immunohistochemistry principles and its role in diagnostics

1. PRINCIPLES OF
IMMUNOHISTOCHEMISTRY AND
ITS USE IN DIAGNOSTICS
EKTA JAJODIA

2. INTRODUCTION
IHC is a method for localizing specific antigens in tissues or
cells based on Ag-Ab recognition
IHC builds on the foundations of histochemistry/special
stains, it doesn’t replace histochemistry
The principle of IHC is a sharp visual localization of target
compounds in tissues , based on satisfactory signal to noise
ratio
Amplifying the signal while reducing non specific b/g
staining (noise) has been a major strategy

3. HISTORY OF
IMMUNOHISTO
CHEMISTRY

4. 1945 – Albert Coons 1st used an Ab labeled with a
fluorescent dye to visualize tissues
1st fluorescent dye attached to an Ab was
fluorescein isothiocyanate (FITC) – it emits green
fluorescence
A fluorescence microscope is required to visualize
this fluorochrome and they have a tendency to
fade . Also morphology is difficult to demonstrate
Due to these limitations – IHC was developed
Here enzymes are used as labels and visualized
with an appropriate chromogen using light
microscope

5. 1966 – 1st developed enzyme labeling instead
of fluorescent label
MC Used enzyme was – horse radish
peroxidase (HRP) – hence also known as
immunoperoxidase technique
It was done on frozen tissues
1974 – IHC was performed for the 1st time on
routine formalin fixed paraffin embedded
sections

6. 1981 – developed avidin-biotin labeling
1991 – Heat induced antigen retrieval
technique in IHC was done
1995 – Polymer technology introduced

7. TERMINOLOGIES
ANTIGENS - Molecules that induces formation of
an Ab and is foreign to the animal into which it is
introduced
Sites on Ag that are capable of inducing Ab
formation are known as – EPITOPES/ ANTIGENIC
DETERMINANT – the exact site on the Ag with
which the Ab combines
Epitopes may be classified as -

8. Continuous – Consisting of
continuum of residues in a
polypeptide chain
Discontinuous – c/o residues from
different parts of a polypeptide
chain brought together by folding of
protein conformation

9. •ANTIBODIES – IgG is the most frequently used Ab for
IHC
•The paratope of Ab binds to the epitope of Ag
•Abs are also proteins - thus any part of the Ab may
itself serve as epitope to induce Ab formation (to which
secondary Ab binds)
•IHC technique prove that Ig molecules can serve both
as Ab and Ag
•Monoclonal Ab is better than polyclonal Ab
(polyclonal Ab gives more non-specific staining)

11. Formalin fixed paraffin
embedded sections
Frozen sections
Smears
Imprints
Cytospins
IHC CAN BE
PERFORMED ON -

13. METHODS
• One step staining method
• Labeled Ab reacts directly
with Ag in tissue
DIRECT conjugate
labeled antibody
procedure
• Unlabeled primary Ab
reacts with tissue Ag
• Conjugated second Ab
reacts against primary Ab
INDIRECT/SANDWICH
PROCEDURE

14. 1. Peroxidase-antiperoxidase method (PAP)
2. Biotin-avidin complex method (ABC)
3. Labeled streptavidin-biotin method (LSAB)
4. Alkaline phosphatase- anti alkaline phosphatase
methos (APAAP)
5. Polymer based labeling

15. IHC PROTOCOL
Fixation and processing
Section cutting
Deparaffinisation and rehydration

16. Blocking endogenous peroxidase
Blocking non-specific antibody binding
Antigen retrieval
Primary antibody

17. Secondary antibody
Chromogen
Chromogen enhancement
Counterstain
Mount
Stringent washing
between reagents

18. IHC PROTOCOL
Fixation and processing
Section cutting
Deparaffinisation and rehydration

19. •Adequate fixation and good processing is required
•Poor and inadequate fixation may lead to incorrect
interpretation of staining patterns – eg – Elution of ER
protein from nucleus to cytoplasm
•There is no ideal fixative
•Any fixative which ensures optimal antigenic
immunoreactivity with preserved morphological details
may be used

