DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: GPHCT

REVIEW OF BASIC HISTOLOGY INTRODUCTION TO GENERAL PATHOLOGY

Histology Study of tissues and their structure. Pathology (pathos: suffering; logos: study)

Germ Layers Group of cells that form during embryonic - study of diseases at cellular, tissue and organ level
A. Ectoderm development. The layers will eventually - bridge between mediZcine and science; it is the scientific
B. Mesoderm differentiate into various tissues and organs. foundation of medicine
C. Endoderm

Types of Tissues Divisions of Pathology

A. Epithelial Covering epithelia: Lines surfaces A. Gross Pathology Macroscopic examination of tissues
Glandular epithelia: secrete substances and organs
B. Microscopic Pathology
B. Connective Most abundant; Binds, protects, supports
i. Anatomic Surgical
•
C. Muscular Highly specialized; Movement Pathology Biopsy (living), Autopsy (dead)
•
 Histopathology
D. Nervous Sensation, integration, response ii. Clinical Pathology • Hematology
• Microbiology
• Clinical Chemistry
• Immunology/Serology
• Clinical Microscopy
Importance of Histology in General Pathology • Parasitology
Rudolf Virchow – father of modern pathology
 Disease processes affect tissues in distinctive ways, which
depend on the type of tissue, and the disease itself. Disease
 These processes may cause them to die, to change their - Any change from a state of health as a result of certain
shape, to divide, to move or to invade other tissues. Any of forms of stimuli and stress, which leads to impaired
these changes also affect the anatomy of the tissue. physiological functioning
 Understanding the changes that are characteristic of a
disease requires a detailed knowledge of the normal histology Four Aspects of a Disease Process
of cells and tissues, and the range of normality. Etiology Cause or origin of the disease; might be
 Knowing the type of tissue and their composition is important genetic factors or acquired factors
in the selection of the appropriate histopathologic technique Pathogenesis Mechanisms of the development of the
and stain to be used. disease
 These changes within cells and tissues can be visualized Sequence of events from initial stimulus to
using histopathologic techniques. ultimate expression of the disease
How etiologic factors trigger cellular &
molecular changes in a disease
Morphologic & Structural, biochemical and molecular
Molecular Changes alterations induced in the cells and organs of
the body as a result of the disease
Clinical Functional consequence of the changes
Manifestations
Signs Effects that can be observed by others
Symptoms Effects apparent only to the patient

Almost all forms of disease start with alterations in cells. Therefore, it

is important to study the causes, mechanisms and morphologic and
biochemical correlates of cell injury.
JALN2020
Stages of the Cellular Response to Stress and Injurious Stimuli CELLULAR ADAPTATION
- Changes made by a cell in response to stress or stimuli
- May be physiologic or pathologic

Forms of Adaptation
1. Hypertrophy
Increased Cell Size  Increased Organ Size
Due to increased protein synthesis
Most common stimulus: Increased Workload

Examples:
 bulging muscles of bodybuilders,
 estrogen-induced enlargement of uterus during pregnancy

Remember: *body builders win trophy*

2. Hyperplasia

Increased Cell Number  Increased Organ Mass

Due to proliferative actions of growth factor, and/or stem cells

Physiologic:
1. Normal cells have defined structure and can perform limited  Hormonal hyperplasia - Breast during puberty or
functions based on their specialization, metabolism, and pregnancy
availability of metabolic substrates. They can handle physiologic
demands through homeostasis.  Compensatory hyperplasia - Liver cells regeneration
Pathologic:
Homeostasis - act of maintaining a steady state
■ Excess Hormonal Stimulation
 Increased Estrogen  Endometrial hyperplasia 
2. When there is a slightly severe stress, or some pathologic
abnormal menstrual bleeding
stimuli, cells undergo adaptation in order to survive and
■ Excess Growth Hormone Stimulation
continue to function.
Papillomavirus mucosal lesions
Adaptation - reversible structural and functional response of 3. Atrophy
cells to stress and stimuli
Decreased Cell Size & Number  Reduce tissue/organ size
3. But if the limits of adaptive response are exceeded, or when cells Due to decreased protein synthesis, and increased protein
are exposed to injurious stimuli (agents or stress), or deprived degradation
of essential nutrients, cell injury occurs.
Physiologic: as in puberty when the thymus and the lymphoid
a. If the stimulus is mild and transient, the injury is
organs decrease in size
reversible. The cell may go back to its normal state.
e.g. Atrophy of uterus after pregnancy
b. If it is severe and progressive, the injury is irreversible.
Cells that undergo irreversible injury will ultimately suffer Pathologic (Types):
cell death, which may be pathological or physiological. 1. Atrophy of disuse - decreased workload, thus
diminished function of organ
Other types of stress can induce responses other than cellular e.g. skeletal muscle atrophy due to bedrest
adaptation, injury and death. The responses are the following:
2. Vascular - Diminished blood supply (ischemia)
A. Autophagy (self-eating) - starved cells eat its own components e.g. brain atrophy during atherosclerosis
during nutrient deprivation 3. Starvation - inadequate nutrition of cell
B. Intracellular accumulation of substances (such as proteins, e.g. muscle wasting (or cachexia) due to use of skeletal
lipids, hyaline, glycogen, pigments) muscle as source of energy during protein malnutrition
C. Pathologic calcification – abnormal tissue deposition of calcum 4. Loss of endocrine stimulation - due to decrease of
salts regulating hormones
D. Cellular aging – progressive decline in the life span and e.g. breast atrophy after menopause due to loss of
functional capacity of cells estrogen stimulation
5. Pressure - as in growth of tumors, atrophy occurs when
tumors suppress the blood supply or by directly putting
pressure to surrounding healthy cells
6. Exhaustion - due to increase in metabolism resulting to
increase of metabolites and loss of the actual cell space

JALN2020
4. Metaplasia APOPTOSIS
Change in one cell type to another - Induced by a tightly regulated suicide program in which
Due to reprogramming of existing stem cells in normal tissue, cells destined to die activate enzymes that degrade the
or of undifferentiated mesenchymal cells in order to cells' own proteins and nuclear DNA
withstand adverse environment - Presence of cleaved, active caspases (cysteine proteases
that cleave aspartic acid residue) is a marker for cells
Example: undergoing apoptosis
> Habitual cigarette smokers - ciliated columnar cells of trachea
- Cells break up into apoptotic bodies, which are tasty
and bronchi are replaced by stratified squamous cells
> Barrett esophagus - squamous cells of esophagus are targets for phagocytes
replaced by intestinal-like columnar cells in response to
refluxed acid Reasons for Apoptosis in Following Conditions:
Physiologic Eliminates cells that are no longer needed, or those
that have served their purposes
Pathologic Eliminates cells that are injured beyond repair
CELLULAR INJURY without eliciting host reaction

- Alteration in cell structure or function due to stress or

pathologic stimuli NECROSIS
- Pathologic cell death
Necrotic - tissue or organs with large numbers of dead cells
Causes
Hypoxia Immunologic Reactions Types of Necrosis According to Morphology
Physical Agents Genetic Abnormalities
Chemical Agents and Drugs Nutritional Imbalances 1. Coagulative Tissue is firm because architecture of dead tissue
Infectious Agents is preserved
Eosinophilic due to denaturation of proteins AND
enzymes
Morphological Alterations in Cell Injury Occurs on affected tissue when vessel is
obstructed, except brain
Generalized swelling of cell and organelles - first manifestation
Blebbing of plasma membrane Infarct - localized area of coagulative necrosis
Detachment of ribosome from ER 2. Liquefactive Tissue becomes liquid viscous mass due to
Clumping of nuclear chromatin digestion of dead cells
Occurs during microbial infection
CELL DEATH Creamy yellow because of pus
- Occurs after irreversible injury Affects CNS
3. Gangrenous Due to ischemia and superimposed bacterial
Difference between the Two Principal Pathways of Cell Death infection
Combination of coagulation and liquefaction
Apoptosis Necrosis necrosis
Cell Size Reduced Enlarged i. Dry Sterile necrosis
Nucleus Fragmentation Pyknosis (clumping)  Gangrene Arterial occlusion, sharp demarcation line, less foul
into Karyorrhexis odor
nucleosome-size (fragmentation)  ii. Wet Nonsterile necrosis (due to bacterial infection)
fragments Karyolysis (dissolution) Gangrene Vein occlusion, no sharp demarcation line, foul
Plasma Intact Disrupted odor
Membrane 4. Caseous Means ‘cheese-like’
Cellular Intact Enzymatic digestion; may Friable white appearance of necrotic area
contents leak out of cell Seen in tuberculosis, granuloma
Adjacent No Frequent (due to leakage of
(because cellular contents) 5. Fat Fat destruction due to pancreatic lipase
Inflammation
 Fatty Acids + Calcium = Chalky-white areas
phagocytes
rapidly devour the (fat saponification)
cells) Seen in acute pancreatitis
Physiologic Physiologic Pathologic 6. Fibrinoid Seen in immune reactions when antigen- antibody
or complexes are deposited in walls of arteries
 Immune complex + fibrin = fibrinoid (bright
Pathologic? Death by destiny Death by disease pink and amorphous appearance in H&E
staining)

JALN2020
 INFLAMMATION
- A protective universal response to tissue Classes of Inflammation
damage (mechanical trauma, tissue 1. According to Duration
necrosis, infection)
- May be beneficial or harmful i. Acute Sudden onset; usually mild and self-
Functions: limiting; Polymorphonuclear (PMNs)
1. contain damage & isolate injury cells
2. destroy cause of injury (microorganism/toxins)
ii. Chronic Involves persistence of injurious
3. destroy resulting necrotic cells and tissues
agent; often severe and progressive;
Mononuclear cells
4. prepare tissue for healing & repair
2. According to Character of Exudate
Harmful effects: i. Serous Out-pouring of relatively protein-poor
1. Digestion of normal tissues fluid; common in cavities
2. Swelling
3. Inappropriate inflammatory response
ii. Fibrinous More severe injury 
more vascular permeability 
more protein leaking (such as
Cardinal Signs:
fibrinogen which is the precursor of
1. Rubor – redness
fibrin)
2. Calor – heat
iii. Catarrhal Increased blood flow to mucosal
3. Tumor – swelling vessels, enlargement of secretory
4. Dolor – pain vessels  discharge of mucus and
5. Functio laesa – loss of function epithelial debris
Components of Inflammation iv. Hemorrhagic Disruption of blood vessel wall 
leakage of large number of RBCs
1. Vascular Reaction
i. Vasoconstriction Occurs first and lasts only for v. Suppurative / Mainly due to bacterial infection or
seconds Purulent secondary condition

ii. Vasodilation Increased diameter of blood Collection of large amount of Pus

vessels  Composed of :
Increased blood flow to area  Neutrophil
Erythema (heat and redness on Necrotic cells
site of infection) Edema fluid
iii. Endothelial Increased vascular permeability 
Activation Edema (extravasation of liquid Abscess = collection of pus
portion of blood)
3. According to Location
2. Cellular Response
i. Neutrophil WBCs enter site of injury i. Localized One site; not widespread
Activation  Kill organism, mop debris
 Release chemokines ii. Generalized / Whole organ; area of tissue; region
(substances that attract other Systemic of tissue
immune substances to site of
inflammation)

Sequential Steps of a Typical Inflammatory Reaction

1. The offending agent is recognized by host cells and
molecules.
2. WBCs and plasma proteins are recruited from the
circulation (Intravascular), and go towards the area where
the offending agent is located.
3. WBCs and proteins are activated and work together to
destroy and eliminate the offending substance.
4. Reaction is controlled and terminated.
5. Damaged tissue is repaired

Note: If inflammatory reaction is not controlled, adverse tissue effects

may occur, such as abscess formation and chronic inflammation.

