This document discusses immunohistochemistry (IHC), which is used to identify tissue antigens through antigen-antibody interactions. It provides details on the IHC process, common antibodies and their targets, and tumor markers. IHC is useful for tumor diagnosis, narrowing differential diagnoses, and detecting unexpected diagnoses. The antibody panels discussed can help determine the primary site of cancers and differentiate between tumor types.

2. IHC needed in 10-25% of malignant tumors for
 Narrowing of possibilities
 Specific diagnosis
 Unsuspected diagnosis
 A correct histopathological diagnosis saves time,
money and inconvenience for the patients and
clinicians.

3.  Likelihood of a given diagnosis.
 Relevant differential diagnoses.
 Optimal selection of antibodies for a diagnostic
algorithm
Primary antibody panels and secondary antibody
panels
 Panel should include not only antibodies that would
expect to be positive in a given tumor , but also
antibodies that would be expected to be negative .

4.  IHC has revolutionized the field of
histopathology .
 Today IHC markers of almost every tumor has
been defined.
 IHC still has the long way to know the
histogenesis of yet unknown lesions.

5.  Immunohistochemistry is a technique for
identifying tissue constituents (antigens) by
means of antigen-antibody interactions.
 The site of antibody binding being identified
either by direct labeling of the antibody, or by
use of secondary antibody labeling method.

6. IMMUNOHSITOCHEMISTRY STEPS
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Tissue sections
Antigen retrieval
Blocking endogenous enzymes
Secondary antibody
Primary antibody
Microscopy Observation
Blocking background staining
Chromogen Substrate
Counterstain
Mounting

7.  Tissue sections are incubated with primary
antibodies against the target antigen.
 The specimens are then washed and a
secondary antibody against primary antibody
is added.
 The secondary antibodies are often
biotinylated, that is, conjugated to biotin, a
vitamin with an extremely high affinity for the
protein streptavidine.

8.  A complex made of streptavidin and the
enzyme peroxidase is used for staining.
 The addition of a chromogenic substrate such
as diaminobenzidine (DAB) or
aminoethylcarbazole (AEC), leads to a color
staining reaction that accurately reflects the
distribution of the target antigen in the tissue
sample.

9.  Tissue fixation on routinely histotechnique can
masked antigens because of formalin cross-
linking or even destroys some antigenic
epitopes.
 The retrieval techniques of unmasked antigens
:
(1) proteolytic enzyme digestion
(2) microwave
(3) microwave and trypsin
(4) pressure cooker

10. 1’Ab : primary antibody
2’Ab : secondary antibody
B : biotin
SA : streptavidine
HRP : horseradish
peroxidase
Target is antigen (protein
argets)
Blocking reagent : BSA
(bovine serum
albumin)
The sandwich of antigen recognition
on tissue samples.

11. Direct labeling of antibody Secondary antibody
labeling

12.  Enzyme labels
 Colloidal metal labels
 Fluorescent labels
 Radiolabels

13.  Horseradish peroxidase
 Alkaline phosphatase
 Bacterial-derived beta-D-galactosidase

14. GENERIC CATEGORIES
 Round-cell tumors
 Undifferentiated tumors
 Metastatic tumors
 Spindle-cell tumors
 Hematopoietic tumors

15.  Small cell carcinoma
 Malignant melanoma
 Lymphoma
 Neuroblastoma
 Ewing sarcoma/Primitive neuroectodermaltumor (ES/PNET)
 Rhabdomyosarcoma(RMS)
 Poorly differentiated synovial sarcoma( PDSS)
 Desmoplasticround cell tumor (DRCT)

16.  KERATIN S100
 EMA HMB45
 CD45 MART-1
 CD15 FLI-1
 VIMENTIN SYN
 DES CD56
 MSA CD99
 MYOGENIN

17. N0N
SARC
OMA
NS NS SARC
OMA
S S S S
Small
cell
carcin
oma
melan
oma
lymph
oma
neurob
lastom
a
rhabdo
myosa
rcoma
ES/
PNET
PDSS DRCT
CK + +/- - - RARE +/- + +
S -100 - + - + - +/- +/- -
Desmi
n
- +/- - - + - - +
CD99
(Mic 2)
- - +/- - -/+ + + RARE
LCA
(CD
45)
- - + - - - - -

18. Lympho
ma
carcinom
a
melanom
a
sarcoma Mesothel
ioma
glioma
LCA (CD
-45)
+ - - - - -
CK - + - - + -
S-100 - -/+ + -/+ - -
Vimentin -/+ -/+ + + + +
GFAP - - - - - +
HMB 45 - - + - - -