20. •MC used – 10% neutral buffered formalin (NBF) (pH-
7-7.6)
•Length of fixation is imp – excessive fixation may
cause Ag denaturation and masking and too little
fixation can cause tissue deterioration
•Generally 6-72 hrs is acceptable
•IHC can be performed on decalcified tissue
•However some markers may not work well on such
tissues – eg – CD43, Ki67, ER, PR
•Paraffin embedding temperature s/b maintained
between 56-60◦C

21. PROCESSING IN SPECIAL
SITUATIONS
•Some Abs act only on frozen sections
•Frozen sections s/b fixed with acetone before storing
•FNAC , blood smears, etc – either
1. Centrifuge and make a pellet that is fixed just as in
tissue fixation, or
2. A fresh smear may be made and fixed in
acetone/alcohol/10% NBF

22. IHC PROTOCOL
Fixation and processing
Section cutting
Deparaffinisation and rehydration

23. • Sections s/b 3-4 microns in thickness
•Thick sections make interpretation difficult and are
also more likely to float during Ag retreival
•Adhesives – Sections are picked up on slides coated
with some adhesives
•MC used – Poly-L-lysine
3-aminoproplytriethoxysilane(APES)

24. IHC PROTOCOL
Fixation and processing
Section cutting
Deparaffinisation and rehydration

25. •Once on the slide, complete deparaffinisation is
needed so that the aqueous Ab solution can adhere
properly and penetrate the tissue

26. Blocking endogenous enzyme
Blocking non-specific antibody binding
Antigen retreival
Primary antibody

27. •Degree of susceptibility of an enzyme to denaturation
and inactivation during fixation varies
•Some enzymes such as peroxidase are preserved in
both paraffin and frozen sections
•Others such as alkaline phosphatase are almost
inactivated by routine fixation and paraffin embedding
•Any residual activity of these endogenous enzymes
must be abolished during immunostaining in order to
avoid false positive reactions when using such enzymes
as labels

28. ENDOGENOUS
PEROXIDASE ACTIVITY
•Peroxidase activity is present in RBCs, neutrophils,
eosinophils and hepatocytes
•So when IHC is performed in such tissues rich in blood
cells , eg bone marrow
•It is recommended that a peroxidase blocking step be
used
•To inhibit peroxidase activity –
• Incubation with 3% H2O2-methanol combination for
15 mins
• Phenylhydrazine + nascent H2O2 + sodium azide
• A mixture of H2O2 + sodium azide
• Cyclopropane hydrate

29. ENDOGENOUS ALKALINE
PHOSPHATASE ACTIVITY
1. Levamisole
2. 20% glacial acetic acid – better blocker

30. ENDOGENOUS AVIDIN-
BIOTIN ACTIVITY
•This step is required if an avidin – biotin detection
system is used
• Can be done by using avidin-biotin blocking reagent
or skim milk
•It is not reqd with polymer based systems
• NOTE – blocking the endogenous enzyme activity
step s/b done anytime before adding the secondary
Ab, or else enzyme label of secondary Ab will be
inactivated by this blocking procedure giving false -ve
result

31. Blocking endogenous peroxidase
Blocking non-specific antibody binding
Antigen retrieval
Primary antibody

32. •Non-specific binding may occur as highly charged Ab
molecules may bind tissue components bearing
reciprocal charge
•This step is performed to block unwanted binding sites
before incubation with primary Ab
•The blocking serum c/o dilute serum from the same
species used for production of secondary Ab
•Proteins in the serum occupy the charged sites within
the tissue section

33. Blocking endogenous peroxidase
Blocking non-specific antibody binding
Antigen retrieval
Primary antibody

34. UNMASKING OF ANTIGEN SITES
When formalin based fixatives are used
Intermolecular and intramolecular cross-bridges are formed
with certain structural proteins
These are responsible for masking of tissue antigens
ANTIGEN RETREIVAL

35. The degree of masking of antigens depends on –
1. Length of time in fixative
2. Temperature
3. Concentration of fixative
• Many labs use automated IHC staining systems –
some of which have the ability to perform on-
board Ag retrieval- using enzymes or heat

36. MANUAL METHODS FOR ANTIGEN RETREIVAL
INCLUDE –
1. Proteolytic enzyme digestion
2. Microwave oven radiation
3. Combined enzyme and microwave
4. Pressure cooker heating
5. Decloaker heating
6. Pressure cooker inside a microwave oven
7. Autoclave heating
8. Water bath heating
9. Steamer heating