JALN2020
 ABNORMALITIES IN CELL GROWTH Classification of Tumor

I. Retrogressive Changes - organs are smaller than the normal Benign Malignant
Death Usually does not Usually causes death
A. Developmental Defects
cause death except
1. Aplasia - incomplete development of the organ
for infants and brain
2. Hypoplasia - failure of an organ to develop fully tumors
3. Agenesia - complete non-appearance of an organ Differentiation Well-differentiated Some lack of
4. Atresia - failure of an organ to form an opening differentiation
Rate of Growth Usually progressive Erratic; may be slow
B. Atrophy - acquired decrease of the size of a normally and slow; may come to rapid
developed organ to a standstill or
regress
Metastasis Absent Frequent
II. Progressive Changes - organs become larger than normal
(spread of tumor
A. Hypertrophy - increase of cell size
to other sites)
1. True - due to increase work load or endocrine
stimulation General Tumor Nomenclature
a. Compensatory - true for paired organs, where Origin Benign Malignant
one is excised and the other incresases in size Epithelial Tissue -carcinoma
to “take responsibility’ for its pair Connective/ -sarcoma
-oma
2. False - due to ECF buildup and CT proliferation Mesenchyme
Tissue
B. Hyperplasia - increase in cell population
1. Physiologic, as in pregnancy
Example: Benign Malignant
2. Pathologic, as in typhoid fever affecting lymphoid
Epithelial lining of Adenoma Adenocarcinoma
follicles gland
Lymph vessels Lymphangioma Lymphangiosarcoma
III. Degenerative Changes – changes in the adult form of cell
A. Metaplasia - replacement of one cell type of cells to
another type in the same site (reversible) Grading of Tumors
B. Dysplasia – means “disordered growth”; development of - Attempts to establish an estimate of the level of
abnormal cell types within a tissue (reversible) malignancy of a tumor
C. Anaplasia – lack of differentiation of cells (irreversible) - Based on cytologic differentiation of tumor cells and
D. Neoplasia – means “new growth”; uncontrolled number of mitoses
proliferation of cells with no purpose; due to carcinogens, - Different from Staging, as staging accounts for size, extent
or DNA alteration (irreversible) of spread to lymph nodes, and presence of metastasis
- Well-differentiated: tumor cells resemble normal cells
 Tumor/Neoplasms – mass of neoplastic cells - Undifferentiated: tumor cells do not resemble normal cells

Broder’s Grading of Tumors

General Characteristics of Tumors
Grade Differentiated Undifferentiated Cells
1. May resemble and function like a normal cell Cells
2. Autonomous; non-responsive to normal growth
I 100% - 75% 0% - 25%
factors
II 75% - 50% 25% - 50%
3. Parasitic nature; competes with cells for metabolic
III 50% - 25% 50% - 75%
needs
IV 25% - 0% 75% - 100%
Parts of Tumor
1. Parenchyma – actively dividing cells Limitation of Grading:
2. Stroma –connective tissue framework and lymphatic
and vascular channels 1. It is subjective
2. Higher grades of tumor have more tendency to
metastasize
3. Most sarcomas cannot be graded

JALN2020
 SOMATIC DEATH
 Refers to the complete cessation of metabolic and functional
abilities of an organism 4. Post-mortem Clotting
 Occurs immediately after death; apparent only in autopsy
Primary Changes of Death (CRC) Post-Mortem Clot Ante-Mortem Clot
 Occurs 4-6 minutes, then death follows Upper layer is clear Has tangled,
1. Circulatory failure – start of death when cardiac function (resembles chicken irregular fibrin
ceases; flat electrocardiogram (ECG), and/or absence of fat); RBC settles at
heartbeat is indicative Appearance the lowest part of
the blood vessel
2. Respiratory failure – decrease O2 and increase CO2; loss of all (resembles currant
processes necessary for life; absence of respiratory sounds and jelly)
movements is indicative
Assumes blood Seldom assumes
Shape
3. CNS failure – loss of coordination and reflexes; absence of vessel shape blood vessel shape
brain stem reflex, and/or electroencephalogram (EEG) activity is Consistency Rubbery Non-rubbery
indicative

Secondary Changes of Death (ARLPDPA)  The next 3 stages of death occurs simultaneously and leads to
1. Algor Mortis the total digestion of cells
 Cooling of the body; decrease in temperature
 Equalizing of the body temperature to the external temp 5. Dessication
 Normal rate of cooling: 7OF/hr  General drying and wrinkling of fluid-filled organs;
 Sped up by: cold environment, malnutrition/dehydration,  most evident in the cornea, and anterior chamber of eye
severe hemorrhage
 Slowed by: fever, extreme physical activity before death 6. Putrefaction
 Decomposition of body carried out by microbial action
2. Rigor Mortis (normal flora from gut migrates to blood vessels and
 Stiffening of muscles due to lack of ATP (ATP is spreads all over the body)
responsible for driving calcium ions back to sarcoplasmic  Principal agent: Clostridium welchii (gram-positive,
reticulum of muscles) anaerobic, rod-shaped)
 First appears in the involuntary muscles of heart  First external sign: Greenish discoloration of skin over the
 Observed in eyelids, followed by neck, then lower right iliac fossa due to bacterial hydrogen sulfide reacting
extremities with hemoglobin, thus forming sulphahemoglobin which
 Starts 2-3 hrs post-mortem, completes 6-12 hrs post- stains the area green
mortem; persists for 3-4 days  Eventual production of foul-smelling gas due to invasion of
 After 3 to 4 days, relaxation occurs due to breakdown of saprophytes, which leads to distension of abdomen,
contracted muscles swelling of face and genitalia, and liquefaction of internal
organs
 Factors: muscle activity by the time of death;
 Most resistant to putrefaction: prostate gland
 Sped up by: warm environment; infancy; thin-layered
muscles
7. Autolysis
 Slowed by: cold environment; obese people
 “Self-destruction”; the self-digestion of the cells by their
own enzymes;
3. Livor Mortis/Sugillation
 First external sign is the whitish appearance of cornea
 Purplish discoloration of skin due to blood stasis
 Lividity of the dependent portions of the body due to
settling of blood to the lowest parts of the body at the time
of death
 Blood vessels dilate due to loss of muscle tone
 Difference of Livor Mortis from Ecchymosis References:
Livor Mortis Ecchymosis 1. Kumar, V., Abbas, A. K., & Aster, J. C. (2015). Robbins
Post-mortem stasis Trauma and Cotran pathologic basis of disease (Tenth edition).
Cause
of blood Philadelphia, PA: Elsevier/Saunder
After application of Discoloration No disappearance 2. Shedge, R., Krishan. K., Warrier, V., & Kanchan, T.
pressure (Blanching disappears (2019). Postmortem Changes. In StatPearls [Internet].
test) StatPearls Publishing
After incision Has oozing No oozing

JALN2020
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: GPHCT

HISTOPATHOLOGY QUALITY IN HISTOPATHOLOGY LABORATORY

- The study of tissues affected by disease
- Useful in making a diagnosis and in determining the
severity and progress of a condition
HISTOPATHOLOGIC TECHNIQUES
- Includes all activities done in the laboratory in order to
produce a suitable specimen slide for viewing by the
pathologist
THE HISTOPATHOLOGY LABORATORY
Quality Assurance
- ensuring that everything is right (test, time, specimen,
patient, diagnosis & price)
- Includes availability of reagents, supplies, preventive
maintenance & monitoring of equipment & evaluation of the
quality of services.
Quality Management Systems
— set of coordinated activities to regulate a lab in order to
continually improve its performance
— skilled personnel
— considers pre-analytic, analytic and post-analytic phase
— proper specimen collection & processing of results &
documentation
— highly quality of reagents & equipment
— continuous professional education of staff

Sample Workflow in the Histopathology Laboratory

HR Management
1. Pathologist
- Head of laboratory

2. Associate Pathologist
- Supports the pathologist

3. Histotechnologisy or Histotechnician
- Provides slides that are properly labeled, processed,
stained, mounted and sequenced
- Ensures that formalin and other agents are fresh and
in good working quality
- Maintains equipment in high quality condition
- Performs preventive maintenance, as well as
troubleshooting procedures

Laboratory Services
1. Tissue Processing
2. Cytology
3. Frozen Biopsy
4. Special Staining
5. Immunohistochemistry

JALN2020
Documents
- numerically, alphabetically, or chronologically arranged

A. Request form
1. Name, Age, Sex, Date of Birth
2. Hospital or Lab Accession #
3. Specimen Type/Source; Clinical Impressions
4. Pertinent History, Operative Findings
5. Test Requested, Procedure performed
6. Date &Time of Request, Collection and Transport
7. Requesting Physician

B. Patient Report: Request Form info + Diagnosis and Gross/

Microscopic findings + Name of pathologist

Types of Results
1. Surgical Pathology
2. Cytopathology
3. Autopsy Report

Turnaround Time (TAT) of Results

• Surgical Pathology and Cytology = 2 days
• Frozen Sections = 5-15 minutes
• Autopsy Report = 7 days

C. Telephone Report - preliminary diagnosis

D. Prelim Report - status of results 48-74 hours from receiving
E. Final Report - conveys results after test is completed
F. Incident Report – documents occurrence of problems

EXAMINATION OF TISSUES
Tissues for examination are usually obtained through biopsy, or autopsy. They range from whole organs or very large specimens to tiny fragments of
tissue.

