QUICK REVIEW Flashcards
-microscopic study of normal tissues
Histology
Gross /Microscopic Pathology:
Gross: macroscopic examination
Microscopic
Anatomic Pathology:
Surgical
Autopsy
Exfoliative
: gross examination of tissue histology obtained during surgery to determine “cause” of disease
Surgical
: gross examination of tissue histology removed from a identification/interpretation/char dead body to determine
“cause” of death
Autopsy
: microscopic study of desquamated epithelial cell surfaces.
Exfoliative
- Diagnosis and monitoring of diseases through examinations of blood, body fluids, secretions, tissue biopsy, chemical, microbiological and immunological abnormalities
Clinical Pathology
-concerned with acterization of changes occurring in cells, tissues and fluids.
Clinical Pathology
-able to support physician’s information for appropriate treatments
Clinical Pathology
- Examination of cells or tissues which are sourced from a living organism
Biopsy
- Requires surgical materials/specimen
Biopsy
Biopsy:
Excisional
Incisional
-entire tumor removal
Excisional
-maybe partial, complete or by pieces removal
Excisional
-portion of tumor only
Incisional
-opening and cutting the tissue to obtain specimen/ through drainage of fluid secretion or exudates
Incisional
METHODS OF SURGICAL BIOPSY:
Fine Needle
Aspiration
Core Needle
Biopsy
Punch Biopsy
Shave Biopsy
Curretings
-simples and least invasive
Fine Needle Aspiration
-remove cells from the area of abnormality
Fine Needle Aspiration
-remove cells from the area of abnormality
Fine Needle Aspiration
- Less pain and may be done conveniently
Fine Needle Aspiration
- Not always adequate
Fine Needle Aspiration
- Disturbs cell architecture
Fine Needle Aspiration
-removes cells and small amount of surrounding tissues
Core Needle Biopsy
-uses vim-silverman needle (cutting needle)
Core Needle Biopsy
Provides additional information in lesion exam without surgery
Core Needle Biopsy
Trauma to surrounding tissues
Core Needle Biopsy
- Preferred for full-thickness skin specimen
Punch Biopsy
- Involves a circular blade, rotated down to epidermis dermis subcutaneous fat
Punch Biopsy
Punch Biopsy
- Yields ________ cylindrical tissue sample
3-4 mm
- No surgery
Punch Biopsy
- May result to reproduction of malignant cells when some portion of mass is removed.
Punch Biopsy
- Small fragments of tissues are scraped from the surface (skin).
Shave Biopsy
- Small fragments of tissues are scraped from the surface (skin).
Shave Biopsy
- For diagnosis of non-cancerous skin tumors
Shave Biopsy
Cosmetically safe
Shave Biopsy
Diagnosis/treatment of benign lesions
Shave Biopsy
- May result to excision if malignant lesion due to possible seeding.
Shave Biopsy
- Tissues are scooped/spooned from tissues that has grown from body cavities.
Curretings
- Most superficial mode of biopsy
Curretings
-scarring is minimal
Curretings
-lack of surgical margins
Curretings
- Incidence of local recurrence of abnormal cells.
Curretings
COMMON ORGANS SUBJECTED TO BIOPSY:
Breast
Lymph Nodes
Liver
Spleen
Exudates from cavities (pleural, peritoneal and pericardial)
Cervix Endometrial mucosa (uterus)
AUTOPSY PURPOSES:
-Determine the [?]
-Determine the [?]
-Preservation of [?] in cases of future examination or scientific study
- [?]
-Disclosure of [?]
