiotin Avidin Procedure
This procedure exploits the high affinity binding betweenThis procedure exploits the high affinity binding between
biotin & avidinbiotin & avidin
Direct i.e. primary Ab is conjugated with biotinDirect i.e. primary Ab is conjugated with biotin
Indirect i.e. secondary Ab is conjugated with biotinIndirect i.e. secondary Ab is conjugated with biotin
Adv –Adv – RapidRapid
DisadvDisadv ––
 Different batches of biotin & different batches of avidinDifferent batches of biotin & different batches of avidin
have differing affinity for each otherhave differing affinity for each other
 Some tissues contain significant amount of endogenousSome tissues contain significant amount of endogenous
biotinbiotin

16. Dr.Sonal AgrawalDr.Sonal Agrawal
Biotin Avidin ProcedureBiotin Avidin Procedure

17. Dr.Sonal AgrawalDr.Sonal Agrawal
Avidin Biotin Conjugate MethodAvidin Biotin Conjugate Method
Standard methodStandard method
One of the widely used techniqueOne of the widely used technique
It involves three layersIt involves three layers
First layer of primary AbFirst layer of primary Ab
Second of biotinylated secondary AbSecond of biotinylated secondary Ab
Third is complex of avidin biotinThird is complex of avidin biotin
peroxidaseperoxidase
Lastly DAB/other substrate isLastly DAB/other substrate is
added to develop colored productsadded to develop colored products

18. Dr.Sonal AgrawalDr.Sonal Agrawal
Indirect Biotin Avidin Method
Avidin Biotin Conjugate Method

19. Dr.Sonal AgrawalDr.Sonal Agrawal
Double Staining MethodDouble Staining Method
Technique to visualizeTechnique to visualize
more than one antigenmore than one antigen
- sequentially- sequentially
-simultaneously-simultaneously
Enzyme labeled antigenEnzyme labeled antigen
method is usedmethod is used
Two antigens are stainedTwo antigens are stained
simultaneously withinsimultaneously within
same section using twosame section using two
labeled antigenslabeled antigens

20. Dr.Sonal AgrawalDr.Sonal Agrawal
Enhanced Polymer One Step Staging MethodEnhanced Polymer One Step Staging Method
Novel technology reported byNovel technology reported by
Pluzek et alPluzek et al
A large number of primaryA large number of primary
antibody molecules &antibody molecules &
peroxidase enzymes areperoxidase enzymes are
attatched to dextran polymerattatched to dextran polymer
Adv: more rapidAdv: more rapid
more sensitivemore sensitive
less time is requiredless time is required

21. Dr.Sonal AgrawalDr.Sonal Agrawal
DAKO EnVision polymeric method
Mirror Image Complementary
Antibody labeling method (MICA)

22. Dr.Sonal AgrawalDr.Sonal Agrawal
Enzyme Bridge Method labeled Antigen Method
Protein A –PAP Method APAAP Method

23. Dr.Sonal AgrawalDr.Sonal Agrawal
2. Amplification Methods2. Amplification Methods
CARD (Catalyzed Reporter Deposition technique)CARD (Catalyzed Reporter Deposition technique)
TSA (Tyramine Signal Amplification)TSA (Tyramine Signal Amplification)

24. Dr.Sonal AgrawalDr.Sonal Agrawal
3a. Blocking of nonspecific antibody3a. Blocking of nonspecific antibody
bindingbinding
 Mainly a problem with polyclonal antibody as multipleMainly a problem with polyclonal antibody as multiple
unwanted antibodies may exist in antiserumunwanted antibodies may exist in antiserum
• Use greater optimal working dilutions of antibodyUse greater optimal working dilutions of antibody
• Preincubate the tissue section with normal serum from the samePreincubate the tissue section with normal serum from the same
species of animal in order to occupy unwanted binding sitesspecies of animal in order to occupy unwanted binding sites
 Antibodies are highly charged molecule & may bindAntibodies are highly charged molecule & may bind
nonspecifically to tissue components bearing reciprocalnonspecifically to tissue components bearing reciprocal
chargecharge
• Preincubate with normal serumPreincubate with normal serum