Specimens Received

Autopsy Biopsy

Dissection Techniques Methods of Examination

Letulle Ghon Rokitansky Virchow Fixed Fresh

Frozen
Smear Prep Crushing Teasing
Sections

Touch Prep Pull-apart Spreading Streaking

JALN2020
 AUTOPSY Proper Handling of Autopsy Specimens

- AKA Necropsy; Thanatopsy 1. Organ block removed from the body cavity should be
- Post-mortem examination of tissues thoroughly washed of blood using cool or cold water to
minimize the blood staining of organs. Never use hot water.
Purposes 2. Organ blocks are placed in a large enameled pot containing
fixative, filled to about one third capacity.
- Determine cause of death and extent of injury
3. Tissues should not be pressed against each other or the
- Uncovering existence of an undetected disease
bottom or walls of the container.
4. Lesions that are encountered during dissection should be
Types of Autopsy according to:
obtained early and placed in fixative before organ is fully
incised.
 Medical/Hospital – performed on a patient
who dies in a hospital during course of treatment
Purpose  Medico-legal – generates evidentiary
document that forms a basis for opinions
rendered in a criminal trial, civil suit and the like

Completeness  Partial
 Complete
 Y-shaped
Manner of Incision  Straight Cut (I-shaped)

Dissection/Evisceration Techniques
Virchow - One by one removal of organs
- most widely used
Rokitansky
- "in situ" (in place) dissection, followed by en bloc
removal
Ghon - "en bloc" removal
- Organs of same group/cavity/region are
removed at the same time
Letulle - "en masse" removal of organs
- All organs are removed at the same time, then
dissected by blocks

Prerequisites to Performing Autopsy

 Written or informed consent from the legal next-of-kin
Order of priority: spouse, adult child, either parent, adult
sibling, grandparent, guardian
 Medical abstract or clinical data
 Autopsy Request (suspicious evidence of foul play)

Personnel
 Coroner – a public official who is empowered to order an
inquest into the manner or cause of death
 Prosector – pathologist who performs the dissection
 Diener – comes from German word “leichendiener” meaning
“servant of the dead”; assists during autopsy, and assumes
many and varied responsibilities in the autopsy laboratory

JALN2020
 BIOPSY compressed between two slides
- May be stained using supravital dye
- Ante-mortem examination of tissues
(ante=before; mortem=death) 3. Smear
- Examination of tissue sample from the living - Method depends on nature of material to be examined
- Useful for cytological examinations
- Cellular materials are spread over a slide using wire loop,
Types of Biopsy (but not limited to the following)
applicator stick or slide
Fine needle Simplest, least invasive
- Vital stains may also be applied
aspiration (FNA) Uses very thin needle attached to syringe to take
- May be made permanent by fixing them
out small amount of fluid and tissue from area
i. Streaking
Core needle Uses slightly larger needle •Rapid, but gentle zigzag application of the material
Remove small column of tissue (1/16 inch in throughout slide
diameter, ½ inch long • Must have a relatively uniform distribution
Incisional Surgical; Small part of a large lesion or tumor is ii. Spreading
taken • Material is gently spread onto the slide, and the mucous
Excisional Surgical; Entire affected area is taken strands are teased apart using an application stick
• ADV: maintains intercellular relationships
Punch For skin; Uses circular blade to obtain deeper skin • For fresh sputum, bronchial aspirates, and thick mucoid
sample that removes a short cylindrical core of secretions
tissue (“apple core”)
iii. Pull-apart
Shave For skin; small fragments of outer layers of skin • A drop of the material is placed into a clean slide, and
are “shaved” or scraped covered with another clean slide. Material is allowed to
disperse evenly
Currettage Tissues are removed from body cavity (or canals)
• Slides are then pulled apart with a single, uninterrupted
using a curette (instrument with a tip shaped like a
movement in opposite directions
small scoop or hook)
iv. Touch Prep
• AKA Impression Smear
Methods of Examination of Biopsy Specimens • Surface of a freshly cut tissue is pressed to a clean slide
Fresh • For Phase-Contrast Microscopy
• ADV: maintains intracellular relationships
Fixed (Preserved)
4. Frozen Sections
- For rapid diagnosis of tissue
FRESH TISSUE EXAMINATION - Requested during intra-operative procedures to help
surgeon in choosing his next plan of action
- Allows examination of cells in their living state - Recommended for demonstration of lipids and nervous
- ADV: View protoplasmic activities (motion, mitosis, tissue
phagocytosis, pinocytosis) - Fresh tissues are frozen using a cryostat or freezing
- DADV: Subject to ischemia, therefore not permanent, and liable microtome
to changes - ADV: rapid processing time with less equipment
requirement, and less need for ventilation
Methods - DADV: relatively poor quality of the final slide; expensive
1. Teasing (Further discussed in “Rapid Processing Techniques” topic)
- AKA Dissociation
- Selected tissue is immersed in petri dish/watch glass
containing isotonic solution, and then carefully dissected
and separated using needle or applicator stick
- Tissue is then transferred to slide and examined under the
microscope (phase contrast or bright field)
- May be stained using supravital dyes
2. Squash Preparation
- AKA Crushing
- Small pieces of tissue with diameter less than 1mm are
JALN2020
 FIXED TISSUE EXAMINATION INSTRUMENTATION

- The end goal is to produce a tissue section of good quality that Before tissues are mounted on slides, they should undergo different
allows for adequate interpretation of microscopic cellular processes. Each step in the process necessitates the use of various
changes (for diagnosis) specific components.
- Accomplished by fixing the tissues and carefully processing I. Microscope – for tissue and cell visualization
them to preserve their structures, then impregnating them with
• Bright Field Microscope – simplest and most popular
hardening substance to permit making thin slices suitable for
staining and microscopic evaluation • Dark Field Microscope
- For unstained and transparent samples
Tissue Processing - Only oblique rays hit the object
- various steps required to take the tissue from fixation to the - Samples are made brightly lit against dark
state where it is completely infiltrated with a suitable background
histological wax and can be embedded ready for section • Phase Contrast Microscope
cutting on the microtome - Phase shifts in light passing through transparent and
1. Fixation colorless specimen are converted into brightness
• Preservation of tissue constituents in a life-like manner as (contrast) changes in the specimen, making them
possible visible
- does not require staining
2. Decalcification (optional)
• Removal of calcium or lime salts from bones following • Polarizing Microscope
fixation - Designed to examine birefringent properties of
• For ease of cutting anisotropic specimens
3. Dehydration - Birefringence: splitting of one ray of light into two
• Removal of water - Anisotropism: substances that exhibit different
properties when measured in different directions
4. Clearing/Dealcoholization - NOTE: Amyloid in Congo Red has Apple Green
• Removal of the dehydrating agent with the use of a birefringence
clearing agent (a solvent miscible with both the dehydrant
and impregnating material)
• Fluorescence Microscope
5. Infiltration/Impregnation - Uses ability of substance to exhibit fluorescence
• Replacement of clearing material with impregnating - Fluorescence: emission of low frequency light of
material substances when they are illuminated with high
• Gives firm consistency to tissue for ease of handling and energy light
sectioning - Only allows observation of the specific structures
6. Embedding which have been labeled for fluorescence
• Placing the infiltrated tissue into a mold filled with molten - NOTE: Acridine Orange stains ssDNA – green
wax to form a solid tissue block fluorescence, and ssDNA or RNA (cytoplasm) – red
fluorescence;
7. Trimming
 Removal of excess wax from tissue block, in preparation for • Electron Microscope
microtomy - Uses a beam of accelerated electrons as source of
illumination
8. Section-Cutting/Microtomy
- Has higher resolving power than light microscopes
• Cutting of tissues into fine tissue sections with the use of a
and can reveal the structure of smaller objects
microtome
9. Staining II. Microtome – for producing tissue ribbons or sections
Main Parts:
 Application of dyes to sections for visualization a) Block Holder
10. Mounting b) Knife Holder
 Use of media to mount a coverslip for ease of handling, c) Pawl and Feedwheel Mechanism
storage and protection of sectioned tissue Kinds of Microtome
11. Labelling
 Indicating of accession number and year for proper [Things to know: Thickness of the tissue produced, Inventor
Microscope to be used, Embedding Medium]
identification
a) Rotary – most commonly used; for routine and serial
(continuous) sections; knife is stationary
JALN2020
 Thickness: 3 to 5 micrometers - others use vacuum in heat to speed up procedures
Inventor: Minot
Microscope: Light Microscope Main Types
Embedding material: Paraffin a. Tissue-Transfer Machine (“dip and dunk”): specimens are
b) Cryostat – consists of a microtome (usually rotary), kept transferred from container to container
inside a cold chamber maintained at -5°C to -30°C b. Fluid-Transfer Machine (“enclosed”): specimens are held
(average: -20°C); capable of freezing fresh tissues, thus in a single chamber, and then fluids are pumped in and
used for STAT diagnosis out as required
c) Sliding – knife is moving
d) Freezing IX. Gross Table
e) Rocking - for gross examination and dissection of submitted
f) Ultrathin specimens. It should have:
a) Sink
(Further discussed in “Trimming and Microtomy” topic) b) Table Top
c) Water Supply
III. Microwave Oven
d) Irrigation System
- best for antigen preservation
e) Fume Ventilation
- used in epitope retrieval for Immunohistochemistry
f) Waste Disposal Unit
- used in speeding up procedures
- agitation and heating will increases fixation rate
(Further discussed in “Gross Examination” topic)
Others: Incubator Oven X. Others
- in situ hybridization and enzyme reactions 1) Wares: Coplin jars, staining racks, staining dishes (all
- Thermal Requirements: 37°C three are for staining) and other glass and plastic materials
(for storage, and preparation of solutions)
IV. Automated Stainers 2) pH meters - measurement of solution and buffers
- Eliminate the need for the laborious manual staining 3) Grossing tools – for gross examination
- May operate through: 4) Freezer (Thermal Requirement: -20°C)
o Dipping the slide into the stain; and/or 5) Refrigerators (Thermal Requirement: 4°C)
o Applying the stain to the slides

V. Slide Dryers
- removing water collected during sectioning (water from
flotation bath)
- Thermal requirement: 5-10°C HIGHER than the melting
point of paraffin
- NOTE: Overheating causes uneven staining, artifacts
formation and tissue destruction
VI. Flotation Bath
- fishing out of tissue section; keeps sections from wrinkling
- has “black” interior, for easy visualization of sections
- Thermal requirement: 5-10°C LOWER than the melting
point of paraffin

VII. Embedding Centers

- System designed for paraffin embedding
- Thermal requirement: 2-4°C higher than melting point of
paraffin
Components:
a) Paraffin dispenser and reservoir
b) Orientation stage
c) Chilling plate
d) Warm storage (for embedding mold)

VIII. Automated Tissue Processor

- may be stationary or moving
- equipped with alarm and warn technicians if high temp
JALN2020
 STORAGE, RETENTION, AND DISPOSAL o Patient confidentiality must still be observed
o Slides may be used by resident pathologists and med
Surgical Specimen students; may be used for further researches
- Storage
o After grossing and cutting sections for fixation, specimen Retention Periods
must be returned to their original (leak-proof and airtight) - As suggested by College of American Pathologists (CAP), 2010
containers, a safety measure.
Records Storage
■ Prevents fume formation
■ Ensures fixative is at right level Requests, Accession logs, 2 years
o Group specimens according to date for ease of finding Maintenance and QC logs
o Storage area must be free from human and animal BB Quality Control 5 years
interference.
BB employee signatures, patient 10 years
- Disposal records, donor and recipient records
o Placed in biohazard bags for incineration or burial Records of indefinitely/permanently Indefinitely
- Returning of Specimen to Patient deferred donors; forensic accession
o Guidelines must be according to the hospital logs
■ Documentation of:
• Release to patient or authorized representative Specimens Storage
• Details of specimen
• Proper info of hazards of the tissue and fixative Urine 24 hours
• Proper disposal
Serum and other body fluids 48 hours
o Limbs and fetuses are buried for religious purposes
o Bullets, breast implants, and foreign bodies may be Microbiology and blood 7 days
evidences for crime; released to appropriate police smears (routine)
authorities and with properly documented with Chain of BB donor/recipient (blood) 7 days post-transfusion
Custody (COC) form
o Gallstones, foreign bodies, orthopedic hardware and teeth Surgical Tissues 2 weeks after final report
o Respect patient confidentiality Cytogenetic slides 3 years

