Histology is the microscopic study of tissues. Key steps in processing tissues for histological examination include fixation, dehydration, clearing, embedding in paraffin wax, sectioning, and staining. Tissues are first fixed in chemicals like formaldehyde to preserve their structure. They are then dehydrated using graded alcohols to remove water. Next, tissues are cleared using solvents like xylene to make them permeable to paraffin prior to embedding. The embedded tissues can then be thinly sectioned and stained for microscopic examination. Proper tissue processing is important for high quality histological analysis.
This is a presentation covering all techniques in histopathology. Comprehensive coverage of all related aspects.. Useful for postgraduate Pathology students and practitioners.
This is a presentation covering all techniques in histopathology. Comprehensive coverage of all related aspects.. Useful for postgraduate Pathology students and practitioners.
Practical Histopathology and cytopathology
Histopathological examination is used to provide diagnostic information that is important for timely diagnosis of disease to determine treatment plan. Fresh tissue is extremely fragile & subject to autolysis.
Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...Indian dental academy
The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.for more details please visit
www.indiandentalacademy.com
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
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
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
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
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
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
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
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
2. Histology is microscopic study of normal tissue of body
Term histology derived from greek word
histos-means tissue……logy –means study coined
by Mayer in 1819
Histopathology means sciences of studying
structural changes in human body by
diseases
3. Histotechnology is concerned with
processing and preparation of tissues in such
a manner that it enables a satisfactory study.
Histotechnique is that branch of biology
concerned with demonstration of minute
tissue structure in diseases.
5. The lab should be well illuminated and well-
ventilated.
Rules and Regulations governing
formalin and
hydrocarbonds
such as xylene
and toluene.
Limits set by the Occupational Safety and
Health Administration (OSHA) that should not
be exceeded.
These limits should be revised and revived to
reduced any mishap.
6. Check the sharpness of scalpel, scissors and
quality of other ones like ruler, probes weighing
machines.
Every instrument used in the laboratory should
meet electrical safety specifications and have
written instructions regarding its use.
Flammable materials may only be stored in
approved rooms and only in storage cabinets that
are designed for this purpose.
7. Fire safety procedures are to be posted.
Safety equipment including fire
extinguishers,
fire blankets,
and fire alarms should be within easy
access.
A shower and eyewash should be readily
available.
No smoking, eating or movements in the labs
Use disposable gloves
8. Laboratory accidents must be documented and
investigated with incident reports and industrial
accident reports.
Specific hazards that you should know about
include:
Bouin's solution is made with picric acid. This acid is
only sold in the aqueous state. When it dries out, it
becomes explosive.
9. Tissue specimens received in the surgical
pathology laboratory have a request form
that lists the patient information and history
along with a description of the site of origin.
The specimens are accessioned by giving
them a number that will identify each
specimen for each patient.
10.
11. Tissues removed from the body for diagnosis arrive in
the Pathology Department and are examined by a
pathologist, pathology assistant, or pathology resident.
Gross examination consists of describing the specimen
and placing all or parts of it into a small plastic
cassette which holds the tissue while it is being
processed to a paraffin block. Initially, the cassettes
are placed into a fixative
When a malignancy is suspected, then the specimen is
often covered with ink in order to mark the margins of
the specimen. Different colored inks can be used to
identify different areas if needed. When sections are
made and processed, the ink will mark the actual
margin on the slide
29. Definition –process by which constituents of
cells and tissues are fixed in a chemical so
that they will withstand treatment with
various reagent with minimum loss or
decomposition.
In simple words—it prevents autolysis of
tissue.
30. 1- To prevent autolysis and bacterial attack.
2- To fix the tissues so they will not change
their volume and shape during processing.
3- To prepare tissue and leave it in a condition
which allow clear staining of sections.
4- To leave tissue as close as their living state
as possible, and no small molecules should be
lost.
Fixation is coming by reaction between the
fixative and protein which form a gel, so
keeping every thing as their in vivo relation to
each other.
33. Inhibition of autolysis
Hardening of tissue
Solidification of colloid material
Optical differentiation
Effects on staining
Loss of material during fixation
Tissue shrinkage
34. 1-Buffer and pH
2-Temperature
3-Penetration capacity
4-Volume change
5-Agitation
6-Osmolarity of fixation solution
7-Concentration of fixation
8-Duration of fixation
35. Fixation is best carried out close to
neutral pH, in the range of 6-8.
