This document discusses immunohistochemistry (IHC), which uses antigen-antibody interactions to identify cellular or tissue constituents to diagnose cancer type and prognosis. IHC utilizes antibodies that bind specifically to antigens on cells. Antibodies are immunoglobulins produced by plasma cells in response to antigens. Antigen-antibody binding is detected using chromogens like DAB, which produce a colored precipitate for visualization under a microscope. The document outlines the IHC process including tissue fixation, antigen retrieval, blocking, primary/secondary antibody incubation, and controls to validate results and avoid artifacts. Automation has improved IHC reproducibility and throughput. IHC is useful for disease diagnosis, prognosis, predicting therapy response, and quantifying markers
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
Basics of immunohistochemistry, steps of histochemistry, primary and secondary antibody use as protein locator and different fixatives with their advantages
Blood grouping of liquid blood: forward typing and reverse typing; Blood grouping of dried blood: Lattes test, adsorption-elution, adsorption-inhibition, mixed agglutination; HLA antigens and HLA typing; Role of sero-genetic markers in individualization and paternity disputes; Pitfalls in red cell typing
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
Basics of immunohistochemistry, steps of histochemistry, primary and secondary antibody use as protein locator and different fixatives with their advantages
Blood grouping of liquid blood: forward typing and reverse typing; Blood grouping of dried blood: Lattes test, adsorption-elution, adsorption-inhibition, mixed agglutination; HLA antigens and HLA typing; Role of sero-genetic markers in individualization and paternity disputes; Pitfalls in red cell typing
Important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment.
pathogenesis, diagnosis, and treatment of diseases are discovered.It is performed without destruction of histologic architecture. It can be used as an effective adjuvant to H & E diagnosis in a majority of tumor cases through the establishment of definitive diagnosis and also gives an insight into tumor histopathogenesis and prognosis.
BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
Histochemistry in pathology and it'sbranches.pptxDrMUSTAFAAlAmeri
Thi is a simple overview about histochemistry and its branches, how the histological diseases can be diagnosed and what methods are used to examine the tissue.
Immobilization of proteins on the solid support of nitrocellulose membrane or polyvinylidinefluoride membrane. Then antibodies bind speciffcally that can be analyzed through Autoradiography
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
Antibody Coupling - Single Coupling Approach To Bind Antibodies Diverse Speci...Anteo Technologies
Traditional covalent chemistries use harsh chemicals and require expertise in the techniques of diverse covalent coupling methodologies. The Antibody Coupling Kit was made to address issues such as: difficulties in binding certain antibodies with traditional covalent chemistries, antibody wastage, and incorrect antibody orientation. Anteo’s technology offers scientists the flexibility to bind any antibody onto a solid support surface through the use of polymeric metal complexes. The polymeric metal nature of the technology allows multi-valent binding of the target antibody through chelation to the electron donating groups located in the Fc region of the antibody. Anteo’s kit promotes gentle monolayer binding, meaning proteins assemble in the correct orientation while reducing the amount of damaged proteins, leading to increased functionality of antibodies and less antibodies used for the experiment.
This application note demonstrates the ability of the Antibody Coupling Kit to bind Mouse IgG, Mouse IgM, Rabbit IgG, Human IgG and Human IgM antibodies onto 200 nm magnetic particles using a particle-based fluorescent antibody loading assay.
Important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment.
pathogenesis, diagnosis, and treatment of diseases are discovered.It is performed without destruction of histologic architecture. It can be used as an effective adjuvant to H & E diagnosis in a majority of tumor cases through the establishment of definitive diagnosis and also gives an insight into tumor histopathogenesis and prognosis.
BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
Histochemistry in pathology and it'sbranches.pptxDrMUSTAFAAlAmeri
Thi is a simple overview about histochemistry and its branches, how the histological diseases can be diagnosed and what methods are used to examine the tissue.
Immobilization of proteins on the solid support of nitrocellulose membrane or polyvinylidinefluoride membrane. Then antibodies bind speciffcally that can be analyzed through Autoradiography
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
Antibody Coupling - Single Coupling Approach To Bind Antibodies Diverse Speci...Anteo Technologies
Traditional covalent chemistries use harsh chemicals and require expertise in the techniques of diverse covalent coupling methodologies. The Antibody Coupling Kit was made to address issues such as: difficulties in binding certain antibodies with traditional covalent chemistries, antibody wastage, and incorrect antibody orientation. Anteo’s technology offers scientists the flexibility to bind any antibody onto a solid support surface through the use of polymeric metal complexes. The polymeric metal nature of the technology allows multi-valent binding of the target antibody through chelation to the electron donating groups located in the Fc region of the antibody. Anteo’s kit promotes gentle monolayer binding, meaning proteins assemble in the correct orientation while reducing the amount of damaged proteins, leading to increased functionality of antibodies and less antibodies used for the experiment.