Paraffin Blocks and Slides Cytology slides (e.g. pap) 5 years

- Storage Tissue, BM, FNA slides 10 years
o Must be in cool, dry place (otherwise, paraffin may melt or
combust)
Paraffin blocks 10 years
o Free from vermin and insects(may be eaten to access
tissue) Forensic: Blocks, Slides Indefinitely
o Paraffin blocks may form amorphous masses that may
obscure ID
o Arranged according to year and surgical pathology Reports (results) Storage
accession number for easy retrieval
Clinical pathology (e.g. CC, Hema) 2 years
- Request for Blocks and Slides
o Borrowing slides/blocks to be viewed by another Anatomical/Surgical pathology 10 years
institution)
o Logbook must be available (Patient data, accession Cytogenetics (final reports and 20 years
number, purpose of borrowing, recipient, quantity, date of photographs)
release and return, proper signatories, pathologist who
screened the slides) Forensic autopsy Indefinitely
o Slides are transported with damage and breakage
prevention; having insulation and shock-proof systems
References:
o Deposit is a customary practice. Charging for new slides 1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
and cuts is also practiced. (2nd Revised Edition). Makati, PH: Katha Publishing
o Preserve the original state of the material (611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
- Disposal (2015). Basic histopathologic techniques. Metro Manila,PH:
C & E Publishing
o Slides should be kept in sharp containers
3. Waters, B. L. (Ed.). (2010). Handbook of autopsy practice.
o Paraffin blocks are treated as infectious, thus stored in Springer Science & Business Media
pathologic waste bags
JALN2020
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: GPHCT

PRE-ANALYTICAL FACTORS
Specimen Accessioning
Ischemia Time
- Time interval between surgical intervention and proper fixation of
the removed specimen
- If prolonged, ischemia will allow activation of tissue enzymes,
autolysis, and degradation of proteins and nucleic acids, which
affects visualization of tissues
- Divided into:
a) Warm Ischemia Time
- Occurs during operation when blood supply of tissue is cut
off
- During this period, the tissue is alive and active, but will
undergo progressive metabolic stress due to hypoxia
- Affected by the whole surgical procedure (complexity of
procedure, ability of surgeon, modality of intervention)
- Beyond the control of the histopathology lab
b) Cold Ischemia Time
- Interval between tissue removal from the patient and arrival
in the pathology laboratory for grossing
- If prolonged, temperature of specimen will gradually reach
the external temperature, and autolysis and drying of the
surface may occur
- Extensions may contribute to poor fixation
- 1st and most important step in HP outside the tissue processing
procedures
- Specimens are given a unique identification number (may be a
barcode, for some laboratories) that will identify each specimen
for each patient
- Also useful for ease of retrieval of specimens, slides, and blocks
- Indicating codes may be used for the following
o Surgical
o Autopsy
o Cytology
- Sample Format of Accession Number:
Indicating Code – Year – ID Number of Specimen
E.g. #S20-12345
- Avoid serial accessioning of similar specimen types to reduce
mix-up of specimens, and cross-contamination.
E.g. gastric biopsies are interspersed with other tissue types

Pre-Analytic Fixation
- All parts to be examined must be initially fixed
- Earlier fixation  better preservation of tissue morphology
- Improper fixation may impede processing (poor fixation has no
remedy
- Proper ratio with tissue must be observed
o 3-5mm thick tissues may be fixed for 6-48hrs
o 5mm thick tissues and large tissues (such as limbs) must be
sectioned prior to fixation, or else, fixation will not be complete
and may occur only at the periphery of the tissue

Specimen Reception
- Submitted specimens must be put in a container labeled with
patient’s name and specimen source/site, and must be
accompanied with a duly accomplished pathology requisition
form.

- Criteria for Rejection of Specimens:

a) Discrepancies between requisition form and specimen
labels
b) Unlabeled, mislabeled, and inappropriately identified
specimens (last resort: DNA identification)
c) Leaking specimen containers
d) Absent clinical data or history, and other necessary info

JALN2020
 GROSS EXAMINATION
- Consists of describing the specimen and placing all or parts of it
into a plastic cassette, in preparation for tissue processing
- One of the basis of pathologists' diagnosis
- Involves selection of elements that appear to be of clinical
significance for histologic examination
Cutting Tools: cleaned before and after use (to avoid carryover)
o Scissors
o Forceps
o Blade holders
o Blades - disposed in sharps container
Gross Table or Gross Workstations
 Sink
 Tabletop
 Water supply
 Irrigation system
 Fume extraction/ventilation system
 Water disposal unit
Specimen Categories
A  Specimens only requiring transfer from container to
tissue cassette. No dissection required
 May need to be placed in filter paper first before
placing in cassette because of their small size
e.g. endometrium, colonic series, breast core biopsies
B  Specimens requiring transfer and routine sample
dissection: sampling, counting, weighing, or slicing
e.g. small lipoma, small skin biopsy, cervical LLETZ
(Large Loop Excision or Transformation Zone)
C  Simple dissection required with sampling needing
a low level of diagnostic assessment and/or
preparation
e.g. Prepuce (fore skin in male) / Folds in clitoris
(female) Gall bladder, hemorrhoids, appendix
D  Dissection and sampling required needing a
moderate level of assessment
e.g. Pigmented skin lesions, skin w/ markers, large
intestine (Crohn's), large glands tumors
E  Specimens requiring complex dissection and
sampling methods
e.g. thyroid, breast cancer, testis (seminoma), uteri
Specimens for Gross Description Only
(because disease is not histologic level)
 Accessory digits  Prosthetic breast implant
 Bunions (aka hallux valgus) &  Prosthetic heart valves
hammer toes without attached tissue
 Extraocular muscle from  Tonsils and adenoids from
corrective surgery children
 Inguinal hernia sacs in adult  Umbilical hernia sacs in
 Nasal bone & cartilage from children
rhinoplasty  Varicose veins
Specimens that may be excluded from mandatory
submission to Histopathology laboratory
• Bone donated to the bone bank
• Bone segments removed as part of reconstructive orthopedic
procedures
• Cataracts removed by pharcoemulsification
• Dental appliances and teeth with no attached soft tissue
• Fat removed by liposuction
• Foreign bodies such as bullets or other medico-legal evidence
given directly to law enforcement personnel
• Foreskin from circumcision
• IUDs without attached soft tissue
• Medical devices (catheters, gastrostomy tubes, stents, and
sutures) that have not contributed to the patient’s illness, injury,
or death
• Middle ear ossicles
• Orthopedic hardware and other radiopaque medical devices,
provided that there is an alternative policy for documentation of
their surgical removal
• Placentas that do not meet institutionally specified criteria for
examination
• Rib segments or other tissues removed only for purposes of
gaining surgical access, provided that the patient does not have
a malignancy
• Saphenous vein segments harvested from coronary artery
bypass
• Skin or other normal tissue removed during cosmetic or
reconstructive procedure (not a lesion or the patient does not
have a history or malignancy)
• Therapeutic radioactive sources
• Normal toenails and fingernails that are incidentally removed
Describing Specimens and Gross Description
- Identify the specimen. Note and verify all anatomical structures.
- Identify orientation markers used by surgeons, if available
a. Inks – used to identify and orient the specimen’s
components, distinguish samples, for embedding
instructions
b. Nicks – indicates laterality
c. Sutures – represented by LL: long lateral; or SS – short
superior
- Describe all notable characteristics: type of specimen, shape,
color, texture, consistency, dimensions, weight
 Weight of intact organs are rounded to the nearest 0.1g. (In
some cases, weight is more important than histopathologic
JALN2020
 characteristics. Examples are hyperplastic tissues.) Hollow Structures
 Dimensions (length, width, depth) are rounded to nearest 1.0 cm.
For multiple pieces, indicate size of the largest piece. - Must be cut open longitudinally and fixed with cottons inside

Sectioning Lymph Nodes

- Taking a representative sample of the tissue.
Small Cut serially about 2 mm thick to look for small
specimens lesions. Lesions are then sampled for
histologic exam. Filter paper may be used in
wrapping small samples
Large Cut at an interval of 1 cm thickness (termed
specimens as breadloafing) to ensure that pathologic
areas or tumoral areas are identified
 Indicate number of sections and blocks on the gross description
 Specimen must fit easily into the standard cassette, which
measures 3 x 2.5 x 0.4 cm
 Thickness: not more than 0.3 cm to allow for closing of cassette
and fixative penetration
 When possible, edges of tissue should be squared
 Paper tags are embedded in the cassette. They should labelled
with accession number using pencil. Markers and pens will
dissolve upon processing.
 If printed, dot matrix must be used.
 Original containers with specimen are saved until case is signed
out (backup evidence in cases of discrepancies)
- most important component of tumor resections because they are
essential for prognosis and planning therapeutic options
- Should be received fresh and not immersed in formalin
- Node is bivalved, and entirely submitted
- Sentinel lymph nodes: usually first lymph node to be involved
during metastasis. Entirely submitted. However, large specimens
may be bisected, and submitted in one or two cassettes
Mastectomy
- Note for weight, size of breast and axillary dissection, skin ellipse,
nipple scar, basal margins
Pediatric SPX
- Additional processes such as IHC, flow cytometry, cytogenetics
and molecular genetics is often done. These may require fresh,
frozen, or specially processed tissues
Specimen with Tumor
- Identify:
► Site & size of tumor
► Location & structure invaded by tumor
► Vascular invasion
► Presence of lymph node
► Distance from resection margin

Other Specimen Considerations

Brain Specimen Worksheet
- Aka “gross worksheet”
- Brain is fixed first before grossed
- Guides histotechnician in assuring that all blocks are processed
 Tied at the Circle of Willis and suspended
 Must not touch side of container to avoid deformity - Must be properly filled up; filled for future reference
 In 10% NBF for 2-3 weeks - Contains the following:
• Accession number
Colon Cancers • Number of sections and blocks
- Polyps: Base (the area where cautery arteries are located) is • “Comments” column (for special requests & etc.)
always inked. • Gross description
o Small polyps: Bisected and placed in one cassette
o Large polyps: sides are trimmed away from the stalk,
and stalk is placed in a separate cassette

Dermatologic SPX References:

1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
- Vertical orientation is always maintained (using markers)
(2nd Revised Edition). Makati, PH: Katha Publishing
- Punch biopsies are submitted whole
(611.0182/B83)
- Tissues greater than 4mm are dissected
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
- Skin ellipses: serially cut along the short axis at 2 to 3 mm interval.
(2015). Basic histopathologic techniques. Metro Manila, PH:
The two most distal sections or tips are submitted in two separate C & E Publishing
cassettes. Remainder is submitted in one or more cassettes

Eyes
- Inject fixative first then gross.
Hard Tissues
- Wash in running water then immerse in tissue softeners

JALN2020
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: GPHCT

FIXATION Factors Affecting Fixation

- First and most critical step in tissue processing because if fixation 1. Fixative of  10% Neutral Buffered Formalin (NBF)
is inadequate, the succeeding tissue processing steps will also Choice  Morphologic criteria for diagnosis have
be inadequate been established based on Formalin-
- Primary purpose: Fixed Paraffin Embedded Specimen
 Preserve morphological & chemical integrity of cell in a life- (FFPES)
like manner as possible by stopping all cellular activities
2. Time  Fixation must be done 20-30mins after
- Performed as soon as tissue is removed from the body
cutting off blood supply (to shorten
- If tissues/cells are exposed to: warm ischemia time)
a. Air  drying of tissue  Prolonged fixation shrinkage
b. Water  swelling of cells
c. Saline  shrinkage of cells 3. Tissue-to-  1:10 or 1:20 (tissue to fixative ratio)
Fixative Ratio  1:20 is more common
 Osmic acid fixatives: 1:5
Effects of Fixatives
■ Hardens soft tissues in preparation for further tissue processing
■ Render cells resistant to damage caused by chemicals used in 4. Penetration Rate  Formalin: 1 mm/hr (but slows down as
further processing it goes deeper into the tissue)
■ Inhibit decomposition caused by bacteria and fungi 5. Thickness of  Larger  Longer fixation time, more
■ Minimize the risk of occupational infection Section fixative
■ Act as mordant for certain stains, thus promoting or hastening  Light Microscopy: 2cm2 x 0.4cm
staining, or inhibit certain dyes  Electron Microscopy: 1-2 mm2