19. LYMPHOMA WHICH CAN BE LCA (CD 45) -NEGATIVE
 Lymphoblastic Lymphoma
 Anaplastic large cell Lymphoma
 Plasmablastic Lymphoma
 Peripheral T-cell type ( Some cases) Lymphoma
 Classic Hodgkin lymphoma ( RS cells)
LYMPHOMA
LCA (CD 45) +
CK -
S -100 -
Vimentin -/+
GFAP -
HMB 45 -

20. CARCINOMAS WHICH CAN BE LMW-CK
NEGETIVE
 Renal cell carcinoma
 Adrenal cortical carcinoma
 Small cell carcinoma
CARCINOMA
LCA (CD 45) -
CK +
S-100 -/+
Vimentin -/+
GFAP -
HMB-45 -

21. SARCOMA WHICH CAN BE CK-POSITIVE
 Synovial sarcoma
 Epitheloid sarcoma
 Malignant rhabdoid tumor
 Myogenic tumor (some)
 Angio sarcoma(some)
sarcoma
LCA (CD45) -
CK -
S-100 -/+
Vimentin +
GFAP -
HMB 45 -

22. If LCA ,CK and S-100 all are negative
Possibilities are
 Angiosarcoma(add CD 31)
 Anaplasticplasmacytoma( add CD79a & Kappa / lambda)
 Hodgkin lymphoma
 Germinoma
 Various sarcomas

23. EMA Desmin p53
Reactive
mesothelial cells
- or focal + + -
Malignant
mesothelioma
+ (membranous) - Or focal + +

24. Msothelioma Adenocarcinoma
Calretinin + (nuclear) -
CK 5/6 + -
WT – 1 + (nuclear) -
CEA - +
MOC – 31 - +
B 72.3 - +

26.  CK & EMA
 S100 protein
 CD15
 PSA
 Thyroglobulin
 TTF-1
 CEA
 CA19.9
 CA-125
 Placental alkaline phosphatase
 ER , PR

27. male
PSA - Prostate
common
Thyroglobulin /
calcitonin-
thyroid
CK 7 & CK 20
female
CA -125 –
Genital tract
adenocarcinoma
GCDFP-15 -
bREAST

29. CK 7 +VE ADENOCA CK 20-VE
ADENOCA

30. Prediction of primary site of a squamouscell carcinoma is a difficult task.
In some cases the following can be done :
 CK20 + ( CK7 +) : Transitional carcinoma of urinary bladder
 EBV + : Nasopharyngeal carcinoma /lymphoepitheliomalike carcinoma
 Mediastinaltumor :
CD 5 : positive = primary thymiccarcinoma
negative = metastatic carcinoma

31. CYTOKERATINPO
SITIVE

33.  Synovial sarcoma
 Fibrosarcoma
 Malignant peripheral nerve sheath tumor(MPNST)
 Leiomyosarcoma
 Solitary fibrous tumor

34.  KERATIN S100 PROTEIN
 ACTIN CD34
 EMA CD56
 VIMENTIN CD31
 DESMIN CD57
 THROMBOMODULIN CD99

35. Synovial
sarcoma
MPNST Fibro
sarcoma
Leiomyo
sarcoma
Solitary
fibrous
tumor
CK + - - Rare Rare
S-100 +/- + - Rare -
CD 34 - +/- - Rare +
Smooth
muscle
actin
- - +/-
(myofibros
arcoma)
+ -

36. S-100

37.  CD15 CD79A
 CD20 LYSOZYME
 CD30
MYELOPEROXIDASE
 CD43 CD3
 CD45 CD5
 CD68 CD163

38.  LCA (CD 45) : to differentiate lymphoid from
other non-lymphoid lesions
 Bcl-2 : + in follicular lymphomas-in follicular
hyperplasia
 Kappa & lambda chain restriction : monoclonal
neoplastic proliferation from polyclonal
reactive plasma cell proliferation

39. CD 45 CD3 CD45
RO
CD20 CD15 CD30 CD68 CD99
B cell
lymph
oma
+ - - + - +/- - -
T cell + + + - - + - -
Hodgk
in’s
dis.
+/- - - - + + - -
lymph
oblasti
c
+/- - - - - - - +
histioc
ytic
+/- - - - - - + -

40. NLPHL ClassicalHL
LCA (CD 45)
+ _
CD20
+ +/_
CD30 _ +
CD 15 _ +
EMA + _
CD- 57
Many small lymphocytes
form rosettes the L &H
cells
Few CD 57+ small
lymhocytes

