PH 156: CLINICAL MICROSCOPY

HISTOPATHOLOGICAL TECHNIQUES
GIANNE EDUARD L. ULANDAY, RMT, MPH, PhD | APRIL 2022
TABLE OF CONTENTS ● Surgical, cytopath, and autopsy reports require at least 3 copies
per report
I. Histotechniques C. Smear Preparation
→ Because these are legal documents
A. Definition D. Frozen Section
B. Biopsy IV. Processing for Preserved B. SIGNATORIES
II. Quality Assurance and Tissue Examination ● Especially true in the histopathology laboratory because it can be
Documentation A. Fixation used legally.
A. Histopathological Reports B. Decalcification ● Request Forms
B. Signatories C. Dehydration → Required before pathological tests in the laboratory are
C. Specimen Handling D. Clearing performed.
D. Routine Turnover of E. Impregnation → Qualified professionals need to sign it.
Results F. Embedding ● Results Forms
E. Storage of Specimens, G.Trimming → Minimum requirement: should be signed by
Tissue Blocks, Specimens H. Sectioning ▪ Examining pathologist, and
III. Fresh Tissue Exam I. Staining ▪ Head of the Laboratory
A. Teasing/Dissociation J. Mounting − A pathologist as mandated by the law.
B. Crushing/Squash V. Questions − (In far flung areas) if non-pathologists, only licensed
Preparation VI. References physicians with basic training in pathology can act as
substitutes.
I. HISTOTECHNIQUES
→ These can have more signatories.
A. DEFINITION C. SPECIMEN HANDLING
● Tissue processing ● Ask first what the request is
→ Histotechniques → If frozen section is requested, there is no need to fix it
▪ The techniques for processing the tissues, whether biopsies, or ▪ You just have to label it directly
larger specimens removed at surgery. → If frozen section is not requested, assume that the specimen will
● Tissue specimens be preserved
→ Received in the surgical pathology laboratory ▪ Add a fixative
▪ a unit of clinical pathology laboratory focused on biopsies ● Fix first!
▪ also known as anatomical pathology laboratory → Using a fixative
→ must have a request form that lists the patient information and ▪ The purpose of the fixative is to freeze/preserve natural state
history along with a description of the site of origin. of the tissue
B. BIOPSY ● Label
● A procedure done to remove tissue from the body to be examined → Important as this is a legal document
microscopically for a precise diagnosis → Needed to avoid data mix-up
Types of Biopsy D. ROUTINE TURN-OVER OF RESULTS
● Excisional ● Depends on the test or request
→ Removal of the entire mass ● Surgical Pathology and Cytology
→ Example: lymph nodes, breast mass → For surgical pathology, it would require a week or more
● Incisional ▪ Depending on the load of the laboratory
→ Removal of a portion of the mass ▪ Needs confirmation from other pathologists
→ Example: soft tissue tumor ▪ Checked twice (i.e. double asterisk **)
● Endoscopic − This means that the result already had a second opinion
→ Removal of tissue using an endoscope ● Frozen Section
→ Example: GI (endoscopy), abdominal (laparoscopy), or bronchial → Done when a patient is in the OR, being operated, and the doctor
(bronchoscopy) unexpectedly sees a tumor
● Fine Needle Aspiration (FNA) → The doctor informs the lab that a tumor is present for them to
→ Cells or tissue removed using a sterile syringe or needle prepare the materials needed for the procedure
▪ done in outpatient setting → Courier from the lab collects the specimen from the OR and
→ Example: tumors in deep structures such as in the lung or liver transfers it back to the lab to be tested immediately
● Punch biopsy → The patient is left unclosed until results are collected
→ Removal of small area of skin → Usually done in less than an hour
▪ for skin lesions ▪ If malignant, the surgeon will completely remove the tumor
→ Examples: ● Autopsy report
▪ Bone marrow biopsy (removal of bone marrow cells or a core → For the dead
of bone → Usually takes more time since speed is not usually important
− Example: abnormal blood counts E. STORAGE OF SPECIMENS, TISSUE BLOCKS, SLIDES
II. QUALITY ASSURANCE AND DOCUMENTATION ● Specimens
→ Never throw the remaining specimen
A. HISTOPATHOLOGICAL REPORTS
→ It needs to be filed stored
● Surgical Pathology
▪ because it can be used in legal proceedings
→ Procedure to remove tissue from the body to be examined
→ Important in cases when a legal case is reopened for trial after
microscopically for a precise diagnosis
years
● Cytopathology Report
● Tissue Blocks
● Autopsy Report
● Slides
→ For dead people
● All should be stored and categorized accordingly