25. Dr.Sonal AgrawalDr.Sonal Agrawal
3b. Blocking of endogenous enzymes3b. Blocking of endogenous enzymes
Enzymes like peroxidase are preserved in both paraffinEnzymes like peroxidase are preserved in both paraffin
& frozen sections& frozen sections
Peroxidase is present normally inPeroxidase is present normally in
erythrocytes,neutrophils ,eosinophils & hepatocyteserythrocytes,neutrophils ,eosinophils & hepatocytes
Peroxidase blocking step should be performedPeroxidase blocking step should be performed
-Incubation in methanol/ H2O2-Incubation in methanol/ H2O2
-Use alternative methods ( immunogold / glucose oxidase-Use alternative methods ( immunogold / glucose oxidase ))

26. Dr.Sonal AgrawalDr.Sonal Agrawal
Fixation for immunocytochemistry andFixation for immunocytochemistry and
cryostat tissue:cryostat tissue:
1 FNAC slides and cytospin sections should be1 FNAC slides and cytospin sections should be
fixed in ether alcoholfixed in ether alcohol
2 Cryostat sections should be fixed in cold2 Cryostat sections should be fixed in cold
acetoneacetone

27. Dr.Sonal AgrawalDr.Sonal Agrawal
IHC REACTION ASSESSMENTIHC REACTION ASSESSMENT
1.1. Qualitative:Qualitative:
a. Pa. Presence or absence of reactionresence or absence of reaction
b. Types of reaction patternb. Types of reaction pattern
Nuclear, cytoplasmic & membranousNuclear, cytoplasmic & membranous

28. Dr.Sonal AgrawalDr.Sonal Agrawal
2. Quantitative (Immunoscores):2. Quantitative (Immunoscores):
Predominantly used in ER PR receptorsPredominantly used in ER PR receptors
Based on intensity of staining & percentage of positiveBased on intensity of staining & percentage of positive
cellscells
Scoring systems: H score, Quick score, Allred scoreScoring systems: H score, Quick score, Allred score
Recently importance of scoring is undermined (weakRecently importance of scoring is undermined (weak
ER stain & staining in 1to 10% cells are enough to startER stain & staining in 1to 10% cells are enough to start
treatment)treatment)
IHC REACTION ASSESSMENTIHC REACTION ASSESSMENT

29. Dr.Sonal AgrawalDr.Sonal Agrawal
REPORTINGREPORTING
1.1. Patient demographics & specimen identification dataPatient demographics & specimen identification data
2.2. Reference to diagnostic problem (that is differential diagnosis)Reference to diagnostic problem (that is differential diagnosis)
3.3. Nature of specimen analyzed (Frozen, FNAC/paraffin section)Nature of specimen analyzed (Frozen, FNAC/paraffin section)
4.4. Statement of all stains used with details of all primary antibodiesStatement of all stains used with details of all primary antibodies
(designate specificity & clone where appropriate)(designate specificity & clone where appropriate)
5.5. Findings both positive & negative, for all stains; sufficient detailsFindings both positive & negative, for all stains; sufficient details
of patterns & controls to justify the interpretationof patterns & controls to justify the interpretation
6.6. The immunohistochemistry should not stand alone but should beThe immunohistochemistry should not stand alone but should be
integrated into the final surgical pathology reportintegrated into the final surgical pathology report

30. Immunohistochemical markers

31. 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

32. 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

33. Vimentin
Intermediate fil.
Mesenchymal tissue
Endothelial cells,
Fibroblast,
vascular
smooth muscle
Mesenchymal
tumors
Renal cell
carcinoma
Mesenchymal markers

34. Muscle Specific Markers

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

36. 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

37. Muscle markers
Actin in myoepithelial cells Myoglobin in MMMT

38. Melanocytic Markers

39. 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,MPN
ST-variable,
Clear cell
sarcoma,
Schwannoma,
Malignant glioma
Rare-
Leiomyosarcoma,
synovial
sarcoma,
chondosarcoma

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

41. HMB-45
immature
melanosomes
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

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

43. Neuroendocrine Markers

44. 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

45. 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

46. Endothelial Markers

47. CD34
Hematopoietic
stem cell Ag
Endothelial cells,
mesenchymal cells
Angiosarcoma,
Kaposi Sarcoma
DFSP, Spindle cell
lipoma, Solitary
fibrous tumor.
CD31
PECAM-1
Endothelial cells,
Megakaryocytes,
Platelets, other
hematopoietic
elements
Angiosarcoma,
Hemangioma,
Hemangioendothel
ioma.
VWF
Factor VIII
related Ag
Endothelial cells
Megakaryocytes
Vascular
tumors