-[?]
cause of death
final diagnosis
tissue from the dead body
Criminal prosecution
public health hazards
Civil justice
PHASES OF AUTOPSY
- External examination
- Determination of _______________ Length/height and weight
- Symmetry observation
- General nutritional status and preservation of tissues
- Inspection of body parts for identification of recent injuries ;scrutiny of unique marks of deeper significance 6. Posterior lividity Rigor mortisBody temperature 7. Microscopic examination
PRE-REQUISITES OF AUTOPSY PROCEDURES
_______________________
_____________________________ is stated and specified according to purpose and completeness.
[?] needed for the procedure
Written consent/permission
Type of autopsy procedure
Apparatuses or instruments needed for the procedure
PRE-REQUISITES OF AUTOPSY PROCEDURES
_______________________
_____________________________ is stated and specified according to purpose and completeness.
[?] needed for the procedure
Written consent/permission
Type of autopsy procedure
Apparatuses or instruments needed for the procedure
Rule 1
The signer must be:
Mentally competent
At least ______ [21]years old or married, or related to the deceased
a. If the deceased was married: ____________
Surviving spouse must sign
b. If no surviving spouse:_______________
Children who are at least 21 yrs old may sign
c. If no surviving spouse and child is either unmarried/under 21:______________
Guardian of the child may sign; if absent, the judge with jurisdiction may sign
d. If no surviving spouse nor child:__________________
• Father of the deceased
• Mother of the deceased
• Guardian of the deceased
• Next of kin
• Person with custody of the body and responsible for the burial
Rule 3
Prior to autopsy, there was an objection from a close relative:
Autopsy will NOT proceed. Inform the pathologist imediately
Rule 4 In cases of absence due to mental incompetency, age or relationship: (?) should be informed and consulted
Hospital administrator/person-in-charge
Rule 5
_______________________________________________must be present when permit is signed.
Two (2) witnesses (hospital employees)
TYPES OF AUTOPSY
According to Purpose
Routine Hospital Autopsy
Medico Legal Autopsy
According to completeness of the procedure
Partial
Complete
According to Incision
Straight-cut
Y-shaped
-conducted in private hospitals
Routine Hospital Autopsy
-conducted in private hospitals
Routine Hospital Autopsy
- Conducted at the NBI or government institutions for purposes of prosecution
Medico Legal Autopsy
-Examination is only on the permitted part/region of the body
Partial
- Examination is done in the whole body.
Complete
- Begins at the midline to suprasternal notch down to the pubis
Straight-cut
- Begins at the midline to suprasternal notch down to the pubis
Straight-cut
- Begins at both of the shoulder regions down to the xiphoid and incised downward to the pubis
Y-shaped
AUTOPSY TECHNIQUES
LETULLE’S
GHON’S
ROKITANSKY’S
VIRCHOW’S
“En masse”
LETULLE’S
VIRCHOW’S
“En bloc”
GHON’S
In Situ
ROKITANSKY’S
E.g. Oral, cervical, thoracic, abdominal and pelvic organs are removed as one large organ block
LETULLE’S
E.g: Cervical and thoracic organs (1 block), abdominal organs (1 block) and urogenital system (1 block) removed as separate blocks.
GHON’S
E.g: infected bodies with HIV, Hepatitis-B. Easy to perform in children.
ROKITANSKY’S
E.g. heart, lungs, uterus
VIRCHOW’S
Commonly done in medical colleges to teach student
LETULLE’S
Block by block dissection
GHON’S
-in combination with en bloc.
ROKITANSKY’S
- Removal of organ one after another.
VIRCHOW’S
TAKE EVERYTHING TOGETHER
LETULLE’S
ONE BLOCK AT A TIME
GHON’S
ALL AT ONCE THEN ONE BY ONE
ROKITANSKY’S
ONE BY ONE
VIRCHOW’S
FRESH TISSUE EXAMINATION:
Teasing/Dissociation
Squash Preparation (Crushing)
Smear Preparation
-dissection of specimen with a needle
Teasing/Dissociation
Teasing/Dissociation -Uses isotonic salt solution (________________________________)
Normal saline/Ringer’s Solution
-can be unstained or stained (supravital dye/Methylene blue)
Teasing/Dissociation
Teasing/Dissociation -permits cells to be examined in a __________________________
Living state
Squash Preparation (Crushing) -done in _____________ small tissues
<1mm
-sample is forcibly compressed with another slide or cover glass
Squash Preparation (Crushing)
-supravital stain is placed at the junction and absorbed through capillary attraction
Squash Preparation (Crushing)
-may be observed as fresh preparation or with supravital staining
Smear Preparation
-can be made permanent by fixing the sample while wet.