Trans # 16 Group I: Fujimori, Montebon, Pedrosa, Timbalopez 1 of 7

 III. FRESH TISSUE EXAM → Uses any microtome
▪ Microtome
A. TEASING/DISSOCIATION − A specialized precision-cutting instrument used to cut the
● Steps specimen
1. Fresh tissue is given − Has blades
2. Placed on a watch glass with isotonic solution − Slicing bread imitates this instrument
▪ Usually normal saline. o Slicing bread thinly using blades is analogous to slicing
3. View Cells using Phase Contrast/Brightfield Microscope the specimen thinly
▪ “That is the basic histotechnique” − Ice-cold CO2 is sprayed on its blade everytime you cut
o Spray-cut-spray procedure to keep the knife very cold
→ Manual/predecessor/original method
▪ More advanced technology already available as replacement
→ Laborious due to its repetitive “spray-cut-spray” nature
● Cryostat Procedure (Cold Microtome)
→ There is a microtome inside a big freezer
→ Optimum working temperature of the big freezer: -18 to -20°C
▪ Cryostat
− A refrigerated cabinet in which a modified microtome is
housed.
− Handles are outside to prevent getting your hands frozen.
▪ Rotary Microtome
Figure 1. Impression smear of a liver biopsy. (A) Blotting gently on filter − Placed inside the refrigerator.
paper. (B and C) The imprint smear is prepared by touching the slide IV. PROCESSING FOR PRESERVED TISSUE EXAMINATION
with the surface of the biopsy in several areas. Retrieved from ● A bulk of the specimens that we get from the patients are actually
(Ulanday, 2021). preserved
B. CRUSHING/SQUASH PREPARATION → “Bihira yung nagffrozen section”
● Steps ● Is the preferred method
1. Tissue is cut ● Main component is the preservative
▪ The cut should be <1mm in thickness ● REMINDER: Before performing the test, check the request form
2. Placed between 2 slides first!
3. Squash/Crush the specimen A. FIXATION
4. A vital stain is applied ● Prevents autolysis by inhibiting the hydrolytic enzymes of
lysosomes
→ If enzymatic activity is being assessed, fixation should NOT be
done.
● Stops bacterial effect and prevents putrefaction.
→ Putrefaction: biodegradation due to bacterial action.
● Preserves fine structure of the tissues.
● Hardens the tissues due to protein coagulation.
● Increases the affinity of tissues to staining effect.
→ Enhances the staining of structures and the cells.
● First and most critical step in histopathologic techniques.
→ Second is proper labeling.
● Ideal time to perform fixation is within 20-30 mins after specimen
Figure 2. Squash Preparation. Retrieved from: Ayele, L., Mohammed, has been taken out of the body.
C., & Yimer, L. (2016). Review on diagnostic cytology: Techniques and ● Rule of thumb: Fixative to tissue ratio is 20:1
applications in veterinary medicine. Journal of Veterinary Science & ● For surgery specimens:
Technology, 08(01). https://doi.org/10.4172/2157-7579.1000408 → Fixation temperature is room temperature (23 to 25°C)
▪ Higher temperatures (e.g. 28 to 30°C) promotes putrefaction.
C. SMEAR PREPARATION
Note: This was shown in the slides but did not have any text and was Aims of Fixation
not discussed. ● Primary aim is to preserve cells in a life-like manner.
→ Characteristics at the moment the specimen was taken is desired
D. FROZEN SECTION for study.
● Used when rapid diagnosis (dx) of a treatment (tx) is required ● Secondary aim is to harden tissue.
● Main Application: preserve the natural state of the specimen or → Histopathologic techniques require visualization under the
tissue microscope, which requires a thinly cut sample.
→ You want to look into the chemistry and the enzymes involved → Hardened tissue preserves the shape when the slice is cut.
● Unfixed ● Most important reaction is stabilizing the proteins.
→ No chemicals will be added → For maintaining tissue morphology.
▪ “When we add chemicals, we automatically cannot use these
specimens anymore for enzymatic histochemistry” Classifications of Fixatives
− Because chemicals would interact with the fixative added ● Note: “You don’t need to memorize all of them, uhm, dadating din
kayo diyan later on sa histopath na subject talaga, pero may
Applications mababanggit akong names para lang mafamiliarize kayo” (Ulanday,
● Rapid Diagnosis (dx) 2021, [PH156] Histopathologic Techniques, 36:20, Verbatim)
● Enzymatic Histochemistry
● Demonstration of Lipids and Carbohydrates (CHOs) According to Composition
● Immunofluorescence and Immunocytochemistry Staining ● Simple Fixatives
● Neuropathology → One component
● Compound Fixatives
Methods of Preparing Frozen Sections → Two or more fixatives in combination
● Cold Knife Procedure
→ The knife is cold According to Action
▪ The cold temperature of the knife should be maintained, thus ● Microanatomical Fixatives
the name of the procedure. → For general microscopic study of tissue structures
→ Uses CO2 → Examples:
▪ 10% Formol Saline
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 ▪ 10% BNF o Usually conducted by pathologist/technologist
▪ Heidenhain's SuSa o Gross examination - Checking for correctly received
▪ Formol sublimate specimen
▪ Zenker's solution = e.g. Checking if specimen labeled placenta is really
▪ Zenker-Formol placenta (can be accidentally switched for liver based
▪ Bouin's on real cases)
▪ Brasil's = Is it the same sample? Is it the correct expected
▪ And many others (enough to dedicate one whole book just for sample? What does it look like? What is the color?
fixatives) What is the size? What is the weight?
● Cytological Fixatives − Fixatives in general, not just metallic ones, can cause
→ Study of specific parts and particular microscopic elements of shrinkage
the cell itself ▪ Decreases amount of demonstrable glycogen
→ Dependent on request/purpose of the test ▪ Corrodes all metals, except for the nickel alloy (Monel)
→ Types: ▪ Produces black granular deposits
1. Nuclear Fixatives − Produces artifacts
− Flemming's fluid − Can cause incorrect interpretation for untrained
− Carnoy's fluid technologist/pathologist
− Bouin's fluid → Examples:
− Newcomer's ▪ Zenker's Fluid
− Heidenhain's SuSa − Has a base of Mercuric chloride
2. Cytoplasmic Fixatives − With glacial acetic acid (gl.HAc)
− Flemming's fluid w/o acetic acid − Recommended for fixing small pieces of liver, spleen,
− Helly's fluid connective tissue fibers, and nuclei
− Formalin with post-chroming ▪ Zenker-Formol (Helly's Solution)
− Regaud's (Moller's) − Zenker's Fluid with Formaldehyde
− Orth's − Used for pituitary gland, bone marrow, and blood
3. Histochemical Fixatives (chemical constituents) containing organs (e.g. spleen and liver)
− 10% Formol Saline ▪ Heidenhain's SuSa
− Absolute ETOH (ethanol) − Recommended for tumor biopsies especially of the skin
− Acetone ▪ Schaudinn's fluid
− Newcomer's fluid ▪ Ohlmacher's fluid
Types of Fixatives ▪ Carnoy-Lebrun fluid
● Fixatives usually have a base component ▪ B-5 fixative
● Concentration and additives are just changed or adjusted depending − Commonly used for bone marrow biopsies
on desired results Picric Acid Fixatives
Aldehyde Fixatives ● Used as a fixative, decalcifying agent, and stain all at once
1. Formaldehyde → Its triple purpose makes it a supposedly desirable material for
→ Formalin/formaldehyde and distilled water as diluent histopathology
→ Most routinely used fixative in the anatomic pathology ● However, it is not commonly used nowadays due to its explosive
laboratory nature
→ Different concentrations but base component is → Highly explosive when dry
formalin/formaldehyde ● Also produces excessive yellow staining
→ General fixative ● Picrates, which colors the specimen, are formed upon reaction with
→ Can also be used for blood fixation protein
→ Highest concentration is 40% ● Excellent fixative for glycogen demonstration
2. 10% Formol-Saline ● Yellow stain
→ Formaldehyde and Normal Saline Solution (NSS) as diluent → Prevents small fragments from being overlooked
→ Recommended for fixation of central nervous tissues → Not permanent
→ Recommended for general post-mortem tissues → Removal process: tissues > 70% ethanol > 5% sodium thiosulfate
3. 10% BNF (Buffered Neutral Formalin) > washed in running water
→ Best fixative for tissues containing iron pigments and for Glacial Acetic Acid
elastic fibers ● Fixes nucleoprotein
→ Best general tissue fixative ● Causes tissues to swell which emphasizes nucleoprotein
4. Formol-Corrosive (Formol-Sublimate) ● However, it destroys mitochondria and Golgi bodies
→ Recommended for routine post-mortem tissues Alcoholic Fixatives
→ Less common ● Generally recommended for glycogen fixation
5. Glutaraldehyde ● Disadvantage: polarization
→ Less commonly used → Viewing under microscope can be painful to the eye
→ Recommended for enzyme histochemistry and electron ● Examples:
microscopy 1. Methanol
▪ Rare 2. Ethanol
▪ Only two working electron microscopes in the PH 3. Carnoy's fluid
6. Formol-calcium 4. Alcoholic Formalin (Gendre's Fixative)
→ For lipids 5. Newcomer's fluid
Metallic Fixatives Heat Fixation
● Mercuric chloride ● Used in undergraduate laboratory
→ Most common metallic fixative 1. Direct flaming fixation
→ Advantages: → Used in microbiology laboratory
▪ Routine fixative of choice for tissue photography ▪ e.g. Sputum sample for bacterial visualization
▪ Permits brilliant metachromatic staining of cells ▪ Bacteria is killed while preserving specimen
− Delineation and outlines of cells can be clearly seen 2. Microwave fixation
→ Disadvantages: → Optimum temperature: 45 to 55°C
▪ Causes tissue shrinkage → Not commonly used due to possibility of contamination of the
− Emphasizes importance of gross examination entire microwave
o Done when specimen is received, prior to fixation