41. CD-5 CD-23 IgD CyclinD1 Bcl-6
B-
Chronicly
mphocytic
leukemia /
small
lyphocyticl
ymphoma
+ + + _ _
Mantle cell
lymphoma
+ _ + + _

43. Minimum markers are : ER, PR & c-erbB2
ER+ ,PR+ tumors are a/w better prognosis
Show better response to hormonal therapy
ER positive in invasive lobular carcinoma of breast

44. Gastric carcinoma Keratin, EMA , CEA +
CK 7 + , CK20 +
Carcinoidtumor ACTH , NSE , Chromogranin+
Synaptophysin, keratin +
Colorectal Carcinoma CK20 + , CK 7 –( D/D lung & ovarian
adenocarcinoma
GIST CD117 + (100%)
Cytoplasmic & membrane
( D/D Fibromatosis)

45. Tumor Antibody panel
Prostate Carcinoma PAP & PSA +
34BE12 –(d/d benign lesion)
P504S +
Seminoma PLAP , CD117 , LDH , Vimentin+
EMA , CD 30 , HMWK –
EmbryonalCa EMA ,CD30 , HMWK +
Choriocarcinoma hCG+

46. Tumor Antibody panel
Hepatocellularcarcinoma AFP , Keratin , EMA +
Hep-par1 +
CEA -
Cholangiocarcinoma EMA , CEA,Keratin +
GB Carcinoma CK 7+ & CK20 + (d/d
cholangiocarcinoma)
Pancreaticcarcinoma Keratin , EMA , CEA , CD 19-9+

47. Tumor Antibody panel
Renalcell carcinoma Coexpressionof keratin + vimentin
A103 , inhibin–(d/d adrenocorticalca)
CA125 -(d/d ovarian ca)
TTF -1 , thyroglobulin–( d/d
thymiccarcinoma)
Transitional carcinoma of bladder CK 7+ & CK20 +
CEA , cathepsinB +

48. Tumor Antibody panel
Pheochromocytoma Catecholamine, neuronal marker +
S-100 +
Neuroblastoma NSE , neurophilaments+
Chromogranin, Synaptophysin+
Adrenocorticalneoplasm Vimentin, Synaptophysin+
Inhibin, A103 +
Chromogranin

49. Tumor Antibody panel
Osteoid Osteonectin,
osteopontin+
Osteocalcin+
Cartilage S-100 +
Ewing’ ssarcoma CD99 , vimentin+
NSE , neurophilaments+

50. Tumor Antibody panel
Papillary carcinoma CK 7 + , CK 20 -,
CK 19 +
HMWK +
Follicular carcinoma Thyroglobulin, TTF –1+
LMWK , EMA +
Medullarycarcinoma Calcitonin, CEA +
Chromogranin+

51. Tumor Antibody panel
Adenocarcinomacervix Keratin , EMA , CEA , HPV +
Endometrial carcinoma ER , PR +
HER2/ neu, GLUT 1 +
Ovarian carcinoma CK 7 + , CK 20 -,
CA 125+
Mesothelioma Calretinin, thrombomodulin+
CK 5/6 +

52. Tumor Antibody panel
Astrocyticneoplasm GFAP +
AE 1/3CK cocktail -
Oligodendroglioma S100 , Leu7+
Ependymoma GFAP , S100 , vimentin+
Meningioma EMA , S 100 +
CK 20-

53. Tumor Antibody panel
MucoepidermoidCarcinoma CK 7 , CK 14 +
Aciniccell carcinoma Keratin , amylase , IgA +
Transferrin, lactoferrin+
Adenoid cystic carcinoma Keratin, CEA,S100 , CD117 +
Lysosome, lactoferrin+
Myoepithelioma Keratin , S100 +
Vimentin, actin, myosin+

54. Tumor Antibody panel
Thymic carcinoma CD5 , CD 70 +
TTF -1 -(d/d from lung carcinoma)
Thymoma Keratin , EMA , CEA+
CD 99, CD 1a +
S100 +

55.  ANTIBODY TO SPECIFICITY
 CK Carcinoma ( epithelial tumors)
 Vimentin Sarcoma (mesenchymaltumors)
 GFAP Gliomas
 HMB -45 Melanoma
 S-100 Neoplasm of neural origin
 CD 68 Proliferationof mast cell
 CD 45 Neoplasm of lymphoid origin
 CD 45 RO Neoplasm of T cell origin
 CD 3 Neoplasm of T cell origin
 CD 20 Neoplasm of B cell origin
 CD 15 Hodgkin’s disease
 CD117 GIST
 CD5 Mantle cell lymphoma
 NB -84 Neuroblastoma
 Chromogranin Neuroendocrinetumors ( pan-endocrine marker)