Hypoxia of tissues lowers the pH, so there
must be buffering capacity in the fixative
to prevent excessive acidity.
Acidity favors formation of formalin-heme
pigment that appears as black,
polarizable deposits in tissue.
Common buffers include phosphate,
bicarbonate, cacodylate, and veronal.
Commercial formalin is buffered with
phosphate at a pH of 7.
36. Penetration of tissues depends upon the
diffusability of each individual fixative, which is
a constant.
Formalin and alcohol penetrate the best, and
glutaraldehyde the worst.
Mercurials and others are somewhere in
between.
One way to get around this problem is sectioning
the tissues thinly (2 to 3 mm).
Penetration into a thin section will occur more
rapidly than for a thick section
37. The volume of fixative is important.
There should be a 10:1 ratio of fixative to tissue.
Obviously, we often get away with less than this,
but may not get ideal fixation.
One way to partially solve the problem is to
change the fixative at intervals to avoid
exhaustion of the fixative.
Agitation of the specimen in the fixative will also
enhance fixation.
38. Increasing the temperature, as
with all chemical reactions, will
increase the speed of fixation,
as long as you don't cook the
tissue.
Hot formalin will fix tissues
faster, and this is often the first
step on an automated tissue
processor.
39. Concentration of fixative should be adjusted down
to the lowest level possible, because you will expend
less money for the fixative.
Formalin is best at 10%;
Glutaraldehyde is generally made up at 0.25% to
4%.
Too high a concentration may adversely affect the
tissues and produce artefact similar to excessive
heat.
40. Also very important is time interval from of
removal of tissues to fixation.
The faster you can get the tissue and fix it, the
better.
Artefact will be introduced by drying, so if
tissue is left out, please keep it moist with
saline.
The longer you wait, the more cellular
organelles will be lost and the more nuclear
shrinkage and artefactual clumping will occur.
41. Penetrate cells or tissue rapidly
Preserve cellular structure before
cell can react to produce
structural artifacts
Not cause autofluorescence, and
act as an antifade reagent.
43. Fix specimens by rapidly changing hydration state of
cellular components
Proteins are either coagulated or extracted
Preserve antigen recognition often.
DISADVANTAGE
Advantages
Disadvantages
• Cause significant shrinkage of specimens.
• Difficult to do accurate 3D confocal images.
• Can shrink cells to 50% size (height).
• Commercial preparations of formaldehyde contain
methanol as a stabilizing agent.
44. Glutaraldehyde
Formaldehyde
Ethelene glycol-bis-succinimidyl succinate (EGS)
Form covalent crosslinks that are determined by
the active groups of each compound
45. Simple fixative
Eg-Formaldehyde,Glutaraldehyde,Ethyl alcohol
Compound fixative
Eg-Carnoys fluid,Zenker’s fluid,Bouins fluid
According to action upon cell and tissue
1-Micro-anatomical fixative
To preserve microscopic structure of tissues.
Eg-Formal-saline,Buffered neutral
formalin,Zenker’s fluid
47. According to action
1-Physical methods ------heating
------microwaving
-------freeze drying
2-Chemical methods(biochemical approach)
48. Tolerant fixative---eg-formalin
Non tolerant—eg carnoy’s fixative
MOST COMMONLY USED FIXATIVE -----
1-10%formalin
2-10%formal saline
3-10%buffered formalin
49. The Process of removing calcium salts from the tissue and making them
suitable for sectioning.
Some tissues contain calcium deposits which are extremely firm and
extremely firm and which will not section properly with paraffin
with paraffin embedding owing to the difference in densities between
densities between calcium and paraffin.
Bone specimens are the most likely type here, but other tissues may
but other tissues may contain calcified areas as well.
well.
This calcium must be removed prior to embedding to allow sectioning.
embedding to allow sectioning.
A variety of agents or techniques have been used to decalcify tissue and
to decalcify tissue and none of them work perfectly.
perfectly.
Mineral acids,
organic acids,
EDTA, and
50. Specimens should be decalcified in
hydrochloric acid/formic acid working
solution 20 times their volume.
Change to fresh solution each day until
decalcification is complete.
It may take 24 hours up to days or months depending
on size of the specimens.