This application note demonstrates the ability of the Antibody Coupling Kit to bind Mouse IgG, Mouse IgM, Rabbit IgG, Human IgG and Human IgM antibodies onto 200 nm magnetic particles using a particle-based fluorescent antibody loading assay.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
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.
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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
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
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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
2. Technique to identify cellular or tissue constituents
(ANTIGENS) by means of ANTIGEN – ANTIBODY interactions.
Used to diagnose the type of cancer and to help determine
the patient's prognosis or as a predictive marker of
therapeutic response.
3. Protein, carbohydrate or lipid molecule on a cell surface
which bears one or more binding sites for antibodies.
These binding sites are highly specific regions made up of
small number of amino-acids or monosaccharide units
4. Group of serum proteins known as Immunoglobulins (Ig’s)
Synthesised by plasma cells
5 types – IgG,IgA,IgD,IgM,IgE
Most common - IgG (used in IHC too)
5.
6. Antigenic epitope combines with a specific binding site on
amino acid chains of antibodies.
Ag-Ab complexes are held together by a combination of
hydrogen bonds,electrostatic forces and van der Waal’s forces
7. Polyclonal Ab’s Monoclonal Ab’s
Produced when a host
animal like mouse/rabbit is
injected with a purified
antigen
Multiple types of Ab’s
produced
Produced when a specific
antibody is produced for a
specific epitope
Produced by a single B-cell
line called a clone
8. Mouse/rabbit injected with purified Ag and Abs are produced
against the Ag
After large number of Abs produced, animal is killed and
spleen is removed as it contains large no of B-lymphocytes
This B-cell suspension is mixed with myeloma cells in a
medium which will cause cells to fuse ---- Hybridoma
9. Antigen – Antibody reactions are detected on tissues with the
help of chromogens like: DAB (3,3 diamino benzidine
tetrahydrochloride) or AEC (3 amino-9-ethyl carbazole)
DAB – insoluble in alcohol
AEC- soluble in alcohol, so water medium like glycerin used
for mounting - temporary
10. These are substances which when incubated with a suitable
chromogen produce a stable coloured end product which can
be visualised on light microscopy.
Various different types of labels can be used.
11. I . ENZYMES :
Horseradish peroxidase – reacts with DAB – Brown colour
3 amino-9-ethylcarbazole – Red colour
Calf intestinal alkaline phosphatase – blue and red colour
with chromogens called Fast blue BB and Fast red TR resp
Glucose oxidase
12. II . COLLOIDAL METALS:
Colloidal gold – pink colour ; silver
precipitation recation used
III. FLUORESCENT LABELS :
FITC (Fluorescein isothiocyanate), tetramethyl
rhodamine isothiocyanate – require fluorescent microscope for
visualisation.
IV . RADIOLABELS :
Needs radiographic facilities.
18. Polymer technology :
Consists of an integrated polymer of active enzyme
and secondary antibody that binds to a primary
antibody target.
This integrated format introduces significantly more
enzyme at the site of localization, thereby generating
a greater reaction with the subsequent chromogen,
compared with a secondary antibody directly
conjugated with enzyme.
24. Ideal Fixative :
Gives good morphological preservation
Doesn’t destroy the immuno-reactivity of the Ag
Prevents extraction, diffusion and displacement of the Ag during
procedural steps after fixation
Doesn’t interfere with the subsequent Ag-Ab reactions employed in
the localisation of the Ag.
Preferred fixative - 10% neutral buffered formalin
25. Slides may be coated with Poly-L-Lysine (PLL) or APES (3-
aminopropyltriethoxysilane) before tissue sections are taken
onto them. This ensures a better fixation of sections on the
slides during all the steps of staining. Commercially available
coated slides can be used.
Alternatively, slides may be heat-fixed well in advance of
staining.
26. Many WBCs, RBCs, muscle cells contain peroxidase. It is
important to block this activity if peroxidase based system
(PAP) is used
Peroxidase blocking done by pre-incubation with 0.3% H2O2
for 10-30 minutes at room temp.