48. Factor VIII related Antigen
epithelioid vascular tumor. Kaposi sarcoma

49. Ulex
Europaeus I
agglutinin
Lectin produced by
a gorse plant
Vascular
neoplasm.
Thrombo-
modulin
Endothelial
cells,
mesothelial
cells,
osteoblasts,
monocytes
Sensitive in
poorly
differentiated
vascular
malignancies.

50. Ulex Europaeus I agglutinin
Vascular invasion of breast ca

51. Neuronal Markers

52. Neurofilament
Protein
Neuronal cells
Small round cell tumors
Neuroblastoma
Medulloblastoma
retinoblastoma
Peripheral
neuroepithelioma
Merkel cell tumors
Carcinoid
Parathyroid tumors
GFAP
Intermediate fil.
Astrocytes
Ependymal
cells
CNS tumors
Schwannoma

53. GFAP in malignant astrocytes. NF-Merkel cell Ca
Chromogranin, NF, S100 in olfactory neuroblastoma

54. CD99-
•Transmembrane Protein
•Encoded by MIC- 2 gene
•Expressed in all hematopoietic cells except
neutrophils, plasma cells, cortical thymocytes.
•Important to diagnose small round cell tumors-
100% expressed in Ewings sarcoma/PNET
100% not expressed in neuroblastoma.
•Other tumors - synovial sarcoma, vascular tumors.

55. CD99 -membrane reactivity in
Ewings/PNET

56. Collagen
type IV
Predominant
component of
basement membrane
Complete BM
around endothelial
cells, smooth
muscle cells,
Schwann cells,
glands
To diff. In
situ/invasive
carcinomas,
To diff. B/W
MPNST & MFH.

57. Malignant soft tissue round cell tumors
• Small cell carcinoma
• Polyphenotypic small cell tumor
• Lymphoma
• Melanoma
• Rhabdomyosarcoma
• Ewing’s sarcoma / PNET
• Desmoplastic small round cell tumor
• Round cell liposarcoma.
• Small cell osteosarcoma.

58. Diagnosis of malignant soft tissue round cell tumors

59. Sarcomas with spindle appearance
• Synovial sarcoma
• Sarcomatoid carcinoma
• Fibrosarcoma, Fibromatosis
• Angiosarcoma
• MPNST, Cellular Schwannoma
• MFH
• Leiomyosarcoma, Spindle cell RMS
• Melanoma
• Clear cell sarcoma

60. Diagnosis of sarcomas with spindle appearance

61. Malignant soft tissue epithelioid tumors
• Alveolar soft part sarcoma
• Angiosarcoma
• Leiomyosarcoma, GIST
• MFH
• Melanoma
• Synovial Sarcoma
• Malignant Mesothelioma
• Malignant Rhabdoid Tumor

62. Diagnosis of malignant soft tissue epithelioid tumors

63. Malignant soft tissue pleomorphic tumors
• MFH
• Pleomorphic liposarcoma
• Pleomorphic RMS
• Pleomorphic MPNST
• Pleomorphic Leiomyosarcoma.

64. Diagnosis of Malignant soft tissue pleomorphic tumors

65. Epithelial Markers

66. Cytokeratin

68. EMA
Epithelial
membrane
antigen
Glycoprotein
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.

69. AFP
Major oncofetal
Protein
Fetal Gut, Liver,
Yolk Sac
HCC, Yolk
Sac Tumor,
Non
seminomatous
germ cell
Tumors
HCG
PLAP
Choriocarcinoma,
Syncytiotrophob-
last cells in
seminomas,
embryonal/yolk
sac tumors.
Most germ cell
tumors-
Seminomas.
Infantile germ
cells

70. Germ cell Tumours
• Seminoma: CAM 5.2 - ve, PLAP +ve, OCT4 +ve,
EMA-ve
• Embryonal carcinoma: CAM 5.2 +ve, PLAP +ve, OCT
4 +ve, EMA -ve, AFP +/-ve
• Yolk sac carcinoma: CAM 5.2 +ve, PLAP +ve, EMA
-ve, AFP +ve, OCT 4 -ve
• Choriocarcinoma: CAM 5.2 +ve, PLAP+/-, EMA +/-,
HCG +ve, OCT 4 -ve