37. PROTEOLYTIC
ENZYME DIGESTION
•MC used enzymes – trypsin and protease
•Others – chymotrypsin
• pronase
• proteinase K
• pepsin
•The digestion by these enzymes breaks down formalin cross-
linking and hence the epitopes are uncovered
• Digestion time needs to be tailored to individual Abs
• - underdigestion results in too little staining
• - overdigestion leads to false +ve staining and high b/g staining

38. Factors responsible are –
1. Duration of enzyme digestion
2. Enzyme concentration
3. Use of coenzyme – such as calcium chloride
with trypsin
4. Temperature
5. pH

39. HEAT INDUCED
ANTIGEN RETREIVAL
(HIER)
MICROWAVE ANTIGEN RETREIVAL
PRESSURE COOKER ANTIGEN RETREIVAL
STEAMER
WATER BATH
AUTOCLAVE

40. Most popular antigen retreival solutions are –
1. 0.01M citrate buffer at pH 6
2. 0.1mM EDTA at pH 8
3. TRIS-EDTA at pH 9
Disadvantage – uneven heating and production of
hot spots
• At no stage should the sections dry out during
antigen retreival
MICROWAVE
ANTIGEN RETREIVAL

41. Major factors that influence the results of AR –IHC are –
1. Heating condition ( temperation and duration of
heating)
2. pH value of the AR solution

42. PRESSURE COOKER
ANTIGEN RETREIVAL
•More uniform than other methods
•Temperature is around 120◦C – this increased
temperature is especially required for nuclear antigens
(ER, PR, p53)
•Preferred – stainless steel domestic pressure cooker
•Aluminium pressure cookers are susceptible to
corrosion from some of the Ag retreival buffers

43. Blocking endogenous peroxidase
Blocking non-specific antibody binding
Antigen retreival
Primary antibody

44. PRODUCTION OF
PRIMARY ANTIBODIES
1. POLYCLONAL ANTIBODIES
Produced by immunizing an animal with a purified specific
molecule (immunogen) bearing the Ag of interest
Animal mounts an immune response to the immunogen
Abs produced are harvested by bleeding the animal to obtain
Ig- rich serum
Animal will produce numerous clones of polyclonal plasma
cells
Each clone will produce different Abs with different specificity
to the variety of epitope on a single antigen
Polyclonal Abs are more sensitive but less specific

45. 2. MONOCLONAL ANTIBODIES–
•One pure Ab with high specificity is produced
•Background staining in such cases is minimal
3. LECTINS –
•Plant or animal proteins that can bind to tissue carbohydrates
with a high degree of specificity
•Carbs may be characteristic of a particular tissue, lectin
binding may have diagnostic significance
•They can be labeled in similar ways to Abs

46. DILUENT for primary Ab – TBS (TRIS BUFFER SALINE )
BSA (bovine serum albumin)

47. Secondary antibody
Chromogen
Chromogen enhancement
Counterstain
Mount

48. •Secondary Ab or a polymer is attached to a label
•It is produced against the primary antigen

49. LABELS
ENZYME
LABELS
COLLOIDAL
METAL LABELS
FLUORESCENT
LABELS
RADIOLABELS

50. ENZYME LABELS
•Mc used labels in IHC are enzymes
•Enzymes used are –
HORSERADISH PEROXIDASE (HRP)
ALKALINE PHOSPHATASE (CALF INTESTINAL)
GLUCOSE OXIDASE
β-D GALACTOSIDASE (BACTERIAL DERIVED)

51. HORSE RADISH
PEROXIDASE (HRP)
•MC used enzyme due to several reasons –
• Its small size does not hinder binding of Abs to
adjacent sites
• Easily obtainable in highly purified form
• Stable enzyme
• Endogenous activity is easily blocked
• Labels are incubated with chromogens that
produce a colored end product suitable for light
microscope