Once the decalcification is complete, rinse specimens
in water briefly and transfer to ammonia solution to
neutralize acids left in specimens for 30 minutes.
Wash specimens in running tap water
thoroughly up to 24 hours.
Routine paraffin embedding.
55. Perenyl’s fluid—
10%nitric acid-----40ml
absolute alcohol---30ml
chromic acid(0.5%)—30ml
Excellent cytological preservation are possible-
--
Chemical test cannot be carried out---x-ray
should be used
56. Nitric and
Hydrochloric acids
rapid
damage cellular morphology,
so are not recommended for delicate tissues such as bone
marrow.
57. Acetic and Formic acid are better
suited to bone marrow, since they
are not as harsh.
However, they act more slowly on
dense cortical bone.
Formic acid in a 10% concentration
is the best all-around decalcifier.
Some commercial solutions are
available that combine formic acid
with formalin to fix and decalcify
tissues at the same time.
58. EDTA can remove calcium and is not
harsh (it is not an acid)
but it penetrates tissue poorly and
works slowly and is
expensive in large amounts.
Electrolysis has been tried in
experimental situations where calcium
had to be removed with the least tissue
damage.
It is slow and not suited for routine daily
use.
59. Most used is EDTA which as ability to bind
calcium forming non-ionized soluble complex
EDTA works best on cancerous bone
Agent of choice for electron microscopy
EDTA solution(hilleman/lee)
----EDTA disodium salt---5.5 g
----Distilled water-----90ml
-----Formalin------10ml
60. Concentration of active reagent
Temperature
Agitation
Density of bone
61.
62. X-ray (the most accurate way)
Chemical testing (accurate)
Physical testing (less accurate and
potentially damage of specimen)
63.
64. Insert a pipette into the decalcifying solution
containing the specimen.
Withdraw approximately 5 ml of the hydrochloric
acid/formic acid decalcification solution from
under the specimen and place it in a test tube.
Add approximately 10 ml of the ammonium
hydroxide/ammonium oxalate working solution,
mix well and let stand overnight.
Decalcification is complete when no precipitate
is observed on two consecutive days of testing.
Repeat this test every two or three days.
65. The Physical tests include bending the
specimen or inserting a pin, razor, or
scalpel directly into the tissue.
The disadvantage of inserting a pin,
razor, or scalpel is the introduction of
tears and pinhole artifacts.
Slightly bending the specimen is safer
and less disruptive but will not
conclusively determine if all calcium salts
have been removed.
After checking for rigidity, wash
thoroughly prior to processing.
66. Once the tissue has been fixed, it must
be processed into a form in which it
can be made into thin microscopic
sections.
The usual way this is done is with
paraffin.
Tissues embedded in paraffin, which is
similar in density to tissue, can be
sectioned at anywhere from 3 to 10
microns, usually 6-8 routinely.
The technique of getting fixed tissue
into paraffin is called tissue processing
67. TISSUE PROCESSING
The aim of tissue processing is to embed the tissue in a solid
medium firm enough to support the tissue and give it sufficient
rigidity to enable thin sections to be cut , and yet soft enough not
to damage the knife or tissue.
Stages of processing:
1- Dehydration.
2- Clearing.
3- Embedding.
68. Wet fixed tissues (in aqueous solutions) cannot
be directly infiltrated with paraffin.
First, the water from the tissues must be
removed by dehydration.
This is usually done with a series of alcohols,
say 70% to 95% to 100%.
Sometimes the first step is a mixture of formalin
and alcohol.
Other dehydrants can be used, but have major
disadvantages.
Acetone is very fast, but a fire hazard, so is safe only
for small, hand-processed sets of tissues.
Dioxane can be used without clearing, but has toxic
fumes
72. Duration of dehydration should be kept to
the minimum consistent with the tissues
being processed. Tissue blocks 1 mm thick
should receive up to 30 minutes in each
alcohol, blocks 5 mm thick require up to 90
minutes or longer in each change. Tissues
may be held and stored indefinitely in 70%
ethanol without harm
73. Removal of the dehydrant with a substance
that will be miscible with the embedding
medium (paraffin).
The commonest clearing agent is xylene.
Toluene works well, and is more tolerant of
small amounts of water left in the tissues,
but is 3 times more expensive than xylene.
Chloroform used to be used, but is a health
hazard, and is slow.
