The document provides information about routine histopathology techniques and staining. It discusses fixation of histology samples, ideal fixatives, changes after fixation, types of fixatives, and the mechanisms of fixation. It also covers tissue processing techniques including dehydration, clearing, infiltration, embedding, and sectioning of tissue blocks using a microtome. Key steps in processing like fixation, dehydration, clearing and infiltration are described in detail. Common fixatives, dehydrating agents, and clearing agents used are also listed.
A STEP IN CASTING OF CAST PARTIAL DENTURE, a precious duplication process and proper wax up of refractory cast results in accurate fitting of the framework of the prosthesis.
A STEP IN CASTING OF CAST PARTIAL DENTURE, a precious duplication process and proper wax up of refractory cast results in accurate fitting of the framework of the prosthesis.
DRILLING FLUIDS FOR THE HPHT ENVIRONMENTMohan Doshi
A BRIEF REVIEW OF THE DRILLING FLUIDS FOR DRILLING HPHT WELLS. HPHT WELLS ARE NOT BUSINESS AS USUAL AND THE SAME APPLIES TO HPHT DRILLING FLUIDS. THE FLUID CHEMISTRY AND THE FLUID COMPOSITION HAVE TO BE TAILORED TO MEET THE RIGORS OF THE HIGH TEMPERATURE ENVIRONMENT
Endodontic sealers a summary and a quick review Rami Al-Saedi
a slideshow presentation lectured and presented in Al-Sadr Specialized dental center in the continuing dental learning weekly lectures.
Rusafa medical institute- Baghdad- Iraq
lecturer: Dr. Rami Ahmed Jumaah (BDS)
Supervisor: Dr. Iman J. Ahmed (BDS: MSc)
It shows methods of gingival retraction and its recent advances.
gingival retraction is done prion to tooth preparation or impression making to widen the gingival sulcus for easy access to the margin around tooth that is prepared.
Methods Of Extractions of crude drugs.pdfArunShah49
This document is made to help different personals to enhance their knowledge about crude drug processing and their proper usage for better therapeutic yeild. In this document one can find the detailed study of some of the popular and effective methods of extraction for crude drugs like as Soxhlet extraction, Countercurrent Extraction and Ultrasonication Assisted extraction methods. Hope this would be helpful for the students and other personals willing to learn about extraction methods related to crude drugs.
DRILLING FLUIDS FOR THE HPHT ENVIRONMENTMohan Doshi
A BRIEF REVIEW OF THE DRILLING FLUIDS FOR DRILLING HPHT WELLS. HPHT WELLS ARE NOT BUSINESS AS USUAL AND THE SAME APPLIES TO HPHT DRILLING FLUIDS. THE FLUID CHEMISTRY AND THE FLUID COMPOSITION HAVE TO BE TAILORED TO MEET THE RIGORS OF THE HIGH TEMPERATURE ENVIRONMENT
Endodontic sealers a summary and a quick review Rami Al-Saedi
a slideshow presentation lectured and presented in Al-Sadr Specialized dental center in the continuing dental learning weekly lectures.
Rusafa medical institute- Baghdad- Iraq
lecturer: Dr. Rami Ahmed Jumaah (BDS)
Supervisor: Dr. Iman J. Ahmed (BDS: MSc)
It shows methods of gingival retraction and its recent advances.
gingival retraction is done prion to tooth preparation or impression making to widen the gingival sulcus for easy access to the margin around tooth that is prepared.
