HISTOPATHOLOGY
preparation, processing and staining of tissue sections of tissue sections for microscopic study to be interpreted by the pathologist
HISTOTECHNIQUES
✓ study of disease at the tissue level
HISTOPATHOLOGY
examined to determine the cause of death
Autopsy Materials:
removal of cells from the area of abnormality
➢ considered as the simplest and least invasive method of collecting biopsy specimens
➢ method of collection for fluid-containing tumors
FINE NEEDLE ASPIRATION
removal of cells and small amount of surrounding tissue
CORE NEEDLE BIOSY
removal of cells with more surrounding tissue
INCISIONAL BIOPSY
EXCISIONAL BIOPSY
removal of the entire area in question
➢ Ensure complete removal of the lesion
➢ Confirm that the diagnosis is correct
OUTSIDE
removal of 3 to 4 mm cylindrical core of tissue samples
➢ small: 2mm; large: 4mm
➢ lesion should be at the center
PUNCH BIOPSY
removal of small fragments of tissue from a surface G.
SHAVE BIOPSY
removal of tissue or growths from body cavities
CURETTINGS
STORAGE 1. Specimen
1 month to 1 year
STORAGE 2. Tissue Blocks
3 to 10 years
STORAGE 3. Slides:
Indefinite
STORAGE 4. Records (request and result forms):
Permanent
FACTORS TO BE CONSIDERED IN CHOOSING A METHOD
1. Structural and chemical components to be studied
2. Nature and amount of sample to be evaluated
3. The need to provide an immediate diagnosis
➢ No fixative required
➢ Examined using a Brightfield or Phase-Contrast microscope
➢ Stained with supravital or differential dyes
METHODS OF PREPARATION AND EXAMINATION
A. Diagnostic Laboratories
1. FRESH TISSUE EXAMINATION
METHODS OF PREPARATION AND EXAMINATION
A. Diagnostic Laboratories
1. FRESH TISSUE EXAMINATION
ADVANTAGES
✓ Observation of physiologic processes or protoplasmic activities (motion, mitosis, phagocytosis and pinocytosis)
✓ Relatively simple and easy to perform
METHODS OF PREPARATION AND EXAMINATION
A. Diagnostic Laboratories
1. FRESH TISSUE EXAMINATION
DISADVANTAGES
✓ Limited use
✓ Liable to develop changes observed after death (putrefaction and autolysis)
✓ Dissection or separation of tissue components in NSS or Ringer’s solution
✓ Examined as stained or unstained
✓ Anatomical relationship is destroyed
TEASING
✓ Tissue (<1mm) is sandwiched between two slides
✓ Stain is applied on one side of the slide and allowed to spread via capillary action
SQUASH
▪ for cytological studies, especially for the diagnosis of cancer
▪ for sections or sediments
▪ performed using a wire loop, applicator stick or another slide
SMEARING
Uniform distribution in a direct or zigzag manner
Streaking
✓ Thick or mucoid specimens
✓ Teasing on a slide
✓ Maintains intercellular relationship
Spreading
For the preparation of blood and bone marrow smears
Pull-Apart
✓ One side of a slide is allowed to touch a surface of the sample
✓ Intercellular relationship is maintained
Touch
Preparation
✓ Prepared using freezing microtome or cryostat
✓ For rapid diagnosis
✓ For delicate specimens
FROZEN SECTION
METHODS OF PREPARATION AND EXAMINATION
A. Diagnostic Laboratories
A.2.PRESERVED TISSUE EXAMINATION
Initial Steps in Tissue Processing
➢ Performed by the medical technologist
➢ Check label and request form
➢ The specimen is given a label (numeric or alpha-numeric) which allows easy accessioning/identification
➢ Request form should have a provisional diagnosis and brief clinical details
A. Specimen Accessioning/Identification
METHODS OF PREPARATION AND EXAMINATION
A. Diagnostic Laboratories
A.2.PRESERVED TISSUE EXAMINATION