Endogenous alkaline phosphatase can be blocked by
levamisole , 20% acetic acid or 0.3% H2O2
27. Non specific binding occurs as Abs are charged molecules
and they can react with substances in tissue bearing the
reciprocal charge (eg: collagen)
This blocking is done to block unwanted sites before adding
primary Ab
Blocking serum consists of dilute serum from the same
species of animals used to produce the secondary Ab
Protein in the serum binds the charged sites in tissue and
doesn’t interfere/participate in the Ag-Ab reaction during
immunostaining
28. Most Ag get partially or extensively masked after
formalin fixation due to the cross linking of amino
acid groups.
2 methods commonly used to retrieve Ag from tissue
are :
i. Proteolytic enzymes
ii. Heat retrieval
29. Sections are exposed to enzymes like trypsin,
protease, proteinase K or pronase for 10-15 min
Enzyme cleaves the protein links allowing the epitope
to return to its normal confirmation enabling more
effective Ab binding
30. After deparaffinizing and rehydrating tissue sections, slides
are immersed in an aqueous solution called the ‘retreival
solution’ and heated
This leads to breaking of the formalin induced cross links and
exposes the epitope of the Ag to the Ab
31. Commonly used solutions are :
i. Citrate buffer at a pH of 6.0
ii. EDTA buffer at a pH of 8.0
iii. TRIS-EDTA buffer at a pH of 9.0
Various methods of heating :
Microwave processing
Pressure cooker
Water bath/steamer
Combination of enzyme and heat retrieval can also be used
(esp if 2-3 Ag’s are being labelled together)
32. Positive control – Slide which contains a tissue known to have
the Ag under study
Positive control slide processed exactly like the sample to be
tested
Negative control – Additional section of the test slide on
which the primary Ab is not added. Rest steps are same. It
helps to differentiate positive staining from non-specific
background staining
34. Deparaffinize in xylene with agitation
Ag retreival (enzyme/heat)
Blocking - 0.3% H2O2
Primary Ab- Incubation
Secondary Ab – Incubation
Add chromogen DAB
Stain with Haematoxylin (light) counterstain
35.
36. Titration is a must
Date Antibody
Used
Dilution Appropriate
dilution
Remarks
04-04-23 CD20 1:50 1:100
1:200
1:100 GOOD
05-05-23 CD3 1:100
1:200
1:300
1:200 BEST
58. Coloured visual product demonstrating an antigen antibody
reaction:
Brown colour (DAB)
Red colour (PAP-APAAP)
Greenish yellow in immunofluorescence
59.
60.
61. Knowledge of Ag location is a must
Knowledge of pattern of staining is essential
Cytoplasmic (diffuse,paranuclear, perinuclear)
Nuclear (diffuse, nucleolar)
Membranous (Continuous, broken)
Interstitial
The cell of interest (larger tumor cell etc)
69. p63 positivity in B-cell lymphomas
C-kit positivity in nasopharyngeal carcinomas
Aberrant CD4 in myeloid leukemias
CD138 in myelomas and carcinomas
……….and the list goes on and on……..
70. Endogenous peroxidase activity
Non specific background staining
Artefacts – precipitates, tissue artefacts, crushing, necrosis,
haemorrhage
Failure of technique – always do with positive & negative
controls.
73. Cross reactivity due to epitope sharing may result in false
positivity
An example is CD79a, a B cell marker cross reacts with
smooth muscle
Error in data entry and mislabelling (MyoD1 and Mib1)
74. Edge and trapping artefacts
Desquamation artefacts
Bubble artefacts
Drying artefacts
Artefacts of poor fixation
Precipitated DAB artefacts
Biotin artefacts
75.
76.
77.
78. Do not call an immunostain negative without
checking out the positive controls
Remember the best control is positive internal
control
80. Vimentin immunostain used to decide the
immunoreactivity/preservation of antigenicity of tissue.
Vimentin is virtually present in all tissues and even smallest of
biopsies usually show some vimentin reactivity, if well
preserved
81. Standardised procedures
Validated antibodies
Sensitivities and specificities of antibodies
Accurate, reproducible and comparable results
Checkpoints for corrective actions
Continuous monitoring and improvement
99. Cell lineage and tissue type – typing of neoplasms
Prognostic markers
Quantification; it is no longer enough that the 'stain' is there;
rather it is a question of 'How much is there?'
Predictive markers for targeted therapy
Benign conditions like infections (HSV, CMV, HPV etc)
100.
101. All the technical staff in IHC laboratory
My colleagues – Dr Katha Rabade, Dr Santosh Menon, Dr
Tanuja Shet
Dr Sumeet Gujral and the whole organizing team