Characteristics of Ideal Fixative 6. Tissue  Longer fixation time:

1. Cheap Components Fibrous tissues
2. Stable Presence of Mucus (wash with NSS)
3. Safe to handle Fat (cut into thin slices fixed longer)
Blood (flushed out with saline)
4. Kill cells quickly to minimize cell distortion
5. Inhibit bacterial decomposition and autolysis  Shorter fixation time:
6. Permit rapid and even penetration of tissues Small or loosely textured tissues
7. Must harden tissues thus easier cutting of tissues
8. Must make cellular components insoluble to hypotonic 7. Hydrogen Ion  Optimal pH: 6 to 8
solutions, and insensitive to subsequent processing Concentration  If outside this pH, ultrastuctural
9. Permit application of staining procedures (pH) changes may occur
 May require the use of buffers
NOTE: No single fixative has all the mentioned characteristics. Each  For Electron microscopy: pH should
of them has own advantages and disadvantages. match physiologic pH
8. Temperature - Higher temp  faster fixation rate and
Mechanism of Fixation autolysis
1. Additive Fixative will forms cross-links between - Cold temp  enzyme inactivation
Fixation soluble molecules, thus gluing them Room temp to 45OC - Optimal Temperature (routine)
together into an insoluble meshwork 40 OC - Tissue processors
Up to 65OC - Microwave processing
2. Non-additive Fixative will not chemically bind with tissue 0-4OC - Electron microscopy
Fixation but removes water from tissue protein 100OC - Tuberculosis
groups thus causing denaturation of cell 60 OC - Rapid biopsy
proteins
9. Osmolality  Hypertonicitycell shrinkage
 Isotonicity and hypotonicity cell
swelling
 Thus, maintain tissues at slightly
hypertonic solution (400-450 mOsm)

10. Agitation,  Hastens fixation

Vacuum
JALN2020
 TYPES OF FIXATIVES I. ALDEHYDES
According to Composition 1. Formaldehyde AKA Formalin
A. Simple made of one component  For routine HP techniques
 Produced from oxidation of methanol
i. Aldehyde  Usually buffered to pH 7 with phosphate buffer
a. Formaldehyde
b. Glutaraldehyde Concentrations:
ii. Metallic Fixatives o 100% - gas form
a. Mercuric chloride o 37-40% - stock concentration (causes overhardening
b. Chromate of the external surfaces of tissues)
c. Lead o 10% - working solution; most commonly used
iii. Picric acid
iv. Glacial acetic acid  ADV: cheap, readily available, easy to prepare, stable,
v. Alcohol compatible with most stains
vi. Osmium Tetroxide
 DADV: nose and eye-irritant, may cause allergic dermatitis
vii. Trichloroacetic acid
viii. Acetone
ix. Heat Effect Cause Remedy
White Prolonged  Filter o
B. Compound 2 or more components or fixatives paraformaldehyde storage  Add 10% methanol
precipitatesturbidity (but denatures
proteins thus
unsuitable for EM)
According to Action
A. Microanatomical (10,10,HFZZBB)
Formation of formic Unbuffered  Buffer
permits general study of tissues without altering the
acid  reduced  Methanol
structure of the subjects of interest
staining quality, and
1. 10% NBF
formation of formalin  10% formol saline +
2. 10% Formol-Saline
pigment Mg++/Ca++ carbonate
3. Heidenhain’s Susa
in jar with marbles
4. Formol-Sublimate/Corrosive
5. Zenker’s
Brown/black Action of  Saturated alcoholic
6. Zenker-formol (Helly’s)
precipitates formic acid picric acid
7. Bouin’s
with excess  1% KOH in 80%
8. Brasil’s
blood ROH
B. Cytological
 Kardasewitsch’s
preserve specific parts of the cell
Method
1. Nuclear Fixatives 2. Cytoplasmic Fixatives
(70% ETOH & 28%
o Preserve nucleus o Other organelles aside
ammonia H20)
from nucleus
o pH ≤ 4-6 o pH > 4-6
o HAc destroys
 Lillie’s MTD
o Glacial acetic acid
mitochondria and Golgi (Acetone, H2O2
has affinity to
bodies 70% ETOH & 28%
nuclear chromatin
ammonia water)
a. Flemming’s with a. Helly’s
glacial acetic acid b. Orth’s
c. Regaud’s/Moller’s 2. 10% Formol-Saline
b. Carnoy’s
d. Formalin with Post-chroming  Formalin diluted with 10% NaCl
c. Bouin’s
e. Flemming’s without glacial  Traditionally, the most common fixative
d. Newcomer’s
e. Heidenhain’s acetic acid  Recommended for CNS tissue and general post-mortem
tissues for histochemical examination
(FCBNH) (HORFF)  ADV: ideal for Silver impregnation staining technique
C. Histochemical  DADV: tissue shrinks during alcohol dehydration [Remedy:
Preserves chemical constituents of cells & tissues Secondary fixation]
1. 10% Formol Saline
2. Absolute ethanol 3. 10% Neutral Buffered Formalin (NBF) or Phosphate
3. Newcomer’s Buffered Formalin
4. Acetone  pH 7
(10FANA)  Best general tissue fixative

JALN2020
  Best for iron-containing pigments and elastic fibers which II. METALLIC FIXATIVES
do not stain well after Susa, Zenker or Chromate fixation, Mercuric chloride Zenker
 DADV: longer to prepare, inert to phospholipids and neutral (ZZCHBS) Zenker-Formol (Helly’s)
fats Carnoy-Lebron
Heidenhain’s Susa
4. Formol-Sublimate/Corrosive B5
 Has HgCl2 Schaudinn’s
 ADV: Excellent for silver reticulum staining method, does
not need washing, fixes lipids Chromates Chromic acid
 DADV: forms mercuric chloride deposits (CROP) Regaud’s/Muller’s
Orth’s
5. Gendre’s (Alcoholic Formalin) Potassium dichromate
 Has 95% ETOH, Picric acid, and GHAc
 ADV: good for microincineration techniques, fixes sputum Lead

6. Hollande’s
 For gastrointestinal (GI) tissues, prostate biopsies, and 1. Mercuric Chloride (HgCl2)
bone marrow (BM)  Most common metallic fixative
 A: penetrates and hardens tissue rapidly
7. Glutaraldehyde  Routine fixative of choice for preservation of cell detail in
 Made up of 2 formaldehyde resides linked by three carbon tissue photography
chains  Conc. 5-7%
 For enzyme histochemistry and electron microscopy  Mostly incorporated in compound fixatives
 ADV: more pleasant and less irritating compared to  DADV: Banned worldwide d/t extreme toxicity, marked cell
formalin shrinkage [Remedy: add acid]
 DADV: less stable and more expensive than formalin  May produce black granular deposits except in
 Container must be refrigerated Heidenhain’s Susa

Concentrations: Remedy: Dezenkerization

o 0.25% - for immunoEM 0.5% Iodine + 70% ETOH  H20  5% Na thiosulfate  H20
o 2.5% - for small TSE fragments
o 3% - most common a. Zenker’s
o 4% - for large TSE  HgCl2 + potassium dichromate + glacial acetic acid
 Good general fixative for adequate preservation of all
8. Paraformaldehyde kinds of tissues
 Polymer of formalin  Good for Trichrome staining
 Powder in form, used in 4%
 Plastic embedding b. Zenker-formol/Helly’s
 For ultrathin and electron microscopy - HgCl2 + potassium dichromate + strong formalin (40%)
 For piituitary gland, BM, blood-containing organs,
9. Karnovsky’s Paraformaldehyde/Glutaraldehyde preserves cytoplasmic granules
 Acrolein in glutaraldehyde or formalin  Brown pigments are removed with saturated alcoholic
 P: for Electron Histochemistry and Electron picric acid or NaOH
Immunocytochemistry
c. Heidenhain Susa
10. 40% Aqueous Glyoxal  Susa: Su = sublimate ; Sa = saure (acid)
 ADV: no smudging of nuclei and distortion of staining  HgCl2 + NaCl + TCA + glacial acetic acid + formalin
compared with formalin  Skin tumor biopsy
 D: reduced staining capacity  ADV: minimum cell shrinkage and tissue hardening
[Remedy: increase staining time] due to counter-balance effect of acids and mercury:
Acids : swelling
Mercury: shrinkage
 Does not produce black pigments
 DADV: Weigert’s staining of elastic fibers not possible

d. B5 Fixative
 HgCl2 + Anhydrous Na acetate
 BM biopsies

JALN2020
2. Chromate Fixatives IV. GLACIAL ACETIC ACID
 Incorporated in compound fixatives
a. Chromic acid  Solidifies at 17OCI
 Conc.: 1-2% aqueous solutions  Important for nuclear fixatives (precipitates nucleoproteins,
 Precipitates all proteins, and preserves carbohydrates chromatins)
 Destroys mitochondria and Golgi elements, thus not for
b. Regaud’s/Muller’s cytoplasmic fixation
 P: chromatin, mitochondria, mitotic figures, golgi
bodies, RBC and colloid-containing TSEs
 DADV: prolonged fixation may lead to blackening of V. ALCOHOL FIXATIVES
tissue pigment  ADV: good for glycogen
[Remedy: Wash in running tap water before  DADV: never for FATs and LIPOPROTEINS (dissolves); causes
dehydration] polarization of glycogen (granules will move towards the poles
or ends of the cells)
c. Orth’s  Effect: rapidly denatures and precips CHONs, preserves nuclear
 P: early degenerative processes and necrosis, stains
demonstration of Rickettsia and other bacteria  (CEMING)
 E: preserves myelin
1. Carnoy’s Fixative
d. 3% Potassium dichromate  Most rapid tissue fixative
 E: preserves lipids, mitochondria, at pH4.5-5.2,  Fixing brain tissues for rabies diagnosis
cytoplasm, chromatin and chromosome are fixed  E: fixes Nissl granules (Tigroid substance) and cytoplasmic
 Corrosive, thus avoid skin contact granules
3. 4% Aqueous Lead 2. 70-100% Ethanol
 P: for acid mucopolysaccharides and mucin  Enzyme studies
 DADV: Prolonged standing  formation of insoluble lead  Does not fix but preserves glycogen
carbonate
[Remedy: add drops of acetic acid to dissolve residue] 3. 100% Methanol/Wood alcohol
 Dry and wet smears, BM smears, bacterial smears
III. PICRIC ACID
4. 95% Isopropyl Alcohol/Rubbing Alcohol
 Used in strong saturated aqueous solution (1%)  Touch prep smears to be Wright-stained
 For Glycogen preservation
 ADV: may be used as a stain as yellowing of tissue will prevent 5. Newcomer’s
small fragments from being overlooked; suitable also with  Mucopolysaccharides and nuclear CHONs
Aniline stains  Better reaction in Feulgen stain than Carnoy’s
 DADV:
1. Explosive when dry 6. Gendre’s (Alcoholic Formalin)
[Remedy: add distilled H2O or 0.5-1% saturated alcohol]
2. Yellowing of tissues  excessive staining
[Remedy: immerse in Li2CO3 with 70%ROH  water  VI. OSMIUM TETROXIDE / OSMIC ACID
70% ethanol  5% Na thiosulfate  water]  Pale yellow powder in water (6% in 20OC)
3. RBC hemolysis
 Ultrathin sections in Electron Microscopy
 (PBB)
 E: Fixes and stains conjugated fats and lipids black
 DADV: very expensive, very volatile, inhibits hematoxylin
1. Bouin’s
 Tissue-to-fixative ratio: 1:5
 P: for embryo and pituitary biopsies, and tissues to be
stained with Masson’s Trichrome  (OFF)
 ADV: minimum cell shrinkage and tissue hardening due to
1. Flemming’s
counter-balance effect of glacial acetic acid (swelling) and
picric acid (shrinking)  Most common osmic acid fixative
 DADV: poorly penetrates large tissue, thus limited to small  P: nuclear structures
fragments of tissues  Effect: permanently fixes fat
 ADV: needs less amount of fixative
2. Brasil’s
 C: TCA 2. Flemming’s w/o acetic acid
 ADV: Better and less messy than Bouin’s  Cytoplasmic structures