71. HCG +ve in seminoma
with trophoblastic giant
cells.
PLAP +ve in tumor cells of
intratubular germ cell
neoplasia.
HCG

72. CEA- +ve in colorectal Ca, Ductal Ca breast,
Lung Ca, HCC- pericanalicular
Hep Par1 Granular cytoplasm staining
Most HCC
Villin- Actin binding protein in brush border of
intestine
+ve in colorectal carcinoma, HCC
(canalicular), Lung Ca.
CDX2- Encodes transcription factor for
intestinal epithelium.
+ve in colorectal Ca, Duodenal Ca, Bladder adenoca,
ovarian mucinous tumors, colloid Ca of lung.

73. Canalicular formation in
HCC -demo. By CEA,
which are diff. From
sinusoids of normal liver.
Luminal staining in glandular
structures of adenosquamous
carcinoma.
CEA

74. TTF-1 Thyroid follicular
& parafollicular C
cells,
Type II
pneumocytes, Clara
cells
Thyroid Ca
Primary Lung Ca
-ve in pulmonary
metastasis
/mesothelioma.
Thyroglobulin Thyroid
follicular cells
Thyroid
carcinoma.
Thyroglobulin staining in
poorly diff. Carcinoma of
thyroid.

75. PSA Formed exclusively by
prostatic epithelial cells.
Prostatic tumors,
BHP,
Salivary, breast,
bladder adenoca.
Intensityœ diff.
PSMA
Highly specific BHP, Prostatic
tumorsIntensity œ1/ diff.

76. GCDFP-15
Breast cystic fluid,
cells with apocrine
features
Breast Ca,
Pagets disease of
skin,
vulva.Salivary
gland Ca,
Prostate Ca.
Fibroadenoma with
apocrine metaplasia

77. Mets-AdenoCa Mesothelioma
B72.3Ab +nt -ve
Ber EP4 Diffuse +ve Focal +ve/- ve
WT 1 -ve +ve
(nuclear (sensitive & specific)
staining) Also +ve in ovarian serous Ca
Calretinin -ve +ve.
(N- neurons, renal convoluted tubules,
steroid producing cells)

78. Calretinin in mesothelioma-
nuclear & cytoplasmic
reactivity.
Reactive mesothelial cells
CK5/6
Mesothelioma

80. CK7
CK20
Transitional cell carcinoma - Bladder

81. Paget’s disease
CK7
CEA
GCDFP

82. Pediatric Neoplasms

83. 1.NBL NSE, SYN, CMG, NF, CD56/57
2.RMS Myogenin, Myo-D, Desmin, Muscle
specific actin, WT
3.Ewings/PNET Vimentin, SYN, CD99, FLI-1,NSE
4.Desmoplatic Cytokeratin, EMA, Vimentin,
small RCT Desmin, WT, CD99, NSE
5.Malignant Vimentin, MSA, synaptophysin
Rhabdoid T. S100, CD99, NSE, Keratin
6.Wilms Tumor Blastemal-vimentin, Desmin
Epithelial- cytokeratin
Mesenchymal- variable acc. To
differentiation pattern

84. Desmoplastic small round cell tumour
Keratin Desmin NSE

85. Diagnosis of head & neck
endocrine tumors.

86. CK
Chromogranin
Synaptophysin
S 100
Paraganglioma
TTF1
TGB
CGN
Calcitonin
PTH
TTF +
TGB +
CGN +
calcitonin +
PTH -ve
Mixed follicular
C cell tumor
Thyroid follicular
cell tumor
TTF +
TGB+
CGN -ve
PTH -ve
Calc -ve
Medullary Ca
TTF+
CGN +
Calc +
TGB -ve
PTH -ve
TTF +
CGN +
Calc +/-
Metast. NE lung Ca
CGN+, PTH+
Parathyroid
Ca
-ve
+ve