Methods Of Extractions of crude drugs.pdfArunShah49
This document is made to help different personals to enhance their knowledge about crude drug processing and their proper usage for better therapeutic yeild. In this document one can find the detailed study of some of the popular and effective methods of extraction for crude drugs like as Soxhlet extraction, Countercurrent Extraction and Ultrasonication Assisted extraction methods. Hope this would be helpful for the students and other personals willing to learn about extraction methods related to crude drugs.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
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
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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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
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
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.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
2. FIXATION OF HISTOLOGY SAMPLES
• APPROPRIATE FIXATION IS CENTRAL TO ALL HISTOLOGY TESTS
AIMS OF FIXATION-
1. TO PRESERVE THE TISSUE NEAREST TO ITS LIVING STATE
2. TO PREVENT ANY CHANGE IN SHAPE AND SIZE OF THE TISSUE AT THE TIME OF
PROCESSING
3. TO PREVENT ANY AUTOLYSIS
4. TO MAKE THE TISSUE FIRM TO HARD
5. TO PREVENT ANY BACTERIAL GROWTH IN THE TISSUE
6. TO MAKE IT POSSIBLE TO HAVE CLEAR STAIN
7. TO HAVE BETTER OPTICAL QUALITY OF THE CELLS
3. IDEAL FIXATIVE -
AN IDEAL FIXATIVE SHOULD HAVE THE FOLLOWING QUALITIES-
1. PREVENTION OF AUTOLYSIS OF THE CELLS OR TISSUE
2. PREVENTION OF DECOMPOSITION OF THE TISSUE BY BACTERIA
3. MAINTAINING THE VOLUME AND SHAPE OF THE CELL AS FAR AS
POSSIBLE
4. CONSISTENTLY HIGH-QUALITY STAINING PARTICULARLY ROUTINE STAIN
5. RAPID ACTION
6. CHEAP
7. NON-TOXIC
4. CHANGE IN TISSUE AFTER FIXATION –
• VOLUME CHANGES – SHRINKAGE OF THE VOLUME BY FORMALIN (33%).
• HARDENING OF TISSUE – MILD DEGREE HARDENING MAY OCCUR.
• INTERFERENCE OF STAINING – INHIBITS ROUTINE STAIN: OSMIUM TETROXIDE
INHIBITS HAEMATOXYLIN AND EOSIN STAINING.
• CHANGES OF OPTICAL DENSITY BY FIXATION – NUCLEI MAY LOOK LIKE
HYPERCHROMATIC.
6. ESSENTIAL PRECAUTIONS FOR FIXATION –
• THE TISSUE SHOULD BE FREE FROM EXCESSIVE BLOOD BEFORE PUTTING IT INTO
FIXATIVE.
• TISSUE SHOULD BE THINLY CUT IN 3–5 MM THICKNESS.
• THE AMOUNT OF FIXATIVE FLUID SHOULD BE 20 TIMES MORE THAN THE
VOLUME OF THE
TISSUE.
• THE TISSUE WITH FIXATIVE SHOULD BE IN A TIGHTLY SCREW-CAPPED BOTTLE.
7. MECHANISM OF FIXATION
• DEHYDRATION AND COAGULATION OF PROTEIN –
• ALCOHOLS REMOVE WATER FROM THE TISSUE, DESTABILIZE THE HYDROGEN
BONDS & DISRUPT THE TERTIARY STRUCTURE OF PROTEIN.
• THE SECONDARY STRUCTURE OF THE PROTEIN IS MAINTAINED.
• ETHANOL IS RELATIVELY STRONGER DEHYDRATING AGENT THAN METHANOL.
• THE ETHANOL AND METHANOL START WORK FROM 60–80% CONCENTRATION,
RESPECTIVELY.
• THE DEHYDRATING FIXATIVE HAS TWO DISADVANTAGES: –
- SHRINKAGE OF THE CELLS
- REMOVAL OF THE SOLUBLE SUBSTANCES FROM THE TISSUE
8. • CROSS-LINKING FIXATIVES:
FORMALDEHYDE-
• IN AQUEOUS SOLUTION IT COMBINES WITH WATER TO FORM METHYLENE
HYDRATE, A METHYLENE GLYCOL
• ON LONG-STANDING, THIS METHYLENE GLYCOL FURTHER REACT WITH WATER &
FORM A POLYMER KNOWN AS POLYOXYMETHYLENE GLYCOL.
• THIS AGAIN DEPOLYMERIZED IN METHYLENE GLYCOL IN A NEUTRAL BUFFER
SYSTEM.
• FORMALDEHYDE REACTS WITH VARIOUS SIDE CHAIN OF THE PROTEIN AND
FORMS HYDROXYMETHYL SIDE GROUP.