JALN2020
 VII. TRICHLOROACETIC FIXATIVES IMPROPER FIXATION
 Incorporated also in compound fixatives Effect Reason
 Marked swelling effect on tissues 1. Failure to arrest early Failure of fixing immediately
 Poor penetrating agent thus for small pieces of tissues or bones cellular autolysis or insufficient fixative
 Weak decalcifying agent, thus has softening effect on dense
fibrous tissues

VIII. ACETONE 2. Too brittle and too hard Prolonged fixation

blocks or tissue
 Used at cold temp -54OC
 For water-diffusible enzymes (Phosphatase, Lipase)
3. Shrinkage and swellings
 For brain tissues (such as in Rabies) of cells in tissue blocks
 DADV: Dissolves fat, evaporates rapidly
4. Soft and feather-like Insufficient or incomplete
tissues fixation
IX. HEAT
5. Presence of artefact Insufficient washing of
 Principle: Thermal coagulation of tissue proteins pigments on sections fixative
 For rapid diagnosis: frozen tissue sections and Bacterial smear 6. Enzyme inactivation and Wrong choice of fixative
prep loss
7. Removal of fixative
Microwave Technique soluble substances
 PCPL: Increases movement of molecules to accelerate
fixation, staining, decalcification
 Electron Microscopy and immunohistochemistry
 ADV: Tissue is heated right through the block in a very
short time; preserves neurochemical substances
(acetylcholine)
 DADV: Penetrates at 10-15mm thickness; spores and References:
pathogen may remain in tissues 1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
 Optimum Temp: 45-55OC (2nd Revised Edition). Makati, PH: Katha Publishing
(611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
SECONDARY FIXATION (2015). Basic histopathologic techniques. Metro Manila, PH:
C & E Publishing
 “Refixation” with another fixative
o Done before dehydration or restaining of deparaffinized
TSEs
o Improve demonstration of substance
o Make special staining techniques possible (with the next
fixative as mordant)
o Ensure further and complete handling
 Post-Chromatization
o Use of 2.5-3% aqueous K2Cr2O7 that will act as mordant

WASHING OUT

 Removal of excess fixative to improve staining and remove

artifacts

1. Tap Water for excess formalin, osmic acid, and

chromates
2. 50-70% ROH for excess picric acid fixatives and
Gendre’s

3. Alcoholic for excess mercuric chloride

iodine

JALN2020
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: GPHCT

DECALCIFICATION ACIDS
- Follows fixation - Most widely used, stable, easily available, relatively inexpensive
- Removal of calcium or lime salts from calcified tissues
- Inadequate decalcification  poor cutting of hard tissues an 1. Nitric acid
damage to the knife edge during sectioning 2. Hydrochloric acid
3. Formic acid
- Hastened by heat and agitation 4. Trichloroacetic acid
- With a common 2-day duration 5. Sulfurous acid
6. Chromic acid
- Calcifications cause a grating sensation during sectioning 7. Citric acid
[Remedy: Remove block from the chuck, and place face down 1. Nitric acid
on cotton or gauze with 10% HCl - Most common and fastest agents
- Hematoxylin-stained microcalcifications = dark purple granular - Removed by 70% ROH during dehydration
masses with light purple halos
- Imparts yellow coloration d/t nitrous acid formation
[Remedy: add Urea or Sodium thiosulfate/sulfate]
Done on:
 Bone a. 10% Aqueous Nitric Acid
 Teeth  Urgent, needle and small biopsies
 Teratoma (means monster)
 Calcified tissues: tuberculous organs, arteriosclerotic vessels b. Formol-Nitric Acid
 Has formalin (allows less destruction)
Grossing:  Urgent biopsies
 Done before fixation
 Double gloves, eye protection glasses, face mask c. Perenyi's fluid
 Specialized table for bone processing  Has chromic acid and ROH (thus, no tissue breakup)
 Bone specimens are grossed in fresh state  Also a tissue softener
 Bone dust particles can be loaded with blood or infectious
malts (osteomyelitis, gangrene) d. Phloroglucin Nitric acid
 Fine fret-saw saw  hand razor  Most rapid decalcifying agent
 Complete decalcification cannot be determined
Factors Affecting Decalcification through chemical means
1. Concentration =faster, but may be more harmful to  Removed with 3 changes of 70-90% ETOH
tissue
2. Tissue-to-volume optimum: 1:20
ratio 2. Hydrochloric acid
3. Temperature =faster, but may be more harmful to - Slower and causes more distortion compared to HNO3
tissue - Provides good nuclear staining
optimum: 18 to 30 degrees Celsius - Recommended for surface decalcification if used in 1%
4. Mechanical Hastens decalcification with 70% ROH
agitation
5. Size & consistency Larger specimens will slow the rate of a. Von Ebner's
of tissue sample decalcification
- HCl + 36% NaCl
- For teeth and small bones
- Complete decalcification cannot be determined
Methods of Decalcification through chemical means
1. Acids
2. Chelating Agents 3. Formic Acid
3. Ion Exchange Resin - Only weak acid used as a primary decalcifying agent
4. Electrical ionization (electrophoresis) - For routine decalcification of post-mortem research tissues,
Most widely used, stable, easily available,
small pieces of bone and teeth, immunohistochemical
staining
- Better nuclear staining, less tissue distortion than HNO3
relatively inexpensive - Addition of citrate  faster decalcifying rate
1. Nitric acid
2. Hydrochloric acid
3. Formic acid acid
JALN2020
 a. Formic acid-Sodium citrate solution MEASURING EXTENT OF DECALCIFICATION
- P: autopsy, BM, cartilage, research tissues
Decalcification must be frequently monitored to avoid maceration of
4. Trichloroacetic acid tissue. The methods of measuring extent of decalcification are as
- Small bone spicules follows:
- Good nuclear staining, does not require washing out
- Weak decalcifying agent and very slow Physical/Mechanical Test
- Touching and bending tissue using fingers; and poking using
5. Sulfurous acid fine needle or a probe
- Very weak agent thus for minute pieces of bone pieces - Rubbery consistency, and soft = Tissue is decalcified
only - DADV: vague and inaccurate artifact production, destruction of
cellular details, small calcified foci may not detected
6. Chromic acid (Flemming's Fluid)
- Both a fixative and decalcifying agent Radiologic Method or X-Ray
- Minute bone spicules - ADV: most ideal, most sensitive, and most reliable method; can
- DADV: highly corrosive to skin, carcinogenic, and detect smallest focus of calcium
environmental toxin - Rinse decal agent from tissue  Put tissue on waterproof
polyethylene sheet on top of X-ray film  Expose to X-ray for 1
7. Citric acid-citrate buffer solution minute at 30kV  Leave until film is developed
- Excellent nuclear and cytoplasmic staining but too slow - Opaque result = Incomplete decalcification
- pH 4.5 - DADV: very expensive, never used with HgCl2-fixed tissues
(radio-opacity, = xrays do not pass through)
CHELATING AGENTS
Chemical Method
- Principle: Use of other salts to form weakly dissociated - Simple, reliable, and convenient
complexes with calcium salts for ease of removal - PCPL: precipitation of calcium hydroxide or calcium oxalate
- For immunohistochemistry, enzyme staining, and electron - Addition of strong ammonia to the discarded decalcifying fluid
microscopy (until solution becomes ALK).
- Duration: 1-3 weeks for small specimens; 6-8 weeks for longer  Cloudiness = presence of Ca in the discarded fluid
& dense bones  If no cloudiness, add saturated aqueous solution of ammonium
- Optimum pH is 7-7.4 oxalate, and let it stand for 30 minutes
- Examples: Cal-Ex & Versene (EDTA)  Cloudiness = incomplete decalcification

ION EXCHANGE RESINS POST-DECALCIFICATION

Removal of decalcifying agent through:
- Hastens decalcification by removing calcium ions from formic
acid-containing decalcifying solutions, thereby increasing 1. Immersion in saturated lithium carbonate or 5-10% aqueous
solubility from the tissue sodium bicarbonate solution for several hours;
- Ion exchange resin (ammonium form of polystyrene resin) is 2. Rinsing in tap water (small spx=30 mins; large spx=1-4 hours);
spread over the bottom of container  put tissue on top  add 3. For frozen section, acid decalcified tissue is stored in formol
decalcifying agent (20 to 30 times the volume of the tissue)
- Not recommended for fluids with mineral acids such as HNO3 saline with 15% sucrose or PO4-buffered saline (PBS) with 15-
and HCl 20% sucrose at 4OC before freezing
- Duration: 1 to 14 days
ELECTROPHORESIS TISSUE SOFTENERS
Performed prior to dehydration or sectioning; for unduly hard tissues
- Calcium ions (positively charged) moves to cathode (negative that may damage microtome knives. The agents are as follows:
electrode) 1. Perenyi's
- For small bone fragments 2. 4% Aqueous Phenol
- Shorter time for calcium removal because of heat and 3. Molliflex (Effect: tissues appear swollen/soapy [not a
electrolytic reaction involved negative effect])
- Uses 88% formic acid 4. 2% HCl
5. 1% NaCl in 70% ROH

