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.
Creative Bioarray introduces the tissue array technology and the procedure of making TMAs. Pre-made tissue array and custom tissue array are both provided. In addition, related services are also available.
Basics of immunohistochemistry, steps of histochemistry, primary and secondary antibody use as protein locator and different fixatives with their advantages
Immunohistochemistry (IHC) is a highly sensitive method that allows the localization of antigen within a cell or a tissue with high resolution. The method is based on the use of a primary antibody that specifically binds to its complementary antigen. The bound antibody may then be visualized by a variety of methods such as colorimetric end points.
Immunohistochemistry (IHC) is the localization of a known antigen in tissues by utilizing antibodies directed towards that (specific) antigen. In this presentation, we will introduce the procedure of IHC and the troubleshooting solutions.
This is a presentation I prepared to demonstrate my mastery of the basics of Immunohistochemistry during my first two months of employment as a Biologist at the Cell Marque Corporation. Please note, there are a few slides that appear to be dysfunctional and overlapping; this is due to the fact that these particular slides included complex animations that I designed to illustrate various scientific concepts related to the practice of Immunohistochemistry. If you wish to view this presentation in its entirety (animations included), feel free to contact me via LinkedIn and I will gladly provide you with a fully-functional version.
This immunohistochemistry presentation discusses assay principles, a general protocol and tips and hints for simplifying your staining procedures.
To view the webinar recording please visit: http://www.innovabiosciences.com/bioconjugation-and-immunoassay-webinars/immunohistochemistry-introduction.html
processing of bone marrow trephine biopsykanwalpreet15
there is no standard method for processing of bone marrow trephine biopsies. there are various fixatives and decalcifying agents . depending upon need of IHC and cytogenetics, we can decide
Creative Bioarray introduces the tissue array technology and the procedure of making TMAs. Pre-made tissue array and custom tissue array are both provided. In addition, related services are also available.
Basics of immunohistochemistry, steps of histochemistry, primary and secondary antibody use as protein locator and different fixatives with their advantages
Immunohistochemistry (IHC) is a highly sensitive method that allows the localization of antigen within a cell or a tissue with high resolution. The method is based on the use of a primary antibody that specifically binds to its complementary antigen. The bound antibody may then be visualized by a variety of methods such as colorimetric end points.
Immunohistochemistry (IHC) is the localization of a known antigen in tissues by utilizing antibodies directed towards that (specific) antigen. In this presentation, we will introduce the procedure of IHC and the troubleshooting solutions.
This is a presentation I prepared to demonstrate my mastery of the basics of Immunohistochemistry during my first two months of employment as a Biologist at the Cell Marque Corporation. Please note, there are a few slides that appear to be dysfunctional and overlapping; this is due to the fact that these particular slides included complex animations that I designed to illustrate various scientific concepts related to the practice of Immunohistochemistry. If you wish to view this presentation in its entirety (animations included), feel free to contact me via LinkedIn and I will gladly provide you with a fully-functional version.
This immunohistochemistry presentation discusses assay principles, a general protocol and tips and hints for simplifying your staining procedures.
To view the webinar recording please visit: http://www.innovabiosciences.com/bioconjugation-and-immunoassay-webinars/immunohistochemistry-introduction.html
processing of bone marrow trephine biopsykanwalpreet15
there is no standard method for processing of bone marrow trephine biopsies. there are various fixatives and decalcifying agents . depending upon need of IHC and cytogenetics, we can decide
Immunohistochemistry (IHC) is the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
https://www.creative-bioarray.com/protocol/immunohistochemistry-protocol.htm
This document describes detailed information about Radio immuno assay (RIA) including its principle, procedure, advantages, disadvantages, application etc
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
2. INDEX
• DEFINITION
• PRINCIPLE
• TECHNIQUE
• CLASSIFICATION OF MARKERS
• APPLICATION IN NEOPLASTIC
CONDITIONS.
• APPLICATION IN INFECTIOUS DIS.
3. 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
4. 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.
5. 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
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 cross linking 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
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
12. 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
13. 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
15. 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
17. 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
19. 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
20. 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
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
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
27. 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
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
44. 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
53. GFAP in malignant astrocytes. NF-Merkel cell Ca
Chromogranin, NF, S100 in olfactory neuroblastoma
54. 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.
56. 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.
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 in seminoma
with trophoblastic giant
cells.
PLAP +ve in tumor cells of
intratubular germ cell
neoplasia.
HCG
72. 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.
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 exclusively by
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,
cells with 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)
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
92. 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
93. 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
94. III. To differentiate b/w in
situ & invasive ductal Ca
SMA
AE 1/AE3
IV. To detect metastasis
sentinel lymph node.
95. 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.
97. 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
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 in neoplastic
cells
CD 3 in non neoplastic
T cells
CD21 in dendritic
follicular cells
101. 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
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- +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.
104. Large T cell lymphoma
CD43 +ve Lysozyme +ve of
reactive histiocytes
105. 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
106. ALK +ve in anaplastic large
cell lymphoma
CD30 in Anaplastic large
cell lymphoma
107. 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
109. 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
115. Advantages-
• Can be done 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.