Appy Akshay Agarwal, profile picture
Uploaded byAppy Akshay Agarwal
PPT, PDF1,729 views

Ihc no dread

This document provides an overview of immunohistochemistry (IHC), including its definition, principle, techniques, and applications. IHC allows for the in situ detection of antigens in tissues through antigen-antibody recognition, with antibodies tagged using visible labels since they cannot be seen microscopically. The document discusses sample preparation, various detection and labeling methods like direct conjugate, indirect conjugate, avidin-biotin, amplification techniques, blocking nonspecific binding, and assessment and reporting of IHC reactions. It also covers common IHC markers for different tissues and cell types.

Embed presentation

Downloaded 87 times
1 / 119
2 / 119
Most read
3 / 119
Most read
4 / 119
5 / 119
6 / 119
7 / 119
8 / 119
9 / 119
10 / 119
11 / 119
12 / 119
Most read
13 / 119
14 / 119
15 / 119
16 / 119
17 / 119
18 / 119
19 / 119
20 / 119
21 / 119
22 / 119
23 / 119
24 / 119
25 / 119
26 / 119
27 / 119
28 / 119
29 / 119
30 / 119
31 / 119
32 / 119
33 / 119
34 / 119
35 / 119
36 / 119
37 / 119
38 / 119
39 / 119
40 / 119
41 / 119
42 / 119
43 / 119
44 / 119
45 / 119
46 / 119
47 / 119
48 / 119
49 / 119
50 / 119
51 / 119
52 / 119
53 / 119
54 / 119
55 / 119
56 / 119
57 / 119
58 / 119
59 / 119
60 / 119
61 / 119
62 / 119
63 / 119
64 / 119
65 / 119
66 / 119
67 / 119
68 / 119
69 / 119
70 / 119
71 / 119
72 / 119
73 / 119
74 / 119
75 / 119
76 / 119
77 / 119
78 / 119
79 / 119
80 / 119
81 / 119
82 / 119
83 / 119
84 / 119
85 / 119
86 / 119
87 / 119
88 / 119
89 / 119
90 / 119
91 / 119
92 / 119
93 / 119
94 / 119
95 / 119
96 / 119
97 / 119
98 / 119
99 / 119
100 / 119
101 / 119
102 / 119
103 / 119
104 / 119
105 / 119
106 / 119
107 / 119
108 / 119
109 / 119
110 / 119
111 / 119
112 / 119
113 / 119
114 / 119
115 / 119
116 / 119
117 / 119
118 / 119
119 / 119
IMMUNOHISTOCHEMISTRY IN HISTOLOGICAL DIAGNOSIS Dr. Sapana Bhandari JR II Pathology Dr. V.M.G.M.C. Solapur
INDEX • DEFINITION • PRINCIPLE • TECHNIQUE • CLASSIFICATION OF MARKERS • APPLICATION IN NEOPLASTIC CONDITIONS. • APPLICATION IN INFECTIOUS DIS.
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
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.
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
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.
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
Principle of cross linking fixatives & Antigen Retrieval Deleterious effects of formaldehyde countered by Antigen retrieval in attempts to retrieve/unmask target Ag.
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
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)
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
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
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
Dr.Sonal AgrawalDr.Sonal Agrawal Peroxidase Antiperoxidase MethodPeroxidase Antiperoxidase Method
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
Dr.Sonal AgrawalDr.Sonal Agrawal Biotin Avidin ProcedureBiotin Avidin Procedure
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
Dr.Sonal AgrawalDr.Sonal Agrawal Indirect Biotin Avidin Method Avidin Biotin Conjugate Method
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
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
Dr.Sonal AgrawalDr.Sonal Agrawal DAKO EnVision polymeric method Mirror Image Complementary Antibody labeling method (MICA)
Dr.Sonal AgrawalDr.Sonal Agrawal Enzyme Bridge Method labeled Antigen Method Protein A –PAP Method APAAP Method
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)
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
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 ))
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
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
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
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
Immunohistochemical markers
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
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
Vimentin Intermediate fil. Mesenchymal tissue Endothelial cells, Fibroblast, vascular smooth muscle Mesenchymal tumors Renal cell carcinoma Mesenchymal markers
Muscle Specific Markers
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
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
Muscle markers Actin in myoepithelial cells Myoglobin in MMMT
Melanocytic Markers
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
S100- Both nuclear & cytoplasmic staining Chondrosarcoma Malignant melanoma
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
HMB 45 staining Isolated melanoma cells in sentinel lymph node- HMB 45
Neuroendocrine Markers
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
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