Smear Preparation
-preferred for thick secretions such as serous fluids, concentrated sputum and enzymatic lavage → cancer diagnosis
Smear Preparation
-accomplished using applicator stick or platinum loop
Streaking
-direct or zigzag line
Streaking
-attempts to obtain relatively uniform distribution of secretion
Streaking
-utilizes selected portion of the material
Spreading
-utilizes selected portion of the material
Spreading
-able to maintain cellular interrelationships of the material
Spreading
-recommended for fresh sputum, bronchial aspirate and thick mucous
Spreading
-a drop of the sample is placed on one slide and disperses upon the placement of another slide.
Pull-apart
-two slides are pulled at opposite direction, uninterrupted.
Pull-apart
-May be fresh or vital stained.
Pull-apart
-the surface of the freshly cut piece of tissue is bought in contact and pressed on to the surface.
Touch preparation (Impression smear)
-allows cells to be directly transferred onto the slide without destroying intercellular relationship
Touch preparation (Impression smear)
FROZEN SECTION
Slice required: thin slices _________________________
10-15 um
Microtome: kept at cold temperature ____________________
-10 to -20 deg celsius
Microtome components:
a.Freezing Microtome
b.Cold Microtome
Effect of slow freezing:
distortion of tissue (ice crystals)
METHODS OF FREEZING:
Liquid nitrogen
Isopentane
Carbon dioxide
Aerosol spray
-most common meyhod of freezing
Liquid nitrogen
-used with non fatty unfixed tissues at -10 to -25 degrees Celsius
Liquid nitrogen
-may result to crack due to rapid expansion; produces ice crystals artifact
Liquid nitrogen
-causes vapor phase which acts as an insulator= uneven cooling
Liquid nitrogen
-damage to the block and blade occurs if done at -70 degrees Celsius
Liquid nitrogen
-liquid at room temperature
Isopentane
-small crystals starts to form at -170 degrees Celsius
Isopentane
-excellent for freezing muscle tissue
Isopentane
-conventional freezing microtome gas
Carbon dioxide
-adequate in freezing small pieces of tissue (except muscle)
Aerosol spray
-e.g. Fluorinated hydrocarbons (Cryokwik) - rapid freezing
Aerosol spray
METHODS OF FROZEN SECTION:
COLD KNIFE PROCEDURE
CRYOSTAT PROCEDURE
-utilizes Carbon Dioxide technique
COLD KNIFE PROCEDURE
COLD KNIFE PROCEDURE -sample must be _________ thick
3-5mm
-applied with drops of gum syrup and several intermittent bursts of CO2 with 1-2 seconds duration, and 4 seconds interval
COLD KNIFE PROCEDURE
COLD KNIFE PROCEDURE **Dew Line: point at which sections may be cut at [?] thickness.
10 um
COLD KNIFE PROCEDURE ** Controlled environment:
Knife: ___
Tissue: ___
Environment : ___
-40 to -60 deg cel
-5 to -10 deg cel
0 to 10 deg cel
CRYOSTAT PROCEDURE
**Consist of: Refrigerated Chamber: __________________
-20 deg cel
CRYOSTAT PROCEDURE
Optimum working temperature of cryostat : __________________
-18 to-20 deg cel
-tissue must be sufficiently cold to prevent compression and displacement of cell.
CRYOSTAT PROCEDURE
-provides the simplest, quickest and least labor intensive
CRYOSTAT PROCEDURE
-routine for intraoperative and rapid diagnosis
CRYOSTAT PROCEDURE
-only provide individual sections, and does not form ribbons.
CRYOSTAT PROCEDURE
MOUNTING OF TISSUE BLOCK (Cryostat)
Recommendation labs:
Optimal Cutting Temperature (O.C.T) compound
Lab-Tek Products
Division of Miles Laboratories
MOUNTING OF TISSUE BLOCK (Cryostat)
Temperature settings:
____ for brain, lymph notes, liver, spleen, uterine, curreting, soft cellular tumors.
____for non-fatty breast tissue, ovary, prostate, tongue, and GI tract
____ for fatty breast and omental tissue
-5 to -15 deg cel (soft tissues)
-15 to -25 deg cel (muscle w/ fat)
-35 deg cel (pure fat)
Mounting media:
Synthetic water-soluble glycols and resins.