• THESE COMPOUNDS ARE HIGHLY REACTIVE AND SUBSEQUENTLY CROSS-LINKING
OCCURS BY FORMING A METHYLENE BRIDGE (PRIMARY REACTION)
9. CONT….
• SUBSEQUENT INTERMOLECULAR AND INTRAMOLECULAR CROSS-LINKING OF THE
MOLECULES OCCURS AS A SLOW-GROWING PROCESS.
• THIS ULTIMATELY PRODUCES AN INSOLUBLE PRODUCT.
• THE FORMALIN CAN BE REMOVED FROM TISSUE BY
PROLONGED WASHING.
• ONCE METHYLENE BRIDGE IS FORMED IN THE TISSUE,
THE REACTION IS STABLE, AND IT IS DIFFICULT TO
REMOVE FORMALIN FROM THE TISSUE.
• FORMALDEHYDE ALSO REACTS WITH THE NUCLEIC ACID
BY REACTING WITH THE AMINO GROUP OF NUCLEOTIDES
10. GLUTARALDEHYDE-
• THE ALDEHYDE GROUP OF GLUTARALDEHYDE REACTS WITH AMINO GROUP OF THE
PROTEIN PREDOMINANTLY LYSINE.
• GLUTARALDEHYDE RAPIDLY AND IRREVERSIBLY CROSS-LINKS THE PROTEIN.
• THE PENETRATION OF GLUTARALDEHYDE IS SLOWER THAN FORMALDEHYDE.
OSMIUM TETROXIDE
• IT CAUSES OXIDATION OF UNSATURATED BONDS IN LIPID.
• IT CONVERTS THE UNSATURATED FATTY ACID INTO A STABLE PRODUCT KNOWN AS
GLYCOL OSMATE.
• THE TETRAVALENT OS BECOMES HEXAVALENT IN THIS REACTION. OSMIC ACID
MONOESTER FORMED IN THIS REACTION IS EASILY HYDROLYSED TO A DIOL AND
OSMIC ACID.
• OSMIUM TETROXIDE MAY REACT WITH TWO UNSATURATED CARBON ATOM OF THE
LIPIDS AND MAY CROSS-LINK
19. PROCESSING OF TISSUE
• AIMS OF TISSUE PROCESSING: TO PROVIDE SUFFICIENT RIGIDITY TO THE TISSUE
SO THAT IT CAN BE CUT INTO THIN SECTION FOR MICROSCOPIC EXAMINATION.
• PRINCIPLE OF PROCESSING: WATER WITHIN THE TISSUE IS REMOVED, AND
ANOTHER MEDIUM (USUALLY PARAFFIN WAX) IS IMPREGNATED IN THE TISSUE
THAT PROVIDES THE ADEQUATE SUPPORT TO THE TISSUE.
• THE ESSENTIAL STEPS IN TISSUE PROCESSING:
20. INFLUENCING FACTORS OF TISSUE PROCESSING
• SIZE OF THE TISSUE: – THE SMALLER THE SIZE, THE BETTER THE PROCESSING.
• AGITATION: – AGITATION FACILITATES THE CONTACT OF TISSUE WITH FRESH
SOLUTION.
• HEAT: – INCREASES THE BETTER PENETRATION OF FLUID.
• VISCOSITY: – THE HIGHER THE VISCOSITY OF THE MEDIUM, LOWER THE
PENETRATION.
• NEGATIVE PRESSURE: – NEGATIVE PRESSURE REMOVES TRAPPED AIR IN THE
TISSUE.
– REMOVAL OF CLEARING AGENT BY INCREASING
VOLATILITY.
21. DEHYDRATION
• REMOVES FREE OR UNBOUND WATER MOLECULE OF THE TISSUE AS THE
SUPPORTING
MEDIUM (PARAFFIN) IS NOT MISCIBLE WITH WATER.
• SHARP DIFFERENCE OF CONCENTRATION GRADIENT OF THE DEHYDRATING
FLUID MAY
DAMAGE THE DELICATE TISSUE.
• GRADUAL DEHYDRATION IS NECESSARY.
• TOO MUCH TIME IN THE DEHYDRATING FLUID: THE TISSUE BECOMES HARD AND
BRITTLE.