Endothelial Markers
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
Factor VIII related Antigen epithelioid vascular tumor. Kaposi sarcoma
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.
Ulex Europaeus I agglutinin Vascular invasion of breast ca
Neuronal Markers
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
GFAP in malignant astrocytes. NF-Merkel cell Ca Chromogranin, NF, S100 in olfactory neuroblastoma
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.
CD99 -membrane reactivity in Ewings/PNET
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.
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.
Diagnosis of malignant soft tissue round cell tumors
Sarcomas with spindle appearance • Synovial sarcoma • Sarcomatoid carcinoma • Fibrosarcoma, Fibromatosis • Angiosarcoma • MPNST, Cellular Schwannoma • MFH • Leiomyosarcoma, Spindle cell RMS • Melanoma • Clear cell sarcoma
Diagnosis of sarcomas with spindle appearance
Malignant soft tissue epithelioid tumors • Alveolar soft part sarcoma • Angiosarcoma • Leiomyosarcoma, GIST • MFH • Melanoma • Synovial Sarcoma • Malignant Mesothelioma • Malignant Rhabdoid Tumor
Diagnosis of malignant soft tissue epithelioid tumors
Malignant soft tissue pleomorphic tumors • MFH • Pleomorphic liposarcoma • Pleomorphic RMS • Pleomorphic MPNST • Pleomorphic Leiomyosarcoma.
Diagnosis of Malignant soft tissue pleomorphic tumors
Epithelial Markers
Cytokeratin
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.
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
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
HCG +ve in seminoma with trophoblastic giant cells. PLAP +ve in tumor cells of intratubular germ cell neoplasia. HCG
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.
Canalicular formation in HCC -demo. By CEA, which are diff. From sinusoids of normal liver. Luminal staining in glandular structures of adenosquamous carcinoma. CEA
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.
PSA Formed exclusively by prostatic epithelial cells. Prostatic tumors, BHP, Salivary, breast, bladder adenoca. Intensityœ diff. PSMA Highly specific BHP, Prostatic tumorsIntensity œ1/ diff.
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
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)
Calretinin in mesothelioma- nuclear & cytoplasmic reactivity. Reactive mesothelial cells CK5/6 Mesothelioma
CK7 CK20 Transitional cell carcinoma - Bladder
Paget’s disease CK7 CEA GCDFP
Pediatric Neoplasms
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
Desmoplastic small round cell tumour Keratin Desmin NSE
Diagnosis of head & neck endocrine tumors.
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
Medullary carcinoma of thyroid. Calcitonin CGN CEA
Applications in carcinoma of breast
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
ER/PR semiquantitation
ER/PR semiquantitation
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
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
III. To differentiate b/w in situ & invasive ductal Ca SMA AE 1/AE3 IV. To detect metastasis sentinel lymph node.
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.
Immunoreactivity for CD117 in GIST CD117- in seminoma
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
Hyperplastic polyp Proliferating cells-MIB1+ve Cells restricted to basal part only. Tubular adenoma Proliferating epithelium also at luminal surface.
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).
Follicular Lymphoma CD20 in neoplastic cells CD 3 in non neoplastic T cells CD21 in dendritic follicular cells
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
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.
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.
Large T cell lymphoma CD43 +ve Lysozyme +ve of reactive histiocytes
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
ALK +ve in anaplastic large cell lymphoma CD30 in Anaplastic large cell lymphoma
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
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
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
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
CD30 in HL EBV/ LMP1 Ag in HL
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.
HbSAg localized in variable quantity in cytoplasm & cell membrane of hepatocytes. Positive cytoplasmic granules in every periportal hepatocytes.
H-pylori in superficial mucus in chr gastritis
Advantages- • Can be done on paraffin embedded tissue • Allows microbiologic & morphologic correlation • Provides diagnosis when fresh tissue not available. • Sensitive & specific.
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.
Facts About IHC • Negative immunoreaction in IHC never rules out a diagnosis. • Key is- to utilise IHC as cost effective tool in patient care.
p-53

