principle of Immunohistochemistry and its use in diagnostics
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Immunohistochemistry (IHC) localizes antigens in tissues based on antigen-antibody recognition. The principle is visualizing target compounds in tissues with high signal-to-noise ratio. IHC was developed in the 1960s using enzyme labels instead of fluorescent labels to visualize targets under a light microscope. Key steps in IHC include antigen retrieval to unmask antigens, blocking endogenous enzymes, primary antibody incubation, secondary antibody or polymer incubation, and signal development with chromogens. IHC is commonly performed on formalin-fixed paraffin-embedded sections and can identify cell types and localization of proteins to characterize tissues.
principle of Immunohistochemistry and its use 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
- 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)
- 48. •Secondary Ab or a polymer is attached to a label •It is produced against the primary antigen
- 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
- 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
- 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
- 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
- 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
Editor's Notes
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