• ROUTINE LABORATORY: 70, 90 AND 100% ALCOHOL FOR 2 H EACH.
• COMMON DEHYDRATING AGENTS: – ETHYL ALCOHOL, METHYLATED SPIRIT,
METHANOL,
BUTYL ALCOHOL, ISOPROPYL ALCOHOL
22. COMPARISON OF DIFFERENT DEHYDRATING AGENTS
Dehydrating
agents
Advantages Disadvantage
Ethyl alcohol • Rapid and efficient
dehydrating agent
• Needs licence from the government
• Inflammable
• Hard and brittle tissue if kept for
long time
Methanol Equally effective as ethanol • Volatile • High cost
Isopropyl
alcohol
• Relatively rapid action
• Non-toxic
• Minimal tissue shrinkage
Not possible to use in celloidin
technique
Dioxane • Rapid action
• No shrinkage of tissue
Highly toxic gas is generated
Ethylene glycol • Rapid
• No graded solution is needed
• Tissue can be kept in it for
long time
• Very expensive
• Clearing agent is needed
Acetone • Rapid action
• Cheaper than ethanol
• Good for fatty tissue
• Quickly evaporates
• Inflammable
• Prolonged use may cause shrinkage
25. INFILTRATION AND EMBEDDING
• AIMS: TO PROVIDE SUPPORT TO THE TISSUE.
• PRINCIPLE: CLEARING AGENT IS REMOVED BY THE PROCESS OF DIFFUSION, AND THE
TISSUE
SPACE IS NOW INFILTRATED WITH THE EMBEDDING MEDIA.
• IDEAL IMPREGNATING MEDIUM:
• MISCIBLE WITH CLEARING AGENT
• LIQUID IN HIGHER TEMPERATURE AND SOLID IN ROOM TEMPERATURE
• HOMOGENOUS AND STABLE • NON-TOXIC AND CHEAP
• TRANSPARENT
• FIT FOR SECTIONING
• THE TISSUE TIME DURATION AND THE NUMBER OF CHANGES OF EMBEDDING
MEDIUM:
• SIZE OF TISSUE: LARGE VERSUS SMALL.
• TYPE OF TISSUE: HARD VERSUS SOFT.
• THE TYPE OF CLEARING AGENT: CEDARWOOD OIL TAKES LONGER TIME.
• TYPE OF PROCESSING: VACUUM PROCESSING ACCELERATES.
26. • PARAFFIN WAX- HYDROCARBON, BY-PRODUCT OF CRUDE PETROLEUM.
• MOST POPULAR EMBEDDING MEDIUM FOR TISSUE PROCESSING.
• THE MELTING POINT VARIES FROM 39 °C TO 70 °C.
• IN INDIAN SUBCONTINENT, THE PARAFFIN WAX WITH MELTING POINT AROUND
60 °C IS THE MOST SUITABLE FOR LABORATORY USE.
• TOTAL 3–4 HR. TIME IN PARAFFIN WAX IS SUFFICIENT FOR IMPREGNATION OF
TISSUE BY WAX.
• ADVANTAGES OF PARAFFIN WAX:
• TISSUE BLOCK CAN BE STORED FOR LONG DURATION.
• NON-TOXIC • CHEAP
• SAFE
• DISADVANTAGES OF PARAFFIN WAX:
• MAY CAUSE TISSUE SHRINKAGE AND HARDENING IN CASE OF PROLONGED
IMPREGNATION.
• PARAFFIN WAX TAKES LONG DURATION FOR THE IMPREGNATION OF THE BONE
27. TISSUE PROCESSING METHODS
• MANUALLY OR BY AUTOMATED PROCESSOR.
• AUTOMATED TISSUE PROCESSOR: THE BASIC PRINCIPLE OF IS TO TRANSFER THE
TISSUE IN DIFFERENT FLUID FOR A SPECIFIED TIME IN A DESIRED ENVIRONMENT.
• TWO TYPES OF PROCESSOR:
1. TISSUE TRANSFER PROCESSOR 2. FLUID TRANSFER
PROCESSOR
28. Overall Precautions of Tissue Processing-
1. The bulk of the tissue should be
optimum for adequate penetration of
fluid.