References:
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
(2nd Revised Edition). Makati, PH: Katha Publishing
(611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
(2015). Basic histopathologic techniques. Metro Manila, PH:
C & E Publishing
JALN2020
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: GPHCT
DEHYDRATION
- Follows fixation, and decalcification (if applicable)
- It is the removal of water from tissue in preparation for
impregnation
- Since most fixatives are aqueous solutions, placing the fixed
tissue in molten paraffin will not achieve impregnation because
paraffin wax and water (from fixative) do not mix. Hence,
dehydration must be done.
- Done as brief as possible and at a tissue-to-fixative ratio of 1:10
Characteristics of Ideal Dehydrating Agents
1. Rapid action, with minimal tissue shrinkage and distortion
2. Should not evaporate fast
3. Able to dehydrate even fatty tissues
4. Should not harden tissues excessively
5. Should not remove stains
6. Non-toxic, and not a fire hazard
Common Dehydrating Agents
I. Alcohol
II. Acetone
III. Dioxane
IV. Cellosolve
V. Triethyl phosphate
VI. Tetrahydrofuran
ALCOHOLS
 Done in ascending grades to avoid distortion of tissue:
70% ROH  90% ROH  100% ROH
 For delicate tissues, start at 30%
 Initial alcohol conc. depends on the size and nature of tissue and
the fixative used
 Strong initial conc.  shrinkage and brittleness
 Prolonged dehydration in less than 70% ROH  tissue
maceration
 37OC will hasten dehydration rate (for urgent exams)
 To ensure complete dehydration:
 Add atleast ¼ deep layer of anhydrous Cu2SO4 at bottom
of container, and cover with filter paper
 Bluing of copper sulfate crystals indicates full saturation of
dehydrating fluids with water, thus alcohol must be changed with
a fresh solution
1. Ethyl alcohol (Ethanol)
- Best dehydrant because it is fast-acting, mixes with water
and many organic solvents, and penetrates tissues easily
- Not poisonous and not very expensive
- Clear, colorless, flammable
2. Methanol
- For blood & tissue films, smears
3. Butanol
- Plant and animal microtechniques
- Less tissue shrinkage and hardening but slow
ACETONE
 Fastest dehydrant (1/2 to 2 hours), thus for rapid biopsies
 Clear, colorless, and more miscible with epoxy resins than
alcohol
 DADV: Flammable, extremely volatile, and not recommended
for routine work because of considerable shrinkage and
brittleness
 Lipids are removed from tissue
DIOXANE (DIETHYLENE DIOXIDE)
 Both a dehydrant and clearing agent
 ADV: Less tissue shrinkage, prolongation is possible
 DADV: extremely dangerous because of toxicity, and risk of
explosion; expensive, tissues will have poor ribbons
 Methods:
1. Graupner's - pure dioxane  paraffin
2. Weiseberger’s - wrapping tissue in gauze and suspension
to bottle containing dioxane with
anhydrous calcium oxide or quicklime
 Note: Tissues treated with chromic acid must be thoroughly
washed with water prior to dioxane treatment.
CELLOSOLVE/ETHYLENE GLYCOL MONOGLYCOL ETHER
 ADV: no shrinkage in prolongation
 DADV: Combustible at 110-120OF, toxic to reproductive, fetal,
urinary, and blood systems
 If cannot be avoided, propylene-based glycol ethers should be
used instead of ethylene-based
TRIETHYL PHOSPHATE
- A:rapid, little distortion and hardening
TETRAHYDROFURAN
 Both a dehydrant and clearing agent
 Dissolves fats
 Most staining procedures give improved results with THF
 Has rather offensive odor, thus room must be well-ventilated
 Toxic to eyes (conjunctival irritation) and skin (Teflon gloves may
be used, but it is recommended to avoid THF use)
ADDITIVES
1. 4% phenol – added to 95% ETOH; softener for hard
tissues
2. Molliflex (glycerol alcohol mixture) - softener
References:
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
(2nd Revised Edition). Makati, PH: Katha Publishing
(611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
(2015). Basic histopathologic techniques. Metro Manila, PH:
C & E Publishing
JALN2020
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: GPHCT
CLEARING  Clove Oil
o For skin and smooth muscle, but tends to be
 Removing dehydrant from tissue, and replacing it with a adulterated
fluid miscible to both dehydrant and embedding agent
 Makes tissues “transluscent” or transparent, hence the  Carbon tetrachloride
term clearing o Properties and disadvantages are similar to
chloroform but is relatively cheaper
 Most are flammable fluids and have low boiling points
 Excessive clearing may cause brittleness  Tetrahydrofuran
o Shortened turnaround time
Characteristics of a Clearing Agent
 Miscible with alcohol, paraffin, and mounting media  Methyl benzoate and Methyl salicylate
 Causes minimum shrinkage o For double embedding
 Should not dissolve Aniline dyes o Slow acting
 Should not evaporate quickly in water bath
 Makes tissues transparent OTHERS
 Gum Syrup & Glycerin used when clearing directly from
Solutions water (as in a frozen section)
 Xylene/Xylol  Oil of Bergamot for skin and smooth muscle
o Most commonly used clearing agent
 Oil of Origanum for skin
o 15-30 mins to 1hr working time (most rapid)
o Colorless  Oil of wintergreen artificial oil, for delicate tissues
o Not for nervous tissues and lymph nodes because  Carbon Disulfide for smooth muscle; has foul odor
will cause hardening and shrinkage  Carbol Xylene for friable tissues
o Becomes milky when tissues are not completely  Terpineol for delicate tissues like eyes
dehydrated  Phenol for smooth muscles
 Benzene  High Test Aviation excellent clearing agent
o Urgent biopsies and routine purposes (15 to 60 Lead-free gasoline
mins); very volatile in paraffin oven
o Damages the bone marrow leading to aplastic
anemia
 Toluene
o Substitute for xylene or benzene, but slower (1-
2hrs) References:
o Does not cause brittleness even when tissues are 1. Bruce-Gregorios, J. H.(2016). Histopathologic
left for 1 day; not carcinogenic techniques. (2nd Revised Edition). Makati, PH: Katha
o Toxic upon prolonged exposure when used in high Publishing (611.0182/B83)
conc. 2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., &
 Chloroform Aguilar, P. (2015). Basic histopathologic techniques.
o For tissue blocks up to 1cm thick; recommended for Metro Manila, PH: C & E Publishing
large & tough tissues; CNS tissues, lymph nodes,
and embryos
o Toxic to liver, tissues tend to float
o Does not make tissues translucent
 Cedarwood oil
o For CNS tissues and cytology, smooth muscle, skin
o Becomes milky in prolonged storage
o Forms crystals with acetic-alcohol fixed tses
(remedy: heat to 200OC prior to using)

 Aniline Oil
o P: for insects, embryos, and delicate tses
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: HPCT

IMPREGNATION
 AKA Infiltration Paraffin Wax Substitutes
 Removal of clearing agent from the tissue and replacing it with 1. Paraplast
the infiltrating media  Mixture of pure paraffin and synthetic plastic polymer
 This infiltrating media will completely fill all tissue cavities, thus (Dimethyl sulfoxide); more elastic and resilient
giving firm consistency, as well as allow easy handling and  MP: 56-57OC
cutting of thin tissue 2. Embeddol – less brittle, and less compressible (MP: 56-57OC)
 Incomplete Impregnation  Airholes in tissue sections 3. Bioloid – semisynthetic; for embedding of eyes
 Main factors: method of impregnation, nature and size of tissue, 4. Tissue Mat – has rubber
clearing agent used 5. Ester Wax
 MP: 46-48OC
Types of Tissue Impregnation and Embedding Media  Harder than paraffin thus used with sliding/sledge-type
1. Paraffin microtome
2. Celloidin (Colloidin)  Water insoluble but soluble in 95% ethanol, thus prior
3. Gelatin clearing is not needed
4. Plastic  But Cellosolve, and xylene may be used if indicated

1. Paraffin 6. Water Soluble Waxes (Polyethylene glycol)

 Simplest, common and best media for routine processing  MP: 38-42OC or 45-56OC
 ADV: sections are cut easily without distortion; very rapid  E.g. Carbowax - most common
(24hrs); permits many staining procedures; o Hygroscopic = absorbs water; no need for
 DADV: not for fatty tissues; must fully impregnate the dehydration and clearing
tissue to avoid tissue crumbling o Easily dissolved in water, thus sections are difficult
 Melting point for routine work: 56OC to float out and mount
o Never overheat (>60OC) – causes brittleness, [Remedy: add soap to water, or 10% polyethylene
shrinkage, hardening; destruction of lymph tissue glycol 900 in water]
 Paraffin oven – maintain at 2 to 5 OC higher than MP of wax o Neutral fats and lipids can be demonstrated
 Used pure o Not exposed to too much heat, thus for enzyme
o Wax must be filtered first using coarse filter paper histochemistry
such as Green’s No. 904 in wax oven at 2OC higher
than MP of wax 2. Celloidin
o Reusable only once, but remove water first by boiling  AKA colloidin
to 100-105OC  Purified form of nitrocellulose/gun cotton
 Specimen with large and hollow cavities which tend to
Methods of Paraffin Impregnation collapse; hard and dense tissues; neurologic tissues
1. Manual  Concentration: in 2%, 4%, 8% dissolved in equal parts of
 Atleast 4 changes of paraffin every 15 minutes ether and ROH
2. Automatic  ADV: Does not require heat for processing; rubbery
 Uses machines like Autotechnicon and Elliot Bench-Type  DADV: very slow (days to weeks)
Processor, which fixes, dehydrates, clears, and infiltrates  Methods:
tissues o Wet Celloidin
 Infiltration is usually at stations 11 and 12  For bones, brain, teeth
 ADV: Has constant agitation  speedy procedure  Procedure:
 NOTE: Any odor in clearing agent indicates that the 1. Fixation & Dehydration
paraffin wax should be changed 2. Place tissue in ether-alcohol
 Wax bath thermostat should be set atleast 3 degrees 3. Thin celloidin
above the MP of paraffin 4. Medium celloidin
3. Vacuum Embedding 5. Thick celloidin
 Fastest (25-75% reduction of usual impregnation time) 6. Remove specimen and the put it in fresh thick
 Uses embedding oven with negative atmospheric pressure celloidin
 rapid removal of air bubbles (e.g. lungs) and clearing 7. Keep in jar or dessicator until ether-alcohol
agent rapid infiltration evaporates
8. Store tissue block in 70%-80% alcohol
 For urgent biopsies, delicate tissues (e.g. CNS, eyes)

JALN2020
  Complete impregnation: No fingerprint marks on o TissueTek – has paraffin reservoir, tissue
surface of tissue block tank, warm plate and cold plate (-5OC)
4. Disposable o Peel-away (thin plastic mold)
o Dry Celloidin molds o Ice tray (inner mold smeared with
 For whole eye sections glycerin or liquid paraffin),
 Uses Gilson’s mixture (equal parts of chloroform o Paper boats
and cedar wood oil) for storage, instead of 70% to
80% alcohol Other Embedding Methods

o Nitrocellulose Method/Low Viscosity Nitrocellulose
(LVN)
 Has lower viscosity, thus can be used in high
concentrations, and rapid tissue penetration
 ADV: Harder tissue block, thus thinner sections are
possible
 DADV: Explosive when dry d/t nitrates
 Plasticizer (e.g. oleum ricini or castor oil) is needed
to prevent tissue cracking in chrome-mordanted
tissues
3. Gelatin
 Rarely used
 For histochemical, enzyme studies, and frozen sections
 ADV: Water soluble (no dehydration and clearing needed)
 DADV: may decay
 TSE must be <2-3mm thick
 Procedure:
1. Wash out of fixative
2. Put tissue in 10% gelatin with 1% phenol
3. 20% gelatin with 1% phenol
4. Fresh 20% gelatin with 1% phenol
5. Cool in refrigerator
6. 10% formalin
 1% Phenol must be added to prevent molds
 TSE:IMPERGNATING AGENT ratio is set at 1:25
EMBEDDING
 AKA Casting, Blocking
 Placing the impregnated tissue into a mold with embedding
media, and then allowing the media to solidify
 Orientation: Arrangement of the tissue in a precise position
in the mold during embedding
 Surface to be cut should be parallel to bottom of the mold
 Molds should bear the accession number
 Procedure:
o Put tissue with label on a mold, immerse them in melted
paraffin at 5-10 OC higher than MP of wax, then rapidly cool
in a refrigerator at -5 OC, or in cold water
Embedding Molds
1. Leuckhart’s L-shaped strips of heavy brass and metal;
Embedding mold reusable and adjustable
1. Double embedding method– celloidin first, then paraffin; for
large blocks of dense tissues; obsolete
2. Plastic (Resin) embedding
 For High resolution light microscopy of thinner than usual
sections, renal biopsies, BM biopsies
Classes:
o Epoxy
 For electron microscopy
 Most widely applied, but carcinogenic due to
vinylcyclohexane dioxide (VCD) component
 Types:
 Bisphenol A (Araldite) – slow
 Glycerol (Epon) – low viscosity
 Cyclohexene Dioxide (Spurr) – very low viscosity;
fastest
o Polyester
 For electron microscopy; seldom used
o Acrylic Plastics
 For High resolution light microscopy
 E.g., polyglycol methacrylate (GMA), methyl metacrylate
(MMA)
 Benzoyl peroxide – catalyst; forms radicals, which are
site for polymerization
 Acrylic plastics must be stored in dark bottles to prevent
radical formation and premature polymerization
 Embedding media may be stained, thus use
hydrophobic MMA
References:
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
(2nd Revised Edition). Makati, PH: Katha Publishing
(611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
(2015). Basic histopathologic techniques. Metro Manila, PH:
C & E Publishing