FREEZING PREVIOUSLY FIXED TISSUES
Cryostat section of [?] -> attaches easily to the slide without adhesive, with good preservation of enzymes.
fresh, unfixed tissue
is recommended for any cold sectioning of fixed material (e.g. Fats and lipids)
Cryostat
DO NOT adhere to slide during staining.
Formalin-fixed tissues
DO NOT adhere to slide during staining.
Formalin-fixed tissues
Formalin-fixed tissues:
-must be coated with [?] or;
-immerse the tissue block in boiling [?] for [?] prior to freezing and sectioning
albumin/ chrome-glycerin jelly
10% buffered formalin ; 1-2 minutes
Special fixatives : [?] Histochemistry and lipid demonstration
10% formol calcium at 4°C
Alcohol fixed tissues: washed in water for [?] before sectioning.
12-24 hours
SPECIAL PROCESSING TECHNIQUE:
FREEZE DRYING
FREEZE SUBSTITUTION
FREEZE DRYING
-done through “quenching” of tissue at _______ then subsequently followed by “desiccation” , then “sublimation” through vacuum chamber at _____________.
-160 deg cel
-40 deg cel
-produces minimum shrinkage and allows tissue to be processed in a fresh state.
FREEZE DRYING
-Used in: Enzyme studies (Hydrolytic) Demonstrating mucous, glycogen, and proteins.
FREEZE DRYING
-involves “dehydration” at low temperature.
FREEZE SUBSTITUTION
-fixed in Rossman’s or 1% Acetone then dehydrated in absolute alcohol.
FREEZE SUBSTITUTION
FREEZE SUBSTITUTION
-rapid freezing (3:1 propane-isopentane) at __________ is followed by substitution of water –free acetone and cooled to ____________
-175 deg cel
-70 deg cel
For best preservation: Substituting fluid should contain fixing agent and solvent for ice (e.g. 1% osmium tetroxide in acetone, mercuric chloride in ethanol or picric acid in ethanol)
FREEZE SUBSTITUTION
For best preservation: Substituting fluid should contain fixing agent and solvent for ice (e.g. 1% osmium tetroxide in acetone, mercuric chloride in ethanol or picric acid in ethanol)
FREEZE SUBSTITUTION
MANUAL TISSUE PROCESSING:
I. FIXATION
II. DECALCIFICATION
III. DEHYDRATION
IV. CLEARING
V. IMPREGNATION/INFILTRATION
VI. EMBEDDING (Casting/Blocking)
➢ Fixation
➢ Dehydration
➢ Clearing
➢ Infiltration
➢ Embedding
➢ Sectioning (+ Floating, Fishing-out, Drying)
➢ Staining
➢ Mounting
➢ Labelling
First and most critical step of tissue processing
FIXATION
FIXATION Purpose:
1. To [?] the tissue by stopping all cellular activity and let it be examined in a “life-like” state
2. To [?] of cellular elements by arresting autolysis and putrefaction.
3. To [?] protoplasmic substances.
preserve
prevent breakdown
coagulate or precipitate
FIXATION Purpose:
1. To [?] the tissue by stopping all cellular activity and let it be examined in a “life-like” state
2. To [?] of cellular elements by arresting autolysis and putrefaction.
3. To [?] protoplasmic substances.
preserve
prevent breakdown
coagulate or precipitate
FIXATION Rationale: To (?) the tissue from trauma of further handling.
harden and protect
Methods of Fixation:
Heat Fixation
Microwave Fixation
Cryopreservation
Chemical Fixation
-rarely used, application is restricted to smears e.g. boiling, poaching
Heat Fixation
-a form of heat fixation,
Microwave Fixation
-utilized best with commercial glycol-based fixatives not evaporate at 55 degrees Celsius.