Methyl salicylate is rarely used because it is
expensive, but it smells nice (it is oil of
wintergreen).
74. Replacing the dehydrating fluid with a fluid that is
totally miscible with both the dehydrating fluid and
the embedding medium.
Choice of a clearing agent depends upon the
following:
- The type of tissues to be processed, and the type
of processing to be undertaken.
- The processor system to be used.
- Intended processing conditions such as
temperature, vacuum and pressure.
- Safety factors.
- Cost and convenience.
- Speedy removal of dehydrating agent .
- Ease of removal by molten paraffin wax .
- Minimal tissue damage .
75. Chloroform – tolerant, no effect on RI
Xylene, Benzene, toluene – rapid, intolerant,
flammable, affects RI
Esters—n butyl acetate----xylene substitute
cedar wood oil – tolerant, expensive
77. When xylene has completely replaced the
alcohol in the tissue, the specimen is ready to be
infiltrated with paraffin.
It is removed from the xylene and placed in a
dish of embedding paraffin, and the dish is put in
a constant temperature of about 60 °C
The exact temperature depend upon melting
point of the paraffin used.
During the course of several hours the specimen
is changed to two or three successive dishes of
paraffin so that all xylene in tissue is replaced by
paraffin
78. Finally, the tissue is infiltrated with the
embedding agent, almost always paraffin.
Paraffins can be purchased that differ in
melting point, for various hardnesses,
depending upon the way the histotechnologist
likes them and upon the climate (warm vs.
cold).
Wax hardness (viscosity) depends upon the
molecular weight of the components and the
ambient temperature.
High molecular weight mixtures melt at higher
temperatures than waxes comprised of lower
molecular weight fractions.
Paraffin wax is traditionally marketed by its
melting points which range from 39°C to
68°C.
82. There are four main mould systems and
associated embedding protocols presently in
use :
1- Traditional methods using paper boats.
2- Leuckart or Dimmock embedding irons or
metal containers.
3- the Peel-a-way system using disposable
plastic moulds and
4- Systems using embedding rings or cassette-
bases which become an integral part of the
block and serve as the block holder in the
microtome.
83. Tissue processing
Embedding moulds:
(A) paper boat;
(B) metal bot mould;
(C) Dimmock embedding
mould;
(D) Peel-a-way disposable
mould;
(E) base mould used with
embedding ring
( F) Cassette
84. General Embedding Procedure
1- Open the tissue cassette, check against worksheet entry to ensure
the correct number of tissue pieces are present.
2- Select the mould, there should be sufficient room for the tissue with
allowance for at least a 2 mm surrounding margin of wax.
3- Fill the mould with paraffin wax.
4 Using warm forceps select the tissue, taking care that it does not cool
in the air; at the same time, correct orientation of tissue in a mould is
the most important step in embedding. Incorrect placement of tissues
may result in diagnostically important tissue elements being missed or
damaged during microtomy.
5- Insert the identifying label or place the labeled embedding ring or
cassette base onto the mould.
6- Cool the block on the cold plate, or carefully submerge it under
water when a thin skin has formed over the wax surface.
7- Remove the block from the mould
.
8- Cross check block, label and worksheet.
85.
86.
87. Turn on the water bath and check that the
temperature is 35-37ºC.
Use fresh deionized water (DEPC treated water
must be used if in situ hybridization will be
performed on the sections).
Blocks to be sectioned are placed face down on
an ice block or heat sink for 10 minutes.
Place a fresh blade on the microtome.
Insert the block into the microtome chuck so the
wax block faces the blade and is aligned in the
vertical plane. Set the dial to cut 4-10 µM
sections.
The blade should be angled 4-6º.
88. Face the block by cutting it down to the desired
tissue plane and discard the paraffin ribbon.
If the block is ribboning well then cut another
four sections and pick them up with forceps or a
fine paint brush and float them on the surface
of the 37ºC water bath.
Float the sections onto the surface of clean
glass slides.
If the block is not ribboning well then place it
back on the ice block to cool off firm up the
wax.
If the specimens fragment when placed on the
water bath then it may be too hot.
Place the slides with paraffin sections in a 65°C
oven for 20 minutes (so the wax just starts to
melt) to bond the tissue to the glass.
Slides can be stored overnight at room
temperature
89.