More Related Content

PPTX
IHC in cancer diagnosis
byzubiya4
 
PPTX
Immunohistochemistry of Prostatic lesions
byAshish Jawarkar
 
PPT
Stratified Medicine in Cancer: The Role of Histopathologist
byDr. Shubhi Saxena
 
PPTX
Ihc
bydamuluri ramu
 
PPTX
Role of ihc
byvellanki sudharshan
 
PPTX
Immunohistochemistry in breast lesions
byAshish Jawarkar
 
PPT
Immunohistochemistry
byAtifa Ambreen
 
PPT
Squash cytology of cns paediatric tumours
bySumanth Deva
 
IHC in cancer diagnosis
byzubiya4
 
Immunohistochemistry of Prostatic lesions
byAshish Jawarkar
 
Stratified Medicine in Cancer: The Role of Histopathologist
byDr. Shubhi Saxena
 
Ihc
bydamuluri ramu
 
Role of ihc
byvellanki sudharshan
 
Immunohistochemistry in breast lesions
byAshish Jawarkar
 
Immunohistochemistry
byAtifa Ambreen
 
Squash cytology of cns paediatric tumours
bySumanth Deva
 

What's hot

PPT
Immunohistochemistry in pathology laboratory
byAppy Akshay Agarwal
 
PPTX
Immunohistochemistry (IHC)
byJessica Aimee White
 
PPTX
Immunohistochemistry
byShankar Bastakoti
 
PPTX
Immunohistochemistry METHODS
byK BHATTACHARJEE
 
PPT
Immunohistochemistry
byOmer ballal
 
PPT
Immunohistochemistry
byAppy Akshay Agarwal
 
PPT
Immunohistochemistry
byAtifa Ambreen
 
PPTX
Special stains in histopathology
byEkta Jajodia
 
PDF
Basics of Immunohistochemistry (IHC)
byBioGenex
 
PPTX
Soft tissue tumor
byNarmada Tiwari
 
PPTX
Special stains
byimrana tanvir
 
PPT
Silver Staining
byDr. Varughese George
 
PPTX
An Introduction to Immunohistochemistry
byCreative-Bioarray
 
PPTX
Fungus in histopathology
byAppy Akshay Agarwal
 
PPTX
principle of Immunohistochemistry and its use in diagnostics
byEkta Jajodia
 
PPTX
immunohistochemistry
bySnehadurugkar
 
PDF
Immunohistochemistry
byJaandeepCheema
 
PPTX
Cytology of bone lesions
bySri Devraj Urs medical college, Kolar, Karnataka , India
 
PPTX
Fnac breast
byDr Neha Mahajan
 
PPTX
Immunohistochemistry in diagnosis of soft tissue tumours seminar
byPannaga Kumar
 
Immunohistochemistry in pathology laboratory
byAppy Akshay Agarwal
 
Immunohistochemistry (IHC)
byJessica Aimee White
 
Immunohistochemistry
byShankar Bastakoti
 
Immunohistochemistry METHODS
byK BHATTACHARJEE
 
Immunohistochemistry
byOmer ballal
 
Immunohistochemistry
byAppy Akshay Agarwal
 
Immunohistochemistry
byAtifa Ambreen
 
Special stains in histopathology
byEkta Jajodia
 
Basics of Immunohistochemistry (IHC)
byBioGenex
 
Soft tissue tumor
byNarmada Tiwari
 
Special stains
byimrana tanvir
 
Silver Staining
byDr. Varughese George
 
An Introduction to Immunohistochemistry
byCreative-Bioarray
 
Fungus in histopathology
byAppy Akshay Agarwal
 
principle of Immunohistochemistry and its use in diagnostics
byEkta Jajodia
 
immunohistochemistry
bySnehadurugkar
 
Immunohistochemistry
byJaandeepCheema
 
Cytology of bone lesions
bySri Devraj Urs medical college, Kolar, Karnataka , India
 
Fnac breast
byDr Neha Mahajan
 
Immunohistochemistry in diagnosis of soft tissue tumours seminar
byPannaga Kumar
 

Similar to Ihc no dread

PPTX
IMMUNOHISTOCHEMISTRY & Steps in IHC in detail
bysoniaakash6572
 
PPTX
subas ihc 1.pptx
byAmadeus Mee
 
PPTX
Basics of Immunohistochemistry
byDr. Devika Kollara
 
PPTX
11. ihc IHC makes it possible to visualize the distribution and localization...
byshilpa939953
 