Microwave Fixation
-usually in the form of freeze drvino
Cryopreservation
-useful in histochemistry (preserve cancer markers)
Cryopreservation
Classification of Fixation:
Additive Fixation
Non-Additive Fixation
- Fixative becomes part of the tissue due to cross linking or formation of molecular complexes = stable proteins
Additive Fixation
-not incorporated to the tissue but alters tissue composition and stabilizes the tissue by means of water removal (H-molecule) from proteins to allow new cross links to occur.
Non-Additive Fixation
-Prevents autolysis and makes tissue unsuitable for bacterial decomposition
Non-Additive Fixation
E.g. Formalin, mercury and osmium tetroxide
Additive Fixation
E.g. Alcoholic fixatives
Non-Additive Fixation
Classification of fixing agents:
- Coagulant Fixatives
- Non-Coagulant fixatives
-result in a permeable meshwork of protein strands
- Coagulant Fixatives
-result in a permeable meshwork of protein strands
- Coagulant Fixatives
-additive in nature; produce a less permeable gel
- Non-Coagulant fixatives
-MOST COMMON fixing agent
- Non-Coagulant fixatives
Mechanism of Fixation:
A. Denaturation
B. Addition and Cross-Link Formation
Most common mechanism of fixation
A. Denaturation
Non-coagulant fixing
B. Addition and Cross-Link Formation
Alcohol-induced/Acetone-induced dehydration
A. Denaturation
Chemicals react with proteins and cells to increase surface area
B. Addition and Cross-Link Formation
Removal of free water, then replacement by dehydrants in tissues
A. Denaturation
Binds to natural chemical in tissues
B. Addition and Cross-Link Formation
Irreversible
A. Denaturation
Staining is stable
B. Addition and Cross-Link Formation
FIXATIVES: General Consideration
1.Fixation of tissue after removal from the body: ____________________
2.Sufficient volume of fixative: _____________________ (best penetration)
3.Removal of anatomical barriers:[?]
4.Sectioning of large specimen: [?]
5. Replacement of contaminated fixatives: [?]
6. Reduction of tissue distortion by filling in the tubular structure with [?]
7. Tissue blocks: [?] for permeation of fixative
8. Avoid [?]: loss of immunohistochemical antigenicity
9. Maintaining the [?] prior to and after fixation is important
<1h
20:1 or 10:1
fascia, bone, feces, thick tissues
GIT tract, lungs (inflated with fixative)
Bile, blood, feces
corkboard or gauze
Small enough ( 4-6mm)
prolonged fixation
shape
FACTORS INVOLVED IN FIXATION:
Volume
Hydrogen ion concentration
Temperature
Thickness of section
Osmolality
Concentration
Time Interval
Fixation Volume __________________________
20:1 or 10:1
enhances fixative penetration
Agitation
Fixation Hydrogen ion concentration __________________________
pH of 6-8
Fixation Acidity: formalin heme pigment (?)
black deposits
Fixation Ideal buffers:
PO4, HCO3, Cacodylate and Veronal
Fixation Temperature
_________________________ _________________________
40 deg cel
0-4 deg cel
Formalin heated at __________________________
60 deg cel: used for urgent biopsy (but distorted)
deteriorate quickly
Brain cells
BM continues to undergo mitosis up to _______________when refrigerated.
30 mins
does not react in room temp
Nucleic acid
Thickness of section
______________ (EM)
______________ (LM)
[?] (solid material, e.g.liver)
1-2mm2
2cm x 0.4mm
10-15 mm
Rate:
______________ (EM)
______________ (LM)
2 cm2x 4mm thick completes at 4-6 hours
2-3 mm/hour
must be removed prior to fixation
Fecal matter and stomach contents
must be removed prior to fixation
Fecal matter and stomach contents
Brain is fixed in _________________for 2-3 weeks due to its structure.
10% buffered formalin
: best penetration
: Worst penetration
: in between
Formalin and Alcohol: best penetration
Glutaraldehyde: Worst penetration
Mercurials: in between
Slightly hypertonic solution:
Isotonic solution:
400-450 mOsm.