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 B. DECALCIFICATION ● Water must be absent in the specimen in order to allow the
● Used to visualize the bone or highly calcified structures embedding medium to enter.
● Done to soften the specimen without compromising its size and → embedding medium is immiscible with water
shape ● Medium for infiltration is also used for embedding
● Best general decalcifying agent → Ratio of infiltrating medium to tissue = 25:1
→ 5% formic acid ● Impregnating solution:
● Ratio of Decalcifying fluid to Tissue = 20:1 → Paraffin wax
● Ideal time required = : 24-48 hours ▪ routinely used
● Optimal Temperature = Room Temp. (18-30C) ▪ Bustschlii
● Extent of Decalcification − introduced paraffin wax embedding
→ 3 ways to measure: ▪ simplest, most common, and best infiltrating/embedding
▪ Physical/Mechanical Test medium
− commonly used and most complicated ▪ not recommended for fatty tissues
− touch the specimen using a probe to determine is the ▪ Temperature of paraffin oven = 55-60°C
specimen is soft enough → Celloidin
▪ X-ray/Radiological Method → Gelatin
− expensive → Plastic
− decrease in Calcium due to decalcification is visualized F. EMBEDDING
▪ Chemical Method (Calcium Oxalate Test) ● Casting/Blocking
− simple and reliable for routine purposes ● Temperature of melted paraffin (embedding medium) = 5-10°C
− rarely used, usually only physical is used above its melting point
● Do you really need to do calcification everytime? ● Steps in embedding
→ No, decalcification is just a supplementary step for highly calcified → the tissue is cut and place in cassettes
tissues. ▪ Cassettes are small containers with holes used to keep the
C. DEHYDRATION samples from floating around in the container
● The purpose is to remove the internal fluid in order to be replaced by ▪ Allow for multiple processing of samples
infiltrating medium (e.g. paraffin wax) → The cassettes are then submerged into the various solutions
● Dehydrating agents: used in the process of tissue processing
→ Alcohol ▪ fixation
▪ Most common ▪ dehydration
▪ Types of Alcohol used: ▪ clearing
− Ethanol ▪ infiltration
o Best dehydrating agent → At the embedding phase, the sample is removed from the
o Sometimes used as both dehydrating agent and fixative cassette and is placed in a mold to be filled with paraffin wax.
depending on the specimen
− Methyl alcohol
o Blood films
− Butyl alcohol
o Plant and animal microtech
− Industrial methylated spirit (denatured alcohol)
− Isopropyl alcohol
→ Acetone
▪ a fixative and dehydrating agent
● Steps in tissue processing
→ Fixation → Dehydration → Clearing → Infiltration → Embedding