2. The amount of fluid should be adequate,
fluid level should be always higher than
the tissue level.
3. The tissue basket and cassettes should
be clean and any spillage of wax should
be cleaned.
4. The temperature of the infiltrating
medium should be optimum, and it is
preferable to keep the temperature 3–
4 °C above the melting point.
5. There should be a proper record of the
change of fluid, number of tissues
30. EMBEDDING OF TISSUE
• THE TISSUE IS SURROUNDED IN A MOLTEN MEDIUM BY USING A MOULD.
• SUBSEQUENTLY THIS MEDIUM IS SOLIDIFIED TO MAKE A BLOCK FOR CUTTING THIN SECTION
OF TISSUE.
• AIMS OF EMBEDDING:
1. TO GIVE SUPPORT OF THE TISSUE
2. TO PREVENT DISTORTION OF THE TISSUE DURING CUTTING
3. TO PRESERVE THE TISSUE FOR ARCHIVAL USE
• THE CHOICE OF THE EMBEDDING MEDIUM:
• PARAFFIN WAX, EPOXY RESIN, METHACRYLATE, CARBOWAX, ETC. ARE USED.
• PARAFFIN WAX IS THE MOST COMMONLY USED EMBEDDING MEDIUM.
• THE CHOICE OF THE EMBEDDING MEDIUM DEPENDS ON :
1. TYPE OF TISSUE: THE DENSITY OF THE TISSUE AND THE EMBEDDING MEDIUM SHOULD BE
CLOSE OTHERWISE TISSUE MAY NOT BE SECTIONED PROPERLY, AND TISSUE WILL BE
DEFORMED.
2. TYPE OF MICROTOME
3. TYPE OF MICROSCOPE
31. DIFFERENT TYPES OF MOULD USED FOR BLOCK
• LEUCKHARD EMBEDDING MOULDS
• STAINLESS STILL MOULD
• PLASTIC MOULD
34. TISSUE MARKING
NEEDED FOR-
1. TO IDENTIFY THE RESECTION PLANE OR OUTER MARGIN OF THE TISSUE
2. TO HELP IN EMBEDDING THE TISSUE
3. ANY AREA OF INTEREST TO IDENTIFY SUCH AS THE AREA OF TRANSITIONAL
ZONE IN CONE BIOPSY OF CERVIX
THE TISSUE MARKERS SHOULD HAVE THE FOLLOWING CHARACTERISTICS
FEATURES:
• THE MARKER SUBSTANCE SHOULD NOT BE DISSOLVED IN FIXATIVE AND TISSUE
PROCESSING
AGENTS.
• THE MARKER SHOULD NOT PENETRATE THE DEEPER TISSUE.
• IT SHOULD BE RECOGNIZABLE IN THE STAINED SECTION BOTH
MICROSCOPICALLY AND
MACROSCOPICALLY.
38. TISSUE MICROTOMY
• MICROTOMES- INSTRUMENT BY WHICH WE CUT THE EMBEDDED TISSUE IN THE
PARAFFIN BLOCK AS THIN SECTION.
• THE DIFFERENT TYPES OF MICROTOMES-
• ROTARY MICROTOME
• ROCKING MICROTOME
• BASE SLEDGE MICROTOME
• SLIDING MICROTOME
• CRYOMICROTOME
• ULTRAMICROTOME
• LASER MICROTOME
39. • ROTARY MICROTOME- MOST COMMONLY USED
• THE CUTTING BLADE IS KEPT IN HORIZONTAL POSITION, AND THE BLOCK CONTAINING TISSUE
MOVES UP AND DOWN WITH THE HELP OF ROTATORY HANDLE ATTACHED WITH THE
MICROTOME.
• IN EACH 360° ROTATION OF THE WHEEL HANDLE, THE BLOCK MOVES DOWN FOLLOWED BY UP,
AND THE TISSUE IS CUT AS THIN RIBBON.
• THIS MICROTOME HAS THE OPTION TO BE SEMIAUTOMATED OR AUTOMATED
• ADVANTAGES:
1. GOOD-QUALITY 2–3-ΜM-THIN SECTION IS POSSIBLE.
2. HEAVY AND STABLE AUTOMATED ROTARY MICROTOME REDUCES HEALTH HAZARD AND
GIVES THE BEST-QUALITY SECTION.