340 mOsm.
: Cell shrinkage
: Swelling, poor fixation
Hypertonic solutions
Hypotonic solutions
added to osmium tetroxide for electron microscopy
Sucrose
Concentration Lowest level possible
Formalin:
Formaldehyde: ______
Glutaraldehyde: _________________(for immuno electron micrsocopy)
10%
35%-40%
3% or 0.25
Too High Concentration:
produces artifacts
Fixation is done immediately (<1 hr) after removal from body/death to prevent
autolysis or putrefaction.
: poor quality of tissue
Longer blood supply interruption
Drying: Artefacts (moist with)
saline
A. SIMPLE FIXATIVES:
- ALDEHYDES
a. Formaldehyde
b. Glutaraldehye - METALLIC
a. Mercuric chloride
b. Chromate fixatives
-Potassium dichromate
-Chromic acid
c. Lead fixative
-Picric acid
-Acetic acid
-Acetone
-Alcohol
-Osmium tetroxide (osmic acid)
d. Heat
-made up of two or more fixative; has combined effect that acts on the cell and tissue constituents
B. COMPOUND FIXATIVES
B. COMPOUND FIXATIVES:
Haidensusa’s Fkuid
Carnoy’s Fluid Bouin’s
Fluid Formol saline
Buffered formalin
B. COMPOUND FIXATIVES:
Haidensusa’s Fkuid
Carnoy’s Fluid Bouin’s
Fluid Formol saline
Buffered formalin
TYPES OF FIXATIVES: According to composition
SIMPLE FIXATIVES
COMPOUND FIXATIVES
TYPES OF FIXATIVES: According to Action
• Lipids:
mercuric chloride or potassium dichromate
• Phospholipids:
Baker’s formol-calcium
• Cholesterol:
digitonin
• Carbohydrates:
alcoholic fixatives
• Glycogen:
Rossman’s fluid or cold absolute alcohol
• Proteins:
neutral buffered formol saline or formaldehyde vapor
• Electron microscopy:
double fixation
Used for electron and light microscopy
Glutaraldehyde
Made up of two formaldehyde residues (larger) slower penetration
Glutaraldehyde
Glutaraldehyde composition:
2% Glutaraldehyde + Osmium Tetroxide= EM
Must be cold and buffered (3 months life span)
Glutaraldehyde
Glutaraldehyde Recommended size:
</=1 mm
Glutaraldehyde Concentrations:
2.5% for small fragments
4% for larger tissues (<4mm)
• Rapid and irreversible changes (fixes quickly and at 4 deg cel)
Glutaraldehyde
• Not good for immunohistochemical
Glutaraldehyde
• Gives best overall cytoplasmic and staining nuclear detail
Glutaraldehyde
• More stable effect, less shrinkage
Glutaraldehyde
• More expensive
Glutaraldehyde
• Makes tissue brittle
Glutaraldehyde
- MOST COMMON FIXATIVE
35-40% Formaldehyde; 10% Formalin
(lung metaplasia with long term expo)
paraformaldehyde
Possible explosion if not neutralized/buffered (Calcium or
Magnesium carbonate)
35-40% Formaldehyde; 10% Formalin
Change regularly to prevent bleaching
Formalin
is added as preservative to formaldehyde
Methanol
: restores natural tissue color after fixation
70% alcohol
Formation of “formic acid”: Counteracted by
alcoholic picric acid or 1% KOH in 80% alcohol/ buffered formalin
Optimal choice
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
Recommended for preservation of surgical post-mortem and
research specimen
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
• Prevent alterations
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
• Less shrinkage
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
Hardens tissue better
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
Tissues are stored indefinitely
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
• Prolonged fixation causes bleach tissues
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
Fixative of choice for immunohistochemistry, molecular tests
10% Neutral Buffered Saline
Readily available, time consuming, stable
10% Neutral Buffered Saline
Compatible with stains
10% Neutral Buffered Saline
Preserves fat, mucin, glycogen
10% Neutral Buffered Saline
Used to preserve cadavers
10% Formol Saline Solution