87. Medullary carcinoma of thyroid.
Calcitonin CGN CEA

88. Applications in carcinoma of
breast

89. I. Hormone Receptors
• ER/PR-nuclear staining , normal breast acini
• varies with menstrual cycle
• Total score= % positive nuclei with intensity of
nuclear staining
• Any nuclear staining = positive, good prognosis
ER +ve invasive breast ca

90. ER/PR semiquantitation

91. ER/PR semiquantitation

92. Her-2/ neu
• Member of tyrosine kinase receptor family
• over-expression-- poor outcome
• current use- predictor of response to doxorubicin
chemotherapy,
• to determine which pts would response to trastuzumab
(herceptin) therapy
• FISH-advantage-
detects gene amplification
• CISH replacing FISH

93. II. E-cadherin
• Calcium dependent trans-membrane protein
• loss mets & poor survival
• crisp, intense cell memb staining
• lobular lesions-in situ / invasive lack it
completely,decreased /focal memb staining
may be seen in high grade ductal ca

94. III. To differentiate b/w in
situ & invasive ductal Ca
SMA
AE 1/AE3
IV. To detect metastasis
sentinel lymph node.

95. CD 117-
Encoded by proto-oncogene C-KIT
Transmembrane tyrosine kinase family.
Expressed normally in mast cells, melanocytes, germ
cells, interstitial cells of Cajal
Expressed in Systemic mastocytosis, small cell lung
cancer, germ cell tumors & sensitive marker for GIST.
Epithelioid GIST - CD117 is -ve
Other markers for GIST- CD34, CD99, smooth muscle
actin.

96. Immunoreactivity for CD117
in GIST
CD117- in
seminoma

97. Ki- 67/ MIB 1- Proliferation Marker
Recognised nuclear protein involved in proliferating
cells.
Expression ass. With p53 exp. Tumor grade, pt.
Prognosis.
Normal cervix HSIL

98. Hyperplastic polyp
Proliferating cells-MIB1+ve
Cells restricted to basal part
only.
Tubular adenoma
Proliferating epithelium
also at luminal surface.

99. B cell markers
CD19- Earlier marker of lineage- not useful.
CD 20- +nt in cell throughout differentiation
+ve in all mature B cell neoplasm(except
Plasma cells). RS cells in 25% cases
CD21- Follicular dendritic cells & some B
lymphocytes
+ve in Follicular lymphoma, Angio-
immunoblastic T cell lymphoma (Dendr-
itic cells).

100. Follicular Lymphoma
CD20 in neoplastic
cells
CD 3 in non neoplastic
T cells
CD21 in dendritic
follicular cells

101. CD 23- +ve in B cell CLL/SLL
-ve in Mantle cell lymphoma
CD 79a- + ve Precursor Bcell LL &
in Mature B cell LL
Ig Light chain-
Lambda in plasmacytoma
B cell markers

102. 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.

103. Other markers
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
B lymph. +nt widespread Myeloid &
T cells.
LCA - AB mixture to CD 45- +ve in all
lymphomas except ALCL, HL.

104. Large T cell lymphoma
CD43 +ve Lysozyme +ve of
reactive histiocytes

105. 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

106. ALK +ve in anaplastic large
cell lymphoma
CD30 in Anaplastic large
cell lymphoma

107. 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

108. 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

109. CD15- Lewis X Ag
Stains membranous paranuclear dot like,
golgi localization.
Specific marker of RS cells of classical HL
-ve in most NHL.
CD30 ALCL,
Classical HL
(TNF receptor family)
Other markers

110. 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

111. CD30 in HL
EBV/ LMP1 Ag
in HL

112. 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.

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

114. H-pylori in
superficial mucus
in chr gastritis

115. Advantages-
• Can be done on paraffin embedded tissue
• Allows microbiologic & morphologic
correlation
• Provides diagnosis when fresh tissue not
available.
• Sensitive & specific.

116. Facts About IHC
• Diagnosis should be based on clinical
history, radiological finding, H & E
morphology with confirmation by IHC
testing.
• Use of a panel of IHC stains rather than
over-reliance on a single Ab is an important
principle.
• Detection of infectious agents/
identification of physiologic substances in
aberrant locations directly determine
diagnosis.

117. Facts About IHC
• Negative immunoreaction in IHC never
rules out a diagnosis.
• Key is- to utilise IHC as cost effective tool
in patient care.

119. p-53