52. CHROMOGENS

53. PRECAUTIONS TO BE TAKEN for chromogens which are
soluble in alcohol
1. Counterstaining with progressive non-alcoholic
hematoxylin (eg mayer’s ) to avoid removal of
alcohol soluble colored end product
2. An aqueous mountant is required – eg – 80%
glycerol or neutral phosphate buffered glycerin jelly
These can be made permanent by sealing the edges of
coverslip by nail varnish

54. ALKALINE
PHOSPHATASE
CHROMOGENS

55. Colloidal metal labels
1. Colloidal gold conjugate – gives pink color
2. Silver – gives yellow color
3. ferritin

56. FLUORESCENT
LABELS
Fluorochromes used are –
1. FITC – green
2. TRITC (rhodamine) – red
3. Texas red - red
4. R- phytoerythrin(PE) – orange/red

57. CHROMOGEN
ENHANCEMENT
•Place staining dish in 25◦C and incubate the slides in
0.5% copper sulfate solution for 1-5 mins
•Then wash under running tap water

58. WASHING
•Washing should be done after each step
•By phosphate buffered saline (PBS)

60. GENERATION OF IHC RESULTS
PRE-ANALYTICAL FACTORS – it includes
1. Time taken to remove the tissue at surgery
2. The ensuing ischemia
3. Interval between surgical resection and
fixation
4. Type of fixative
5. Length of fixative
ANALYTICAL FACTORS –
It pertains to lab procedures

61. POST ANALYTICAL EVALUATION –
•Very crucial
•Interpreting immunostains as merely +ve or –ve
without appreciating the following is inappropriate
STAINING PATTERN
% OF CELLS SHOWING POSITIVITY
INTENSITY OF STAINING

62. Nuclear Cytoplasmic Membranous
OCT 4
ER/PR
Myogenin
Myo D1
P53
P63
TTF 1
MIB-1
Actin
Alpha fetoprotein
Chromogranin
Factor VIII related antigen
Desmin
GFAP
Hep-Par 1
HMB 45
Melan A/MART
CK, vimentin, NFP
Her-2/neu
CEA
CD 99
CD20
EMA
LOCATION ! LOCATION ! LOCATION !

63. •Sometimes the staining pattern of a single stain
could be different in different diagnostic contexts –
CD3 (T-cell marker)
Cytoplasmic
positivity – in
precursor T cell
neoplasms
Membranous
positivity – in
peripheral T cell
neoplasms

64. MARKERS OF
DIFFERENTIATION
EPITHELIAL DIFFERENTIATION
MUSCLE DIFFERENTIATION
NERVE SHEATH DIFFERENTIATION
NEUROENDOCRINE AND NEUROECTODER,AL DIFFERENTIATION
MELANOCYTIC DIFFERENTIATION
VASCULAR DIFFERENTIATION
GLIAL DIFFERENTIATION

65. MARKERS OF EPITHELIAL
DIFFERENTIATION
1.CYTOKERATINS –
• Currently used CK designation syatem is known as
MOLL’s catalogue
• 20 CKs known
• Divided into –
• Type I – acidic keratins – 12 types – CK9 to CK20
• Type II – basic keratins – 8 types – CK1 to CK 8
• Also divided into –
• High molecular weight CK (HMWCK)
• Low molecular weight CK (LMWCK)

67. •HMWCK – aa squamous keratins
•Exp. In squamous epithelium
•Ultrastructurally/EM – known as tonofilaments –
hallmark of squamous cell carcinoma
•LMWCK – aka simple/non squamous keratins
•Exp. In glandular epithelium and visceral parenchyma
(liver, kidney)
•Intermediate molecular weight CK – aka basal keratins
•exp. In basal cells

68. ANTIBODY COCKTAIL

69. •AE1/AE3 –is a pan-CK
•Can recognise both LMWCK and HMWCK
•Cannot identify HCC – as the combination do not
contain CK18 which is exp by HCC
•Cam5.2 – can recognise LMWCK (including CK18)
•But may miss squamous cell carcinoma – as these exp
HMWCK
•So, AE1/AE3 and Cam 5.2 are complimentary for
screening purposes
•35β E12 – HMWCK – stains basal cells – used to
distinguish well differentiated prostate adenocarcionma
from benign lesion of prostate

