PRINCIPLES OF IMMUNOHISTOCHEMISTRY AND ITS USE

IN DIAGNOSTICS

EKTA JAJODIA

INTRODUCTIONIHC 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

HISTORY OF IMMUNOHISTOCHEMISTRY

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

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

1981 – developed avidin-biotin labeling

1991 – Heat induced antigen retrieval technique in IHC was done

1995 – Polymer technology introduced

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 -

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

•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)

Formalin fixed paraffin embedded sections

Frozen sectionsSmearsImprints

Cytospins

IHC CAN BE PERFORMED ON -

METHODS

DIRECT conjugate labeled antibody

procedure

•One step staining method•Labeled Ab reacts directly with Ag in tissue

INDIRECT/SANDWICH PROCEDURE

•Unlabeled primary Ab reacts with tissue Ag•Conjugated second Ab reacts against primary Ab

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

IHC PROTOCOL

Fixation and processing

Section cutting

Deparaffinisation and rehydration

Blocking endogenous peroxidase

Blocking non-specific antibody binding

Antigen retrieval

Primary antibody

Secondary antibody

Chromogen

Chromogen enhancement

Counterstain

Mount

Stringent washing between reagents

IHC PROTOCOL

Fixation and processing

Section cutting

Deparaffinisation and rehydration

•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

•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

PROCESSING IN SPECIAL SITUATIONS

•Some Abs act only on frozen sections•Frozen sections s/b fixed with acetone before storing

•FNAC , blood smears, etc – either1. Centrifuge and make a pellet that is fixed just as in

tissue fixation, or2. A fresh smear may be made and fixed in

acetone/alcohol/10% NBF

IHC PROTOCOL

Fixation and processing

Section cutting

Deparaffinisation and rehydration

• 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)

IHC PROTOCOL

Fixation and processing

Section cutting

Deparaffinisation and rehydration

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

Blocking endogenous enzyme

Blocking non-specific antibody binding

Antigen retreival

Primary antibody

•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

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

ENDOGENOUS ALKALINE PHOSPHATASE ACTIVITY

1. Levamisole

2. 20% glacial acetic acid – better blocker

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

Blocking endogenous peroxidase

Blocking non-specific antibody binding

Antigen retrieval

Primary antibody

•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

Blocking endogenous peroxidase

Blocking non-specific antibody binding

Antigen retrieval

Primary antibody

UNMASKING OF ANTIGEN SITESWhen 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

The degree of masking of antigens depends on –1. Length of time in fixative2. Temperature3. 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

MANUAL METHODS FOR ANTIGEN RETREIVAL INCLUDE – 1. Proteolytic enzyme digestion2. Microwave oven radiation3. Combined enzyme and microwave4. Pressure cooker heating5. Decloaker heating6. Pressure cooker inside a microwave oven7. Autoclave heating8. Water bath heating9. Steamer heating

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

Factors responsible are –

1. Duration of enzyme digestion2. Enzyme concentration3. Use of coenzyme – such as calcium chloride with

trypsin4. Temperature5. pH

HEAT INDUCED ANTIGEN RETREIVAL (HIER)

MICROWAVE ANTIGEN RETREIVAL

PRESSURE COOKER ANTIGEN RETREIVAL

STEAMER

WATER BATH

AUTOCLAVE

Most popular antigen retreival solutions are –

1. 0.01M citrate buffer at pH 62. 0.1mM EDTA at pH 83. 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

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

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

Blocking endogenous peroxidase

Blocking non-specific antibody binding

Antigen retreival

Primary antibody

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

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

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

Secondary antibody

Chromogen

Chromogen enhancement

Counterstain

Mount

•Secondary Ab or a polymer is attached to a label

•It is produced against the primary antigen

LABELS

ENZYME LABELS

COLLOIDAL METAL LABELS FLUORESCENT LABELS

RADIOLABELS

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)

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

CHROMOGENS

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

ALKALINE PHOSPHATASE

CHROMOGENS

Colloidal metal labels

1. Colloidal gold conjugate – gives pink color 2. Silver – gives yellow color3. ferritin

FLUORESCENT LABELS

Fluorochromes used are –

1. FITC – green2. TRITC (rhodamine) – red3. Texas red - red4. R- phytoerythrin(PE) – orange/red

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

WASHING

•Washing should be done after each step

•By phosphate buffered saline (PBS)

GENERATION OF IHC RESULTS

PRE-ANALYTICAL FACTORS – it includes 1. Time taken to remove the tissue at surgery2. The ensuing ischemia3. Interval between surgical resection and

fixation4. Type of fixative5. Length of fixative

ANALYTICAL FACTORS –It pertains to lab procedures

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

Nuclear Cytoplasmic Membranous

OCT 4ER/PRMyogeninMyo D1P53P63TTF 1MIB-1

ActinAlpha fetoproteinChromograninFactor VIII related antigenDesminGFAPHep-Par 1HMB 45Melan A/MARTCK, vimentin, NFP

Her-2/neuCEACD 99CD20EMA

LOCATION ! LOCATION ! LOCATION !