3. GOOD TISSUE RIBBON PRODUCTION.
4. EASY-TO-CUT VARIOUS TYPES OF TISSUE: FIRM, FRAGILE, SMALL BIOPSY, ETC.
• DISADVANTAGES:
1. EXPENSIVE.
2. UNSUITABLE TO CUT LARGE BLOCK.
3. KNIFE FACES UP AND SO MAY BE DANGEROUS TO THE TECHNICAL STAFF.
41. • SECTIONING THE PARAFFIN BLOCK
• THE FOLLOWING INSTRUMENTS ARE ESSENTIAL –
1. MICROTOME WITH BLADE
2. WATER BATH
3. PARAFFIN BLOCK WITH EMBEDDED TISSUE TO CUT
4. ICE TRAY
5. A BLUNT FORCEPS OR CAMEL BRUSH
6. SLIDE RACK WITH SLIDES WATER BATH (FLOATATION CHAMBER)
7. ADHESIVE- USED FOR BRAIN SECTIONS, DECALCIFIED TISSUE, USING STRONG ALKALI AT THE
TIME OF STAINING
THE MOST COMMONLY USED ADHESIVES INCLUDE:
• MAYER’S EGG ALBUMIN AND GLYCEROL
• POLY-L-LYSINE
• 3-AMINOPROPYLTRIETHOXYSILANE (ACEP)
46. FROZEN SECTION
• INDICATIONS-
• RAPID DIAGNOSIS OF THE LESION FOR INTRAOPERATIVE MANAGEMENT
• TO KNOW THE EXTENT OF THE LESION
• TO DO ENZYME IMMUNOCYTOCHEMISTRY
• TO DO IMMUNOFLUORESCENCE STUDY
• TO STAIN LIPID AND CERTAIN CARBOHYDRATE IN THE TISSUE
• PRINCIPLE-
• RAPID FREEZING OF THE TISSUE SAMPLE CONVERTS THE WATER INTO ICE. THE
FIRM ICE WITHIN THE TISSUE ACTS AS EMBEDDING MEDIA TO CUT THE TISSUE.
• THE CRYOSTAT IS THE INSTRUMENT THAT HAS THE ARRANGEMENT TO FREEZE
THE TISSUE AND ALSO TO CUT THE FROZEN TISSUE FOR MICROSCOPIC SECTION.
50. • STAINING- HAEMATOXYLIN AND EOSIN (H&E) STAINING-
• RINSE THE SLIDE IN TAP WATER.
• PUT IN HAEMATOXYLIN FOR 1 MIN.
• RINSE IN TAP WATER FOR 5 S.
• RINSE IN SCOTT’S TAP WATER FOR 5 S FOR BLUING.
• DIP IN EOSIN FOR 20 S.
• RAPIDLY RINSE IN TAP WATER.
• 95% ETHANOL FOR 10 S.
• 100% ETHANOL FOR 10 S.
• 100% ETHANOL FOR 10 S.
• DIP IN XYLENE FOR 20 S.
• MOUNT BY DPX.
Additives and Modification of Paraffin Wax To alter the physical characteristics of paraffin wax, the following modifications may be done: 1. To increase hardness: addition of stearic acid 2. Reduction of melting point: addition of phenanthrene 3. Improving adhesiveness with tissue and wax: addition of 0.5% of ceresin Dimethyl Sulphoxide (DMSO) The addition of small amount of DMSO in paraffin wax reduces the infiltration time of the wax and removes the residual clearing agent. It produces a homogenous matrix and better support.
Microwave Processing Microwave processing in histopathology reduces the time of processing significantly [2]. It is suitable for small number of delicate tissues. The microwave oven usually has: 1. System to control the temperature 2. System to control the time duration of particular temperature 3. Proper exhaust to remove the toxic gas The microwave processing may be used for all the steps of processing.