70. 2. EMA
3. CEA
4. p63 – HMWCK equivalent
Marker of squamous and urothelial epithelia
5. BerEp4 –expressed in adenocarcinoma
•To differentiate lung adenocarcinoma (+ve) from
mesothelioma (-ve)
•Favored marker in effusion cytology – selectively
labels adenocarcinoma, whereas b/g mesothelial
cells are –ve (CK would label both)
LMWCK EQUIVALENTS – MARKERS
OF GLANDULAR EPITHELIA

71. •Some CK are aberrantly expressed by
mesenchymal cells and tumors – CK8, 18 and 19
•Mesenchymal neoplasms that are CK +ve are –
1. Synovial sarcoma
2. Epithelioid sarcoma
3. Chordoma

72. MARKERS OF MUSCLE
DIFFERENTIAION
There are 3 types of muscle differentiation –
1. Skeletal muscle differentiation – as in
rhabdomyoma and rhabdomyosarcoma (RMS)
2. True smooth muscle differentiation – as in
leiomyoma and LMS
3. Partial smooth muscle differentiation – as in
myofibroblasts – these constitute a significant
population of cells in healing wounds and stromal
reaction to tumors . And also in nodular fascitis and
myofibroblastoma

73. 1. DESMIN – ass. with both skeletal and smooth
muscle
Not exp by myofibroblast
NON muscle cells that exp desmin are –
A. fibroblast reticulum cells of lymph node
B. Endometrial stromal cells
C. Submesothelial fibroblast

74. 2. ACTIN – divided into muscle and non-muscle
isoforms
• Interpretation is quantitative rather than qualitative
• Muscle cells have far more actin than many other
cells
• Smooth muscle isoform – also expressed by
myofibroblast – show a characteristic tram track
pattern - (exp only in the periphery of their
cytoplasm)
• This distinguish them from smooth muscle cells
(uniform cytoplasmic +vity)

75. ACTIN– left – tram track pattern in myofibroblast
right – cytoplasmic +ve in true smooth muscles

76. 3. MYOGLOBIN – O2 binding heme protein
•Found in skeletal and cardiac muscle
•Not in smooth muscle
RECOMMENDATION FOR USE OF MUSCLE MARKERS –
•For identifying smooth muscle differentiation –
myogenin and myoD1
•For identifying skeletal muscle differentiation – desmin
and smooth muscle actin
•For identifying myofibroblasts desmin –ve and SMA –
tram track +vity

77. 1. S-100 protein – calcium binding protein
• Named as such due to its 100% solubility in
ammonium sulfate
• 2 subunits –α and β – they combine to form 3
isotypes
• α-α isotype – found in myocardium , skeletal muscle
and neurons
• α - β isotype – melanocytes, chondrocytes, glia and
skin adnexae
• β - β isotype – langerhan cells and schwann cells
MARKERS OF NERVE
SHEATH
DIFFERENTIATION

78. • Is of most value as a marker of benign and
malignant nerve sheath tumors and melanoma
• MPNST – show patchy and weak exp of S-100
• Benign nerve sheath tumors – strong and uniform
+vity
• Perineural cells are -ve

79. 2. CLAUDIN-1 –
•+ve in perineural cells
•Useful marker in perineuromas – granular membrane
+vity
•-ve in NF and schawannomas
2. GLUT-1 – perineural cells +ve
3. CD57 – found in NK cells , T cells, oligodendroglial
cells and schwann cells

80. MARKERS OF
MELANOCYTIC
DIFFERENTIATION
1. HMB-45 (human melanoma black) – monoclonal
Ab HMB-45 identifies Pmel17 gene product gp100
(present in premelanosomes)
+ve in immature melanocytes and –ve in mature
melanocytes
+ve in - -ve in -
a. melanoma a. Nevi
b.PEComa b. Resting melanocyte
Desmoplastic and spindle cell melanomas – usually
negative

81. •Less sensitive than melan-A and S-100
•But more specific
2. Melan-A
•Product of MART-1 gene
•Also +ve in nevi and resting melanocytes
•+ve in 50% cases of desmoplastic melanoma
•Also +ve in – adrenal cortical tumors and other steroid
producing tumors