Initial Steps in Tissue Processing
➢ Performed by the pathologist
➢ Describing the sample macroscopically
➢ Weight and dimensions of the sample are determined
B. Gross Examination and Sampling
METHODS OF PREPARATION AND EXAMINATION
A. Diagnostic Laboratories
A.2.PRESERVED TISSUE EXAMINATION
Initial Steps in Tissue Processing
➢ Fixation
➢ Dehydration
➢ Clearing
➢ Infiltration
➢ Embedding
➢ Sectioning (+ Floating, Fishing-out, Drying)
➢ Staining
➢ Mounting
➢ Labelling
Tissue Processing
METHODS OF PREPARATION AND EXAMINATION
B. Research Laboratories
✓ Used to locate the presence and position of mineral elements in the tissue
✓ Two duplicate sections of alcohol-fixed tissues
MICROINCINERATION
METHODS OF PREPARATION AND EXAMINATION
B. Research Laboratories
✓ Injection of radioactive isotopes into organs
✓ Determines the relationship and location of the isotopes and cells to be studied
✓ Provides qualitative and quantitative information
AUTORADIOGRAPHY
✓ first and most critical step in tissue processing
✓ fixing or preserving fresh tissue for examination
✓ should be done immediately to preserve cellular and tissue morphology
FIXATION
Two Purposes of Fixation
-PRIMARY PURPOSE
1. Preservation
Two Purposes of Fixation
-SECONDARY PURPOSE
2. Protection
✓ capable of forming cross-links between proteins
➢ stabilizes tissue components making them insoluble to lysosomal enzymes
✓ capable of inactivating lysosomes
Fixative agents
Types of Fixative: becomes part of the cross-link itself
1. Additive
Types of Fixative: facilitates the removal of water in order for cross-links to form
2. Non-Additive
FIXATIVES RETARDED BY:
• Increase in size and thickness
• Presence of mucus
• Presence of fats
• Presence of blood
• Decrease in temperature
FIXATIVES ENHANCED BY:
✓ Presence of heat
✓ Decrease in size and thickness
✓ Presence of agitation
FACTORS TO BE CONSIDERED IN CHOOSING FIXATIVE
✓ pH
✓ Temperature
✓ Size and Thickness
✓ Osmolality
✓ Concentration
✓ Volume
FACTORS TO BE CONSIDERED IN CHOOSING FIXATIVE
✓ pH
6 to 8
FACTORS TO BE CONSIDERED IN CHOOSING FIXATIVE
▪ Routine Manual
▪ Routine Automated:
▪ Electron Microscopy:
▪ Formalin at 60 oC:
▪ Formalin at 100 oC:
▪ DNA:
▪ RNA:
▪ Routine Manual: Room Temperature (20-22'c)
▪ Routine Automated: 40'c
▪ Electron Microscopy: 0-4'c (Freezing Temprature)
▪ Formalin at 60'C: Very Urgent Biopsy
▪ Formalin at 100'C: Diagnosis for Tubercullosis
▪ DNA: 65'c
▪ RNA: 45'c
FACTORS TO BE CONSIDERED IN CHOOSING FIXATIVE
✓ Size and Thickness
Light Microscopy:
Electron Microscopy:
Lung Edema:
▪ Light Microscopy: 2cm2 by 0.4cm thick
▪ Electron Microscopy: 1 to 2 mm2
▪ Lung Edema: 1 to 2 cm thick
FACTORS TO BE CONSIDERED IN CHOOSING FIXATIVE
✓ Osmolality
-Light microscopy:
-Electron Microscopy:
✓ Osmolality
▪ Light microscopy: slightly hypertonic (400-450 mOsm)
▪ Electron Microscopy: more or less isotonic (340 mOsm)
FACTORS TO BE CONSIDERED IN CHOOSING FIXATIVE
✓ Concentration
▪ Formaldehyde:
▪ Glutaraldehyde:
▪ Pure Stock of Formalin:
▪ Formaldehyde: 10%
▪ Glutaraldehyde: 3%
▪ Pure Stock of Formalin: 40%
FACTORS TO BE CONSIDERED IN CHOOSING FIXATIVE
✓ Volume
▪ Routine:
▪ Museum:
▪ Routine: 10-25 x the volume of the specimen
▪ Museum: 50-100 x the volume of the specimen
Tissue/Organ Preparations:
✓ Cover with several layers of gauze
Air-filled Lungs
Tissue/Organ Preparations:
✓ Dilate with cotton soaked in fixative
✓ Completely open specimen
Hollow Organs
Tissue/Organ Preparations:
✓ Fix first before sampling
✓ Suspended by a cord tied under the Circle of Willis