PPTX
IHC interpretation- and its quality control .pptx
byDeepshikhaSinghmar
 
PDF
hdjwkihsnsjsjjshsbshishwbwnwjwijwbsbsjsijsje
byAbigailBagay1
 
PPT
Immunohistochemistry
byGanesh Aderao
 
PPTX
Immunohistochemistry
byShahzebHUSSAIN5
 
PPT
ABC method of IHC
byAtifa Ambreen
 
PPTX
Immunohistochemistry, the basics and applications.pptx
byAmirRaziq1
 
PPTX
9_Dr Poonam Panjwani Basic principles of IHC.pptx
byaditisikarwar2
 
PDF
AN OVERVIEW OF IMMUNOHISTOCHEMISTRY(IHC)
byBiswajit Das
 
PPT
lisa.Final NSH IHC Part 2
byLisa Ryan
 
PPTX
Immunohistochemistry description of the fluorescence mehodes and enzymetic m...
byHadeelAlboaklah
 
PPTX
Lectures slide for the HISTOCHEMISTRY.pptx
byAbdulwahabShehu1
 
PPTX
IMMUNOHISTOCHEMISTRY TECHNIQUES-converted.pptx
byaamanimutakani
 
PPTX
GROUP 9 HISTOPATHOLOGY coursework on INDIRECT IMMUNOHISTOCHEMISTRY
byNuwemuramuziIanMarti
 
PPTX
Immunohistochemistry
byCreative-Bioarray
 
PPTX
Immunohistochemistry
byCreative-Bioarray
 
PPT
Overview of Immunohistochemistry.ppt
bykmsave
 
IMMUNOHISTOCHEMISTRY & Steps in IHC in detail
bysoniaakash6572
 
subas ihc 1.pptx
byAmadeus Mee
 
Basics of Immunohistochemistry
byDr. Devika Kollara
 
11. ihc IHC makes it possible to visualize the distribution and localization...
byshilpa939953
 
IHC interpretation- and its quality control .pptx
byDeepshikhaSinghmar
 
hdjwkihsnsjsjjshsbshishwbwnwjwijwbsbsjsijsje
byAbigailBagay1
 
Immunohistochemistry
byGanesh Aderao
 
Immunohistochemistry
byShahzebHUSSAIN5
 
ABC method of IHC
byAtifa Ambreen
 
Immunohistochemistry, the basics and applications.pptx
byAmirRaziq1
 
9_Dr Poonam Panjwani Basic principles of IHC.pptx
byaditisikarwar2
 
AN OVERVIEW OF IMMUNOHISTOCHEMISTRY(IHC)
byBiswajit Das
 
lisa.Final NSH IHC Part 2
byLisa Ryan
 
Immunohistochemistry description of the fluorescence mehodes and enzymetic m...
byHadeelAlboaklah
 
Lectures slide for the HISTOCHEMISTRY.pptx
byAbdulwahabShehu1
 
IMMUNOHISTOCHEMISTRY TECHNIQUES-converted.pptx
byaamanimutakani
 
GROUP 9 HISTOPATHOLOGY coursework on INDIRECT IMMUNOHISTOCHEMISTRY
byNuwemuramuziIanMarti
 
Immunohistochemistry
byCreative-Bioarray
 
Immunohistochemistry
byCreative-Bioarray
 
Overview of Immunohistochemistry.ppt
bykmsave
 

More from Appy Akshay Agarwal

PPTX
Urine analysis in pathology clinical
byAppy Akshay Agarwal
 
PPTX
Pre leukemia MDS
byAppy Akshay Agarwal
 
PPTX
IgG4 related disorders of the eye
byAppy Akshay Agarwal
 
PPT
Necrosis & gangrene pathology calcification
byAppy Akshay Agarwal
 
PPT
Hypersensitivity reaction pathology microbiology immunity
byAppy Akshay Agarwal
 