82. 3. MiTF – Micropthalmia transcription factor
Product of micropthalmia (mi) genenuclear +vity
4. TYROSINASE – enzyme involved in synthesis of
melanin
5. S-100 –
Highly sensitive for melanoma
-ve for S-100 makes melanoma highly unlikely
Only 2-3% melanomas are –ve for S-100
6. SOX10
7. PNL2
8. MUM1
RECENT MARERS

84. NEUROECTODERMAL AND
NEUROENDOCRINE MARKERS
1. CD99 –
• Product of MIC2 gene
• Transmembrane gp
• Most imp use is in diagnosis of ES/PNET –
membrane +vity
Other SRCBT that are CD99 +ve–
• lymphoblastic lymphomas
• PD synovial sarcoma
• Mesenchymal chondrosarcoma
• Small cell OS
• DSRCT
never seen in neuroblastoma (NB)

85. 2. CD56 – mediates calcium independent cell-cell
binding
•Exp in Normal cells like –
•Neurons, astrocytes, glia, NK cells
•+ve in high grade NE neoplasms – esp small cell
carcinoma(which may be –ve for all other NE
markers)
•+ve in NB
3. NB-84
Highly sensitive for neuroblastoma

86. 4. SYNAPTOPHYSIN (SYN) and CHROMOGRANIN A
(CHR)-
•1st line markers for NE differentiation
•Mark neurosecretory granules – show granular
cytoplasmic positivity
•SYN is more sensitive
•CHR is more specific
•NON NE neoplasms which are SYN +ve but CHR –ve
are-
•Adrenocortical neoplasms and pancreatic solid
pseudopapillary tumors

87. 5. NSE (neuron specific enolase)
6. CK in NE neoplasms –
NE neoplasms fall in 2 categories-
a. Epithelial –carcinoid, pancreatic NE tumor, small cell
carcinoma
b. Non epithelial/neural – pheochromocytoma,
paraganglioma , NB
Epithelial NE neoplasms – CK+ve
Non epithelial NE neoplasms – CK-ve

88. • Distinctive feature of high grade NE carcinoma (small
cell carcinoma and merkel cell carcinoma ) – CK
shows dot like (punctate) perinuclear pattern
• CK20 – esp +ve in merkel cell Ca

89. 1. CD31 – more sensitive and specific
2. CD34
3. Factor –VIII (actual Ag is vWF)
4. Ulex europaeus I
5. CD141 (thrombomodulin)
6. Fli-1
7. ERG – a new promising vascular marker which is
also positive in prostate adenocarcinoma
8. D2-40 (podoplanin) – novel marker for lymphatic
endothelial cells
MARKERS OF VASCULAR
DIFFERENTIATION

90. KEY APPLICATIONS OF VASCULAR MARKERS
a. To identify vascular nature of Poorly differentiated
neoplasms – angiosarcoma, epithelioid
hemangioendothelioma, hemangiopericytoma, kaposi
sarcoma
b. To highlight vessels to help identify LV invasion in
tumors
CD34+ve non vascular tumors –
a. Solitary fibrous tumor
b. DFSP
c. GIST
d. Epithelioid sarcoma
e. Nerve sheath tumors
f. Granulocytic sarcoma

92. APPLICATION OF IHC IN ROUTINE
SETTINGS
DIAGNOSIS OF TUMORS
PROGNOSTIC MARKER
PREDICTIVE OR THERANOSTIC MARKERS
IDENTIFICATION OF INFECTIOUS ORGANISMS

93. DIAGNOSIS OF
TUMORS
1. Maximum utility of IHC is in distinguishing
carcinoma from lymphoma, sarcoma and melanoma
2. Workup of hematolymphoid neoplasms
3. Metastatic carcinoma of unknown primary (CUP)
4. Soft tissue neoplasms – 4 common diagnostic setting
a. Small round cell tumors
b. Monomorphic spindle cell tumors
c. Epithelioid soft tissue tumors
d. Pleomorphic spindle cell tumors

94. 5. In bone – to differentiate primary from metastatic
non –osseous tumors
6. CNS tumors
7. Germ cell tumors

95. 1. Loss of myoepithelial or basal cells or
basement membrane/collagen type IV – these
allow assessment of microinvasion
2. Endothelial markers – assist in identification of
lymphovascular spaces to ascertain tumor
embolism
3. ER, PR and her2/neu
4. Ki-67 /MIB-1 – proliferation markers
PROGNOSTIC
MARKERS