90. A microtome is a mechanical
instrument used to cut biological
specimens into very thin segments for
microscopic examination. Most
microtome use a steel blade and are
used to prepare sections of animal or
plant tissues for histology.
95. The embedding process must be reversed
in order to get the paraffin wax out of the
tissue and allow water soluble dyes to
penetrate the sections.
Therefore, before any staining can be
done, the slides are "deparaffinized" by
running them through xylenes (or
substitutes) to alcohols to water.
There are no stains that can be done on
tissues containing paraffin.
96. The staining process makes use of a
variety of dyes that have been chosen for
their ability to stain various cellular
components of tissue.
The routine stain is that of hematoxylin
and eosion (H and E).
Other stains are referred to as "special
stains" because they are employed in
specific situations according to the
diagnostic need.
98. Frozen sections are stained by hand, because
this is faster for one or a few individual sections.
The stain is a "progressive" stain in which the
section is left in contact with the stain until the
desired tint is achieved.
99.
100. The stained section on the slide must be covered
with a thin piece plastic or glass to protect the
tissue from being scratched, to provide better
optical quality for viewing under the microscope,
and to preserve the tissue section for years to
come.
The stained slide must go through the reverse
process that it went through from paraffin
section to water.
The stained slide is taken through a series of
alcohol solutions to remove the water, then
through clearing agents to a point at which a
permanent resinous substance beneath the glass
coverslip, or a plastic film, can be placed over
the section.
101. Exfoliative Cytology – In this method, cells are collected
after they have been either spontaneously shed by the body
("spontaneous exfoliation") or manually scraped/brushed off
of a surface in the body ("mechanical exfoliation"). An
example of spontaneous exfoliation is when cells of
the pleural cavity or peritoneal cavity are shed into the
pleural or peritoneal fluid. This fluid can be collected via
various methods for examination. Examples of mechanical
exfoliation include Pap smears, where cells are scraped from
the cervix with a cervical spatula, or bronchial brushings,
where a bronchoscope is inserted into the trachea and used to
evaluate a visible lesion by brushing cells from its surface
and subjecting them to cytopathologic analysis
102. Suitable for hard structure like bone and
teeth.
Similar to paraffin embedding technique and
the only difference is infiltration of
embedding is done in parlodion instade of
paraffin.
103. It is a diagnostic procedure used to investigate
superficial (just under the skin) lumps or masses.
In this technique, a thin, hollow needle is
inserted into the mass for sampling of cells that,
after being stained, will be examined under
a microscope. There could be cytology exam of
aspirate (cell specimen evaluation, FNAC) or
histological (biopsy - tissue specimen evaluation,
FNAB). Fine-needle aspiration biopsies are very
safe, minor surgical procedures. Often, a major
surgical (excisional or open) biopsy can be
avoided by performing a needle aspiration biopsy
instead. Now a day, this procedure is widely used
in the diagnosis of cancer.
104. Micrograph of a needle aspiration biopsy
specimen of a salivary gland showing adenoid
cystic carcinoma.
105. It 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.
IHC takes its name from the roots "immuno," in
reference to antibodies used in the procedure,
and "histo," meaning tissue (compare
to immunocytochemistry).
Immunohistochemical staining is widely used in
the diagnosis of abnormal cells such as those
found in cancerous tumors. Specific molecular
markers are characteristic of particular cellular
events such as proliferation or cell death
(apoptosis).
106. Immunohistochemistry labels individual proteins,
such as TH (green) in the axons of
sympathetic autonomic neurons
107. It consist of paraffin blocks in which up to 1000
,separate tissue cores are assembled in array fashion to
allow multiplex histological analysis.
In the tissue microarray technique, a hollow needle is
used to remove tissue cores as small as 0.6 mm in
diameter from regions of interest in paraffin-embedded
tissues such as clinical biopsies or tumor samples. These
tissue cores are then inserted in a recipient paraffin block
in a precisely spaced, array pattern.
Sections from this block are cut using a microtome,
mounted on a microscope slide and then analyzed by any
method of standard histological analysis. Each microarray
block can be cut into 100 – 500 sections, which can be
subjected to independent tests. Tests commonly
employed in tissue microarray
include immunohistochemistry, and fluorescent in situ
hybridization. Tissue microarrays are particularly useful in
analysis of cancer samples