PPTX
Histotechnique for practicals pathology
byAppy Akshay Agarwal
 
PPT
Fungal infections in histopathology
byAppy Akshay Agarwal
 
PPT
Diseases of blood vessels
byAppy Akshay Agarwal
 
PPTX
Coagulation profile and its uses
byAppy Akshay Agarwal
 
PPT
Cerebrovascular disease pathology stroke
byAppy Akshay Agarwal
 
PPTX
Blood products separation and quality control
byAppy Akshay Agarwal
 
PPTX
Blood component preparation blood banking
byAppy Akshay Agarwal
 
PPTX
Blood coagulation and physiology
byAppy Akshay Agarwal
 
PPTX
Benign inclusions in lymph nodes histopathology
byAppy Akshay Agarwal
 
PPTX
Barrett oesophagus journal club pathology
byAppy Akshay Agarwal
 
PPTX
Atherosclerosis pathology mbbs
byAppy Akshay Agarwal
 
PPTX
Antibody mediated rejection pathology histopathology
byAppy Akshay Agarwal
 
PPT
introduction of cytopathology
byAppy Akshay Agarwal
 
PPT
Metastasis pg activity
byAppy Akshay Agarwal
 
PPT
Embryonal brain tumours in children
byAppy Akshay Agarwal
 
Urine analysis in pathology clinical
byAppy Akshay Agarwal
 
Pre leukemia MDS
byAppy Akshay Agarwal
 
IgG4 related disorders of the eye
byAppy Akshay Agarwal
 
Necrosis & gangrene pathology calcification
byAppy Akshay Agarwal
 
Hypersensitivity reaction pathology microbiology immunity
byAppy Akshay Agarwal
 
Histotechnique for practicals pathology
byAppy Akshay Agarwal
 
Fungal infections in histopathology
byAppy Akshay Agarwal
 
Diseases of blood vessels
byAppy Akshay Agarwal
 
Coagulation profile and its uses
byAppy Akshay Agarwal
 
Cerebrovascular disease pathology stroke
byAppy Akshay Agarwal
 
Blood products separation and quality control
byAppy Akshay Agarwal
 
Blood component preparation blood banking
byAppy Akshay Agarwal
 
Blood coagulation and physiology
byAppy Akshay Agarwal
 
Benign inclusions in lymph nodes histopathology
byAppy Akshay Agarwal
 
Barrett oesophagus journal club pathology
byAppy Akshay Agarwal
 
Atherosclerosis pathology mbbs
byAppy Akshay Agarwal
 
Antibody mediated rejection pathology histopathology
byAppy Akshay Agarwal
 
introduction of cytopathology
byAppy Akshay Agarwal
 
Metastasis pg activity
byAppy Akshay Agarwal
 
Embryonal brain tumours in children
byAppy Akshay Agarwal
 

Recently uploaded

PPTX
Mental Health Awareness Campaign 2025 PPT.PPTX
byBirendra Bhatt
 
PPTX
Understanding Autoimmune Endocrine Diseases
byArchitMehta11
 
PDF
Regal Best Kidney Multispecialty Hospital
bysekhardigitalmarketi
 
PPTX
OBSTRUCTED LABOR - Edward Mwansa.LGH.pptx
byedwardmwansa4
 
PPTX
"SUPER FLU": TRUTH VS HYPE - WHAT CLINICIANS MUST KNOW
byArchitMehta11
 
PPTX
Unit- II (Bioavaliability & Bioequivalence).pptx
byUditiHanda
 
PPTX
Spirituality and Mental Health - therapy perspectives and formulation pptx
bySaju Pj
 
PPTX
Drug Distribution Explained | Pharmacokinetics, Barriers, Vd & Redistribution...
byWasim Akram Rajoor
 
PPTX
case control study for Community Medicine.pptx
byDr.Bharti Koria
 
PPT
Elevating NMIBC Care: Putting the Latest Innovations for Intravesical Therapi...
byPVI, PeerView Institute for Medical Education
 
PPTX
SPS_PE_LAB 2_Liquid Operation-Centrifugation.pptx
bysupriyabhagwatsps
 
PPTX
EYELID TUMOURS . CLINICAL FEATURES . DIAGNOSIS . SURGICAL MANAGEMENT
byDr Aarushi Choudhary
 
PPT
RSV Prophylaxis Unveiled: What Healthcare Providers Need to Know
byPVI, PeerView Institute for Medical Education
 
PPTX
Historical background and development of profession of pharmacy pptx
byGulam Muheyuddeen
 
PPTX
Types of Communication Disorders SLP lecture 3.pptx
byDr Zonera Khalid PT
 
PPTX
Seminar on Ebola virus , سیمینارێکی ئامادەکراو لەسەر Ebola virus
bydanaShalal2
 
PPT
New Kids on the Block in mHSPC: Personalising Approaches Through Emerging Pre...
byPVI, PeerView Institute for Medical Education
 