•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

MARKERS OF DIFFERENTIATION

EPITHELIAL DIFFERENTIATION

MUSCLE DIFFERENTIATION

NERVE SHEATH DIFFERENTIATION

NEUROENDOCRINE AND NEUROECTODER,AL DIFFERENTIATION

MELANOCYTIC DIFFERENTIATION

VASCULAR DIFFERENTIATION

GLIAL DIFFERENTIATION

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)

•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

ANTIBODY COCKTAIL

•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

2. EMA3. CEA

4. p63 – HMWCK equivalentMarker 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

•Some CK are aberrantly expressed by mesenchymal cells and tumors – CK8, 18 and 19

•Mesenchymal neoplasms that are CK +ve are –1. Synovial sarcoma2. Epithelioid sarcoma3. Chordoma

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

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

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)

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

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

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

• 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

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

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

•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

3. MiTF – Micropthalmia transcription factorProduct 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 unlikelyOnly 2-3% melanomas are –ve for S-100

6. SOX107. PNL28. MUM1

RECENT MARERS

NEUROECTODERMAL AND NEUROENDOCRINE MARKERS1. CD99 –

• Product of MIC2 gene• Transmembrane gp• Most imp use is in diagnosis of ES/PNET –

membrane +vityOther SRCBT that are CD99 +ve–• lymphoblastic lymphomas• PD synovial sarcoma• Mesenchymal chondrosarcoma• Small cell OS• DSRCT never seen in neuroblastoma (NB)

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

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

5. NSE (neuron specific enolase)

6. CK in NE neoplasms – NE neoplasms fall in 2 categories- a. Epithelial –carcinoid, pancreatic NE tumor, small cell

carcinomab. Non epithelial/neural – pheochromocytoma,

paraganglioma , NB

Epithelial NE neoplasms – CK+veNon epithelial NE neoplasms – CK-ve

• 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

1. CD31 – more sensitive and specific2. CD343. Factor –VIII (actual Ag is vWF)4. Ulex europaeus I5. CD141 (thrombomodulin)6. Fli-17. ERG – a new promising vascular marker which is

also positive in prostate adenocarcinoma8. D2-40 (podoplanin) – novel marker for lymphatic

endothelial cells

MARKERS OF VASCULAR DIFFERENTIATION

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

APPLICATION OF IHC IN ROUTINE SETTINGS

DIAGNOSIS OF TUMORS

PROGNOSTIC MARKER

PREDICTIVE OR THERANOSTIC MARKERS

IDENTIFICATION OF INFECTIOUS ORGANISMS

DIAGNOSIS OF TUMORS

1. Maximum utility of IHC is in distinguishing carcinoma from lymphoma, sarcoma and melanoma

2. Workup of hematolymphoid neoplasms3. 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

5. In bone – to differentiate primary from metastatic non –osseous tumors

6. CNS tumors7. Germ cell tumors

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/neu4. Ki-67 /MIB-1 – proliferation markers

PROGNOSTIC MARKERS

PREDICTIVE OR THERANOSTIC MARKERS

1. ER/PR – tamoxifen in Ca. breast2. Her 2 – herceptin in breast cancer3. C-kit – gleevac/imatinib in GIST, CML4. CD20 – rituximab in B-cell NHL5. EGFR – erlotinib in lung cancer

IDENTIFICATION OF INFECTIOUS ORGANISMS

1. Viruses – HSV, CMV, EBV2. Others – toxoplasma, pneumocystis

DIAGNOSIS OF TUMORS

In distinguishing carcinoma from lymphoma, sarcoma and melanoma

METASTATIC CARCINOMA OF UNKNOWN PRIMARY (CUP)

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

EPITHELIOID SOFT TISSUE TUMORS

GERM CELL TUMORS

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

Classic HL NLPHL

LCA -ve +ve

CD30 +ve -ve

CD15 +ve -ve

CD20 -ve +ve

EMA -ve -/+

HODGIN LYMPHOMA – BASIC PANEL

PROGNOSTIC MARKERS

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 carcinomas3. Racemase (AMACR) – for prostate carcinomaExp in malignant acinar cells but is –ve in benign

acinar cells

ERPRHER 2/neu

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

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

Recent ASCO/CAP HER2 Testing Guideline

RECENT ADVANCES FUTURE DIRECTIONS

1. Genogenic IHC for diagnosis2. Sequential double staining method3. Develop better monoclonal Abs with recombinant

technology4. Technician free automation of IHC procedures5. “Pathologist-free” microscope image analysis

technology for interpretation of IHC

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) gene2. BRCA-1 gene3. DNA repair genes (microsatellite instability)4. Loss of E-cadherin

5. ALK overexpression to recognise t(2;5) in ALCL6. FLI-1 overexpression for t(11;22) in ES7. WT-1 overexpression for t(11;22) in DSRCT

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

REFERENCES –

1.Diagnostic immunohistochemistry – Dabbs2.Enzinger and Weiss3.Handbook of surgical pathology4.Handbook of practical histochemistry5.Immunohistochemistry in surgical pathology

practice : nirmala jambhekar

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