✓ Intravascular perfusion using Ringer’s lactate
Brain
Tissue/Organ Preparations:
✓ Fixed whole
✓ Inject formol-alcohol
Eyes
Tissue/Organ Preparations:
✓ Lendrum’s Method: washed out with running water overnight and immersed in 4% aq. phenol solution for 1-3 days
Hard Tissues
Tissue/Organ Preparations:
✓ Stretched with sutures on each end
✓ Laid flat in a moist filter paper
Muscles
CHARACTERISTICS OF A GOOD FIXATIVE
1. Cheap & economical
2. Stable and safe to handle
3. Fast acting, permits rapid an even penetration
4. Inhibits bacterial decomposition & autolysis
5. Must harden tissues
6. At least isotonic
7. Must render tissues insensitive to subsequent processing
8. Must be compatible with many staining procedures
2 TYPES OF FIXATIVES:
According to Composition
According to Action
TYPES OF FIXATIVES:
According to Composition
1. Simple Fixatives:
2. Compound Fixatives:
TYPES OF FIXATIVES:
According to Composition: only one component of fixative
Simple Fixatives
TYPES OF FIXATIVES:
According to Composition: combination of 2 or more fixative
Compound Fixatives
TYPES OF FIXATIVES:
According to Action
Microanatomical Fixatives:
Cytological Fixatives
Histochemical Fixatives:
TYPES OF FIXATIVES:
According to Action
➢ permit general microscopic study of tissue structures and normal intercellular relationship of tissues
1. Microanatomical Fixatives:
TYPES OF FIXATIVES:
According to Action
➢ preserve specific cellular components
Cytological Fixatives:
TYPES OF FIXATIVES:
According to Action
type of Cytological Fixatives:
✓ preserve nuclear structures
✓ contain glacial HAc
▪ high affinity to nuclear chromatin
▪ destroys mitochondria and Golgi bodies
✓ pH 4.6 or less
Nuclear Fixatives:
TYPES OF FIXATIVES:
According to Action
type of Cytological Fixatives:
✓ preserve cytoplasmic structures
✓ do not contain glacial HAc
✓ pH of more than 4.6
Cytoplasmic Fixatives:
TYPES OF FIXATIVES:
According to Action
➢ preserve chemical constituents
a. Lipids:
b. Phospholipids:
c. Cholesterol:
d. Carbohydrates:
e. Glycogen:
f. Proteins:
g. Electron microscopy: double fixation
3. Histochemical Fixatives:
3. Histochemical Fixatives:
➢ preserve chemical constituents
a. Lipids:
b. Phospholipids:
c. Cholesterol:
d. Carbohydrates:
e. Glycogen:
f. Proteins:
g. Electron microscopy:
a. Lipids: Mercuric Chloride, Potassium dichromate
b. Phospholipids: Baker's Formol - Calcium
c. Cholesterol: Digitonin
d. Carbohydrates: Alcoholic Fixative
e. Glycogen: Rossman's Fluid, Cold Absolutr Alcohol (100% concentration)
f. Proteins: Neutral Buffered Saline/ Formaldehyde Vapor
g. Electron microscopy: double fixation
Mixture of Fixatives:
1. Electron Cytochemistry:
2. Acrolein:
1. Electron Cytochemistry: Karnovsky’s paraformaldehydeglutaraldehyde solution
2. Acrolein: mixture of glutaraldehyde or fomaldehyde
✓ rapid penetration, preserves morphology and enzyme activity at low concentrations
✓ immersion fixation of surgical biopsies
examples of fixative MICROANATOMICAL
• 10% Formol saline
• 10% Neutral buffered formalin
• Heidenhain’s SuSa
• Formol sublimate/corrosive
• Zenker’s solution
• Zenker-formol/Helly’s solution
• Bouin’s solution
• Brasil’s solution
examples of fixative CYTOLOGIC
NUCLEAR
• Heidenhain’s SuSa
• Newcomer’s
• Bouin’s
• Flemming’s
• Carnoy’s
examples of fixative CYTOLOGIC
CYTOPLASMIC
• Helly’s
• Orth’s
• Regaud’s/Muller’s
• Flemming’s
• Formalin with post-chroming
examples of fixative HISTOCHEMICAL
• 10% Formol saline
• Absolute ethanol
• Newcomer’s
• Acetone