PPTX
WCPCCS 2025 Intelligent Digital Agents Final.pptx
byJAI NAHAR, MD MBA
 
PPTX
Transport of Oxygen& CO2.pptx by dr ambareesha
byDr K Ambareesha Goud PhD
 
PPTX
Imaging of benign focal lesions of the Liver
byDr. Aryan (Anish Dhakal)
 
Mental Health Awareness Campaign 2025 PPT.PPTX
byBirendra Bhatt
 
Understanding Autoimmune Endocrine Diseases
byArchitMehta11
 
Regal Best Kidney Multispecialty Hospital
bysekhardigitalmarketi
 
OBSTRUCTED LABOR - Edward Mwansa.LGH.pptx
byedwardmwansa4
 
"SUPER FLU": TRUTH VS HYPE - WHAT CLINICIANS MUST KNOW
byArchitMehta11
 
Unit- II (Bioavaliability & Bioequivalence).pptx
byUditiHanda
 
Spirituality and Mental Health - therapy perspectives and formulation pptx
bySaju Pj
 
Drug Distribution Explained | Pharmacokinetics, Barriers, Vd & Redistribution...
byWasim Akram Rajoor
 
case control study for Community Medicine.pptx
byDr.Bharti Koria
 
Elevating NMIBC Care: Putting the Latest Innovations for Intravesical Therapi...
byPVI, PeerView Institute for Medical Education
 
SPS_PE_LAB 2_Liquid Operation-Centrifugation.pptx
bysupriyabhagwatsps
 
EYELID TUMOURS . CLINICAL FEATURES . DIAGNOSIS . SURGICAL MANAGEMENT
byDr Aarushi Choudhary
 
RSV Prophylaxis Unveiled: What Healthcare Providers Need to Know
byPVI, PeerView Institute for Medical Education
 
Historical background and development of profession of pharmacy pptx
byGulam Muheyuddeen
 
Types of Communication Disorders SLP lecture 3.pptx
byDr Zonera Khalid PT
 
Seminar on Ebola virus , سیمینارێکی ئامادەکراو لەسەر Ebola virus
bydanaShalal2
 
New Kids on the Block in mHSPC: Personalising Approaches Through Emerging Pre...
byPVI, PeerView Institute for Medical Education
 
WCPCCS 2025 Intelligent Digital Agents Final.pptx
byJAI NAHAR, MD MBA
 
Transport of Oxygen& CO2.pptx by dr ambareesha
byDr K Ambareesha Goud PhD
 
Imaging of benign focal lesions of the Liver
byDr. Aryan (Anish Dhakal)
 