Paraffin wax: As described in the previous chapter, paraffin wax is a solid polycrystalline hydrocarbon. The paraffin wax is sold in the market with different melting point. Paraffin wax with melting points ranging from 56 to 62 °C is used in our laboratory. Paraffin wax is cheaper and easy to use. Little supervision is needed to make block by it. (b) Epoxy resin: Epoxy resin is mainly used in electron microscopy as it provides better resolution and greater details of tissue. (c) Acrylic medium: Methacrylate monomer is miscible with ethanol. In the presence of catalyst (benzoyl peroxide 2%), methacrylate monomer is polymerized and provides a hard and clear block. Methacrylate monomer is available in the market along with hydroquinone which should be removed by treating with weak alkali solution followed by thoroughly washing with water. The presence of water may lead to small bubbles within the block. (d) Agar gel: Agar gel helps in cohesion of friable and fragmented tissue particularly in cytology sample and also endometrial curetting and small endoscopic biopsies. It does not provide good support of the tissue for section cutting. Agar-paraffin wax double embedding is more suitable technique than agar alone
Gelatin: It is also used in small friable tissues and frozen section containing friable and necrotic tissue. The melting point of gelatin is 35–40 °C, and this low melting point makes it unsuitable for embedding. (f) Celloidin medium: Celloidin is nitrocellulose and was mainly used for embedding hard tissue. Nowadays it is not used in the laboratory.
India ink: This is the most commonly used marker in the routine surgical pathology laboratory. It takes 15 min time to mark the tissue. • silver nitrate: This is also a good marker. It produces brown-black colour. •
Usually 3% acetic acid or 50% white vinegar is used as fixer.
The strong acids: • Hydrochloric acid • Nitric acid Weak acids: • Formic acid • Trichloroacetic acid
Nitric acid may give yellow colour to the tissue that can be removed by urea
Nitric acid formaldehyde (10%) Nitric acid 10 ml Formalin 10 ml Distilled water 80 ml
Von Ebner’s fluid Saturated solution of sodium chloride: 175 g Hydrochloric acid (concentrated): 15 ml Distilled water: make it up to 1000 ml Advantages: 1. Rapid action 2. Ideal decalcifying agent for the tooth
Perenyi’s fluid Nitric acid (10%) 40 ml Chromic acid (0.5%) 30 ml Absolute alcohol 30 ml Advantages: 1. Provides excellent result 2. Softens the fibrous tissue 3. Cellular morphology well-preserved Disadvantages: 1. Slower in action. 2. End point detection is difficult
Weak acids Gooding and Stewart solution Formic acid 5 ml Formalin (40% formaldehyde) 5 ml Distilled water 90 ml
Water bath is used to float the tissue after cutting (Fig. 5.5). The temperature of the water bath is usually controlled automatically by a thermostat. The temperature of water in the water bath should be 10 °C below the melting point of the embedded paraffin wax and is usually kept in 40–50 °C. It is necessary to prevent formation of any air bubbles within the water bath. For adequate floating of the tissue, one can add a few drops of alcohol or little amount of detergent. This reduces the surface tension of the water and tissue floats smoothly.
White part of egg: 100 ml. – Glycerol: 100 ml. – Homogenize the mixture thoroughly, and filter it by gauze piece. Add few crystals of thymol to prevent bacterial growth.
This medium is used to hold the tissue over the chuck. Presently optimum cutting temperature (OCT) compound is used as embedding medium. The OCT is made of water-soluble glycols and resin
Toluidine Blue Stain This is a very simple stain and takes only a few seconds. The drops of toluidine blue stain are put on the section, and the coverslip is put on the section. The slide is now ready to see. The histopathologist feels more comfortable in H&E stain than this unfamiliar toluidine blue stBrain, liver, spleen −7 °C to −10 °C Rectum, uterus, adrenal, muscle, skin −12 °C to −15 °C Heart, lung, intestine, pancreas, ovary, cervix, prostate −16 °C to −20 °C Bone marrow, breast −20 °C to −25 °Brain, liver, spleen −7 °C to −10 °C Rectum, uterus, adrenal, muscle, skin −12 °C to −15 °C Heart, lung, intestine, pancreas, ovary, cervix, prostate −16 °C to −20 °C Bone marrow, breast −20 °C to −25 °ain.