2. Compound Interlocking plates resting on flat metal

embedding unit base; for batch embedding
3. Plastic Special stainless steel base mold fitted with
embedding rings a plastic embedding ring (serves as block
and base molds holder during cutting)

JALN2020
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: HPCT

TRIMMING
 Removal of excess wax using knife or cutter after the wax block Types of Microtome
is removed from the tissue cassette or paper boat The cutting rate depend upon the type of tissue, the size of the block
 Formation of truncated pyramid and exposure of the tissue and the model of the microtome that is used.
surface for ease of sectioning
 Allow tissue blocks to fit into the block holder of microtome 1. Rocking or Cambridge  Simplest
 At least 2mm should surround the tissue block Inventor: Paldwell Trefall  Cutting section: 10-12µm
o Coarse Trimming  For small and large paraffin
o Fine Trimming blocks
 not for serial sections
MICROTOMY because of slightly curved
 AKA Sectioning planes
 Formation of uniformly thin slices/sections/ribbons from the 2. Rotary  Most common
tissue block with the use of a microtome in order to facilitate Inventor: Minot  Media: Paraffin
studies under the microscope  Excellent for serial sections
 Sections usually form ribbons due to slight heat generated 3. Sliding  Media: Celloidin
between the block and the knife edge during the process of Inventor: Adams  Very hard and rough tissue
cutting. blocks
 Complete ribbons are picked up with camel hair brush, forceps  Types:
or fingers. o Base-sledge
o Standard sliding – more
Microtome dangerous d/t moving
Principle: Spring-balanced or pawl is brought into contact with, and knife
turns a ratchet feed wheel connected to a micrometer screw, which is 4. Freezing  Frozen sections
in turn rotated, moving the tissue block at a predetermined distance Inventor: Queckett (undehydrated tissues like
towards the knife for cutting sections at uniform thickness. fat) for rapid diagnosis
 Stage for block holder is
hollow and perforated,
Microtome Basic Parts attached to a flexible lead
 Block holder/chuck/cassette clamp – where the tissue is held in pipe containing carbon
position dioxide.
 Knife Carrier and Knife – for actual cutting of tissue sections  CO2 as propellant/freezing
 Pawl, Ratchet Feed Wheel and Adjustment Screws – to line up agent (2 to 3 minutes)
the tissue block in proper position with the knife, and to adjust
the proper thickness of the tissue 5. Cryostat/Cold  More common than freezing
Microtome microtome
 STAT frozen section
Care of the Microtome (intraoperative diagnosis)
 Brush away accumulated paraffin and small pieces of  Chamber maintained -5 to -
tissues with soft brush after sectioning 30OC with rotary microtome
 Excess paraffin and tissues may later on interfere inside
with the cutting of tissue blocks  Cutting Section: 4 µm
 Xylene – may also be used for cleaning some
parts of the microtome
 Oil movable parts to prevent rusting. 6. Ultrathin  Cutting section: 0.5µm
 Cover microtome to prevent accumulation of dust.  Media: Plastic
 For electron microscopy,
tissues fixed with osmic acid
 Uses special diamond knife
(ADV: very sharp and no
easy dulling)

JALN2020
 Microtome Knives SHARPENING
Plane-Concave Biconcave Plane-  Badly nicked knives with blunted ends have to undergo sharpening
Wedge in order to ensure optimum sectioning of tissue blocks.
Length Length: 25mm 120mm 100mm  Sharpening of the knives involves 2 stages, namely:
1) Honing
Characteristic One side is flat Both sides Both 2) Stropping
Other side is concave sides
concave straight
Honing
Embedding Less More Paraffin Paraffin
Removal of nicks and irregularities on the knife edges
Medium concave concave
Materials:
Celloidin Paraffin
 Hones
- Natural sharpening stone or hard grinding surface
Microtome Sliding Base- Rotary Base-
- Long enough to allow the whole length of knife edge to be
sledge, sledge
sharpened in a single stroke
Rotary
- Wide enough to sufficiently support and prevent the
or
rocking of the knife.
Rocking
o Belgium Yellow – most common; best result
NOTE:
o Arkansas – has more polishing effect
 A good cutting edge must be able to cut good sections from a
o Fine Carborundum – coarser; for badly nicked knives
paraffin wax block about 2-3microns thick, without any serration
o Plate Glass (8x3x1in) – excellent
 Too soft cutting edges - likely to become easily dull
 Lubricants
 Too hard cutting edges – likely to produce nicks or jagged edges
o Soapy water
Angles o Mineral oil
o Clove oil
1. Bevel Angle: 27° to 32°
o Xylene
 Angle between the cutting edges
o Liquid paraffin
 Maintained by slide-on back (spring-loaded, semi-circular
metal sheet slipped onto the knife)  Knife sharpeners
1. Cutting angle (15°) - angle between the face of a cutting tool and  Flat Glass plate with finely powdered aluminum oxide
the surface of the work  May be used for grinding and removing nicks
2. Clearing Angle - knife should be inclined at 5-10° from the cutting  Diamantine
plane so that the cutting facets (bevel angle) will not compress the  For final polishing
block during the process of cutting. Procedure: Heel to Toe movement, Edge first
1. Clean hone with xylene to remove scattered particles of
stones and metal
Other Knives and Blades 2. Cover with lubricant
3. Knife is fitted to its corresponding back, placed on one end
1. Disposable o Widely used now because cheaper; of the hone with cutting edge first
Blades honing and stropping are no longer 4. With cutting knife edge first, the “heel” (handle end) is drawn
common practice; obliquely or diagonally towards the operator on the stone
o coated with polytetrafluoroethylene (for until the “toe” (head portion) is reached.
ease of ribboning) 5. Honing is then continued until all the teeth in the knife edge
o E.g. Magnetic knives in cryostat have been eradicated”
6. Washed with water after using to simply remove the metal
2. Glass Knives/ o For ultrathin microtomes collected during the process
Ralph Knives 7. After honing, wipe off the oil or soap from the knife with
3. Diamond o For resin blocks on ultrathin xylene, then strop it thoroughly……
knives microtomes; brittle and expensive
10-20 strokes per surface for Minot or Plane-wedge knife
4. Safety razor o For partially calcified materials, paraffin • For plane-concave knives, only the concave surface
blades and frozen sections. should be rubbed on the hone
o Easily replaced when dull and produces • Plane-wedge & plane-concave is provided with “backs” to
good tissue sections same as with maintain the correct bevel angle (27-32°) throughout
microtome knives honing
o Unsatisfactory for sections less than
10micra NOTES:
Mechanical Honing
 Utilizes a machine that make use of vibrating frosted slide
plate or wheel driven by electrical motor.
 The knife is pressed against the flat side of a rotating glass
wheel.
JALN2020
  30 double strokes - given to each side of the knife.
 Advantages: Time-saving; produce well sharpened knives
with uniform bevels
 Disadvantage: Expensive
Stropping
Removal of burrs and polishing of cutting edge
Materials:
 Paddle strop made of horse leather attached to a solid back,
in order to prevent sagging.
- Usually dry thus require oiling
- Vegetable oil (e.g. castor oil) applied on the back of the
horse leather
- Not mineral oil because it tends to blister and destroy
the leather
Procedure: Toe to Heel movement, Edge Last
- The procedure is the reverse of honing
1. The knife is fitted with its appropriate knife back
2. Knife is laid obliquely on the strop and with the cutting
edge behind
3. Edge last is pushed backward and drawn forward
Precautions:
 The knife should always be wiped clean with a “soft” cloth
before and after series of stropping (NEVER use paper or
cloth)
 The knife edge is the oiled or greased to preventing it
from rusting
 Pressure during the first stropping strokes should be quite
light since the natural compressibility of the leather is
what actually does the work
 Speed in stropping should be avoided
 Wax must not be allowed to come in contact with the
strop.
40-120 double strokes
 Plane wedge knife: both sides are required for
stropping
 Plane – Concave knife: only the concave surface
should be stropped.
TYPES OF TISSUE SECTIONS
1. Paraffin (4-6µm)
• Successive sections will usually stick edge-to-edge (knife)
due to local pressure with each cutting stroke, thereby
forming a ribbon. (remedy: cut slowly)
• Sections are removed in ribbons of ten to allow easy
location of serial sections.
2. Celloidin (10-15µm)
• The blocks are trimmed in the same manner as in paraffin
blocks
• To avoid dehydration and shrinkage, sections are usually
cut by the wet method, both the sections and the block
being kept moist with 70% alcohol during cutting.
• Celloidin sections do not come off in ribbons and have to
be collected into 70% alcohol immediately.
3. Frozen Sections
• Methods of preparing frozen section
1) Cold knife procedure
2) Cryostat procedure (cold microtome)
(NOTE: More on RAPID TISSUE PROCESSING topic)
FLOTATION/FLOATING-OUT
• For paraffin sections
• Sections are floated out on a water bath set at 45-50°c
(approx. 6-10°C lower than the melting point of the wax used
for embedding the tissue.)
• Flotation Bath
• 5 to 10OC↓MP of Wax
• Inside is specifically colored enamel black
• TSEs flatted after 30sec; removes tse
wrinkling
• Dimensions: d=11in, h=4in, 2L capacity
• Regulated temp. to flatten the sections and prepare them for
mounting into the slides/slider
• Slides (76x25mm, 1-1.2mm thick, frosted)
• Sections should not be left on the water bath for a long
time (30 seconds will be enough) to avoid undue expansion
and distortion of the tissue.
• Adhesives may be used for adhesion of tissue to the slide
(more on ADHESIVES topic)
• Folds or creases sections – may be removed by stretching the
sections gently
• Bubbles – may be teased out beneath the sections by means
of needle
• Selected sections for staining should be fished out in a vertical
position
DRYING THE SLIDES
• Mounted sections are placed in a paraffin oven to dry.
• 45 – 55°c for:
• enzyme digestion
• chemical extraction
• metallic impregnation
• enzyme localization technique
• Hot plates are not recommended because they can cause:
 Overheating
 Dust falling – onto the section during drying period
• Metal racks with 25-slide divisions are used to store the
mounted sections during the drying process which usually
takes 5 minutes in the heated oven. Once dry, the whole rack
of slides can be taken for manual staining.
References:
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
(2nd Revised Edition). Makati, PH: Katha Publishing
(611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
(2015). Basic histopathologic techniques. Metro Manila,
PH: C & E Publishing
JALN2020