96. PREDICTIVE OR
THERANOSTIC
MARKERS
1. ER/PR – tamoxifen in Ca. breast
2. Her 2 – herceptin in breast cancer
3. C-kit – gleevac/imatinib in GIST, CML
4. CD20 – rituximab in B-cell NHL
5. EGFR – erlotinib in lung cancer

97. IDENTIFICATION OF
INFECTIOUS
ORGANISMS
1. Viruses – HSV, CMV, EBV
2. Others – toxoplasma, pneumocystis

98. DIAGNOSIS OF TUMORS

99. In distinguishing carcinoma from
lymphoma, sarcoma and melanoma

100. METASTATIC CARCINOMA OF
UNKNOWN PRIMARY (CUP)

106. Soft tissue neoplasms –
a. Small round cell tumors
b. Spindle cell tumors
c. Epithelioid soft tissue tumors

109. EPITHELIOID SOFT TISSUE
TUMORS

110. GERM CELL TUMORS

111. IMMUNOPHENOTYPIC ALGORITHM FOR CLASSIFICATION
OF THE MAJOR MATURE B-CELL LYMPHOID NEOPLASMS.

112. Classic HL NLPHL
LCA -ve +ve
CD30 +ve -ve
CD15 +ve -ve
CD20 -ve +ve
EMA -ve -/+
HODGIN LYMPHOMA –
BASIC PANEL

113. PROGNOSTIC MARKERS

114. MARKERS ON ASSESSMENT
OF INVASION
1. Collagen type IV – component of basement
membrane
2. Basal cells and myoepithelial cells – in prostate
and breast carcinoma respectively
• These are absent in invasive carcinomas
3. Racemase (AMACR) – for prostate carcinoma
Exp in malignant acinar cells but is –ve in benign
acinar cells

116. ER
PR
HER 2/neu

117. Interpretation
*IHC results
(% of tumor cells with nuclear
staining)
**Control
Positive 1% or more -
Negative < 1% Positive
Uninterpretable No tumor cells show nuclear staining No staining
•% of tumor cells with nuclear staining, and staining intensity should be provided.
Allred score should be provided.
** Nuclear staining of internal control (benign ductal epithelium) or external control
JCO, 2010: ASCO/CAP guidelines for ER PR Testing
How much is needed for a
Positive Result

118. Allred Scoring for ER and PR – Guidelines
>0 to 1% >1 to 10% >10 to 33% >33 to 67% >67 to 100%
Modified from: Allred, Mod Pathology, 1998

119. Recent ASCO/CAP HER2 Testing Guideline

120. RECENT ADVANCES FUTURE
DIRECTIONS
1. Genogenic IHC for diagnosis
2. Sequential double staining method
3. Develop better monoclonal Abs with recombinant
technology
4. Technician free automation of IHC procedures
5. “Pathologist-free” microscope image analysis
technology for interpretation of IHC

121. GENOGENIC IHC
Identification of underlying molecular changes by IHC
–
1. Markers to monitor drug resistance –
P-glycoprotein which is the product of mdr (multidrug
resistance) gene
2. BRCA-1 gene
3. DNA repair genes (microsatellite instability)
4. Loss of E-cadherin

122. 5. ALK overexpression to recognise t(2;5) in ALCL
6. FLI-1 overexpression for t(11;22) in ES
7. WT-1 overexpression for t(11;22) in DSRCT

123. SEQUENTIAL DOUBLE
STAINING TECHNIQUE
•The procedure involves sequential application of 2
staining systems
•To demonstrate more than 1 Ag in a single section
•Must produce contrasting colors to be effective
•Eg -In 1st staining system – secondary Ab may be
conjugated with HRP and AEC as substrate
•In 2nd system – secondary Ab may be conjugated with
Alkaline phosphatase and fast blue as substrate

124. REFERENCES –
1.Diagnostic immunohistochemistry – Dabbs
2.Enzinger and Weiss
3.Handbook of surgical pathology
4.Handbook of practical histochemistry
5.Immunohistochemistry in surgical pathology
practice : nirmala jambhekar