Ihc no dread

  • 1.
    IMMUNOHISTOCHEMISTRY IN HISTOLOGICAL DIAGNOSIS Dr. SapanaBhandari 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- Themethod 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 • Thebasic 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 SAMPLESFOR 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 crosslinking 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 DirectConjugate 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 Indirector 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 PeroxidaseAntiperoxidase 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 PeroxidaseAntiperoxidase MethodPeroxidase Antiperoxidase Method
  • 15.
    Dr.Sonal AgrawalDr.Sonal Agrawal BiotinAvidin 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 BiotinAvidin ProcedureBiotin Avidin Procedure
  • 17.
    Dr.Sonal AgrawalDr.Sonal Agrawal AvidinBiotin 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 IndirectBiotin Avidin Method Avidin Biotin Conjugate Method
  • 19.
    Dr.Sonal AgrawalDr.Sonal Agrawal DoubleStaining 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 EnhancedPolymer 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 DAKOEnVision polymeric method Mirror Image Complementary Antibody labeling method (MICA)
  • 22.
    Dr.Sonal AgrawalDr.Sonal Agrawal EnzymeBridge 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 Fixationfor 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 IHCREACTION 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.
  • 31.
    Common Panels ofIHC 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 Endothelialcells, Fibroblast, vascular smooth muscle Mesenchymal tumors Renal cell carcinoma Mesenchymal markers
  • 34.
  • 35.
    Desmin Intermediate filament Smooth muscle- cytoplasmic dense bodies Striatedmuscle- 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 inmyoepithelial cells Myoglobin in MMMT
  • 38.
  • 39.
    S100 Intracellular Ca binding protein, Solublein 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, cellularnevi, 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 Isolatedmelanoma cells in sentinel lymph node- HMB 45
  • 43.
  • 44.
    Neuron specific Enolase (NSE) Gammagamma 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 Neuroendocrinaltumors Chromo. A-Gastric & appendiceal carcinoid Chrom. B-Rectal carcinoid, prolactinoma To diff b/w adrenal cortical Ca & pheochromocytoma Carcinoid tumour stomach Paraganglioma
  • 46.
  • 47.
    CD34 Hematopoietic stem cell Ag Endothelialcells, 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 relatedAntigen epithelioid vascular tumor. Kaposi sarcoma
  • 49.
    Ulex Europaeus I agglutinin Lectin producedby a gorse plant Vascular neoplasm. Thrombo- modulin Endothelial cells, mesothelial cells, osteoblasts, monocytes Sensitive in poorly differentiated vascular malignancies.
  • 50.
    Ulex Europaeus Iagglutinin Vascular invasion of breast ca
  • 51.
  • 52.
    Neurofilament Protein Neuronal cells Small roundcell tumors Neuroblastoma Medulloblastoma retinoblastoma Peripheral neuroepithelioma Merkel cell tumors Carcinoid Parathyroid tumors GFAP Intermediate fil. Astrocytes Ependymal cells CNS tumors Schwannoma
  • 53.
    GFAP in malignantastrocytes. NF-Merkel cell Ca Chromogranin, NF, S100 in olfactory neuroblastoma
  • 54.
    CD99- •Transmembrane Protein •Encoded byMIC- 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.
  • 56.
    Collagen type IV Predominant component of basementmembrane 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 tissueround 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 malignantsoft tissue round cell tumors
  • 59.
    Sarcomas with spindleappearance • Synovial sarcoma • Sarcomatoid carcinoma • Fibrosarcoma, Fibromatosis • Angiosarcoma • MPNST, Cellular Schwannoma • MFH • Leiomyosarcoma, Spindle cell RMS • Melanoma • Clear cell sarcoma
  • 60.
    Diagnosis of sarcomaswith spindle appearance
  • 61.
    Malignant soft tissueepithelioid tumors • Alveolar soft part sarcoma • Angiosarcoma • Leiomyosarcoma, GIST • MFH • Melanoma • Synovial Sarcoma • Malignant Mesothelioma • Malignant Rhabdoid Tumor
  • 62.
    Diagnosis of malignantsoft tissue epithelioid tumors
  • 63.
    Malignant soft tissuepleomorphic tumors • MFH • Pleomorphic liposarcoma • Pleomorphic RMS • Pleomorphic MPNST • Pleomorphic Leiomyosarcoma.
  • 64.
    Diagnosis of Malignantsoft tissue pleomorphic tumors
  • 65.
  • 66.
  • 68.
    EMA Epithelial membrane antigen Glycoprotein Apical surface of most glandular epithelialcells. 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 inseminoma with trophoblastic giant cells. PLAP +ve in tumor cells of intratubular germ cell neoplasia. HCG
  • 72.
    CEA- +ve incolorectal 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 exclusivelyby 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, cellswith 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.
  • 81.
  • 82.
  • 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 roundcell 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 ofthyroid. Calcitonin CGN CEA
  • 88.
  • 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.
  • 91.
  • 92.
    Her-2/ neu • Memberof 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 • Calciumdependent 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 differentiateb/w in situ & invasive ductal Ca SMA AE 1/AE3 IV. To detect metastasis sentinel lymph node.
  • 95.
    CD 117- Encoded byproto-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 inGIST CD117- in seminoma
  • 97.
    Ki- 67/ MIB1- 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 Cellsrestricted 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 inneoplastic cells CD 3 in non neoplastic T cells CD21 in dendritic follicular cells
  • 101.
    CD 23- +vein 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- +vein 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 celllymphoma CD43 +ve Lysozyme +ve of reactive histiocytes
  • 105.
    ALK +ve inALCL (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 inanaplastic large cell lymphoma CD30 in Anaplastic large cell lymphoma
  • 107.
    Bcl 6- Nucleusof 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 Mantlecell 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 XAg 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 CD20 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 Infectiousdiseases • 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 invariable quantity in cytoplasm & cell membrane of hepatocytes. Positive cytoplasmic granules in every periportal hepatocytes.
  • 114.
  • 115.
    Advantages- • Can bedone 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.