Figure 4. Embedding
G. TRIMMING
● Removal of excess wax
● Standard shape of the block:
→ Four-sided prism or truncated pyramid

Figure 3. Steps in tissue processing and microtome.

D. CLEARING
● Dealcoholization
● Replacing the dehydrating fluid w/ fluid miscible with dehydrating
fluid and impregnating/embedding medium.
● Clearing substances:
→ xylene/xylol
▪ Most common
→ Toluene
→ Chloroform Figure 5. Paraffin holder where the excess wax is removed.
→ Methyl benzoate and methyl salicylate H. SECTIONING
→ Cedarwood oil and clove oil ● Cutting or Microtomy
E. IMPREGNATION ● Processed tissue is cut into uniformly thin slices (sections)
● Infiltration → Routine tissue microtomy = 4-6 μm
● Replacing the clearing agent w/ the infiltrating medium → Frozen sections = 10-15 μm
→ Electron microscopy = 0.5 μm

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 Figure 9. Standard Sliding Microtome

Figure 6. Sectioning
→ Rotary Rocking Microtome
→ Vibrotome
● Kinds of Microtomes: − Used for unfixed, unfrozen specimen
→ Rocking Microtome
▪ Inventor: Paldwell Trefall in 1881
▪ Simplest

Figure 10. Vibrotome

→ Ultrathin Microtome
▪ For electron microscopy
▪ Uses diamond knives
▪ or broken plate glass
→ Freezing Microtome
Figure 7. Rocking Microtome ▪ invented by Queckett in 1848
→ Rotary/Minot Microtome ● After cutting, the sections are placed into a warm water bath in order
▪ Inventor: Minot In 1885-1886 to melt off the paraffin.
▪ Most common ● The sections of the tissue are then fished out of the water bath and
placed onto a slide.
I. STAINING
● Tissues are very difficult visualize after being obtained
→ Due to lack of color/dull
→ Pathologist or microscopist might miss important details
● Staining
→ Used to help guide microscopist in what he or she is looking at
Types of Dyes
● Natural Dyes
→ Obtained from plants and animals
→ Hematoxylin
→ Cochineal dyes
▪ Extracted from female cochineal bug (Coccus cacti)
→ Orcein
→ Saffron
▪ Usually used for thesis as it is cheaper
Figure 8. Rotary Microtome
● Synthetic Dyes
→ Sliding Microtome → AKA “Coal Tar Dyes”
▪ Most dangerous type due to movable exposed knife → Derived from benzene
▪ inventor: Adams in 1789 → Collectively known as “Aniline Dyes”
▪ 2 types: Dye-to-Tissue Mechanisms
− Base-Sledge
● Tissues will bind dyes through the following mechanisms
o Block holder: moving
● Electrostatic
o Knife: stationary
→ Majority of tissue-dye reactions
− Standard sliding microtome
→ Difference in charges
o Block: stationary
→ Examples:
o Knife: moving
▪ Neutral red
▪ Light green
● Hydrogen Bonding
→ Examples:
▪ Congo red
▪ Carmine
▪ Weigert-type resorcinol dye
→ Basically just chemistry
● Van der Waals Forces
→ Example:

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 ▪ Aluminum hematoxylin solutions ▪ Eukitt
● Physical Staining ▪ Entallan
→ Example: ▪ DPX (1.532)
▪ Sudan dyes ▪ Histomount
→ Sudanophilia ▪ XAM (1.52)
▪ Property of tissues to be stained with fat or oil-soluble dyes ▪ Paramount
regardless of type of dye used ▪ Canada Balsam - Abus Balsamea (1.524)
▪ Due to essential lipid nature: ▪ Clarite (1.544)
− Cells are lipid in nature V. QUESTIONS
● Note: Questions listed include both between-discussion questions
− Lipids are present in outer part and post-discussion questions
● Are fixatives bacteriostatic instead of bactericidal?
− As long as it is a sudan dye, it will stain
→ Context: if tissue has bacterial infection, bactericidal fixatives
● Natural Affinity would cause the bacteria to die and, thus, cause a false negative
→ Example: Janus Green reaction
H and E Staining → No, the target of the fixative is the tissue, not the bacteria.
● Most widely used histological stain Assumption is bacterial infection is confirmed with a separate test,
● Hematoxylin not histopathology unless specific organism is being targeted (i.e.
→ Heartwood of a Mexican tree visualizing tuberculosis using lung tissue with acid-fast staining).
→ Waldeyer (1862): First person to use hematoxylin in histology Otherwise, most common and generic fixatives are actually
● Eosin bactericidal.
→ Red acid dye → Bacterial infection is not tested using preserved specimens, so
→ Routinely used in histopathology as a counterstain the specimen is not even fixed. Fresh samples are used instead
▪ Something that would contrast the primary stain for microbiology.
▪ After hematoxylin → This is why histopathology is usually the last laboratory process
▪ Before methylene blue conducted on specimen
→ Three Forms ● What is the difference between Surgical Pathology and the
▪ Eosin Y (yellowish) : Most commonly used Frozen Section?
▪ Eosin B (bluish): Deeper red color → Surgical Pathology
▪ Eosin S (Ethyl eosin) ▪ Branch of medicine
▪ Department that usually belongs to the Anatomical Pathology
Department
▪ They are the ones doing the Frozen Sections
→ Frozen Section
▪ Not a department
▪ Procedure being done by Surgical Pathology/Anatomical
Pathology Department
● In what cases are specimens used?
→ It depends; very wide
→ Common routinely for checking malignancy
▪ Whether mass is benign or malignant (cancer-forming)
▪ Just by looking at cell, you won’t be able to see without
staining or other gross examinations
▪ Needs histopathology
→ Autopsy
Figure 11. Sample Tissue Dye. ▪ More uncommon
● Figure 11: ▪ Insurance purposes
→ Seen in histology classes; commonly used in laboratory ▪ Court litigation
→ Observe contrasting colors that provide divisions or delineations ▪ Cause of death
→ Help in identifying structures → Legal Reasons
▪ To see if it is “ballistic”
J. MOUNTING
▪ MedTechs have to assist in an autopsy at least three (3) times
● Mounting in a span of six (6) months
→ Last step ▪ Confirm cause of death: if suicide or murder
→ Placing it in a slide to be visualized ▪ Rape cases
● Refractive Index − Vaginal samples
→ Ratio of speed of light in air and speed of light in a specific − Part of anatomic pathology
medium ● What is being looked for in tissues to determine malignancy?
→ Refractive index of glass: 1.518 → Pathologists are trained to look
● Goal ▪ Familiar with normal structures
→ To have a mounting medium with a refractive index close to glass, ▪ Based on what you know and think is normal, you compare the
or greater than that (1.518) sample
● Principle of Mounting − Does it look familiar?
→ When you put something in between the medium to mount it, − Does it look like the normal ones you’re used to?
sometimes we put water or normal saline to block refraction or → If abnormal
bending of light ▪ Large
→ If it bends, an error in the identification of structure will occur (bent ▪ Aggregated
structures) ▪ Lessened borders
→ To negate the effect and to see the structure as close to the ▪ Increases risk for malignancy
original, we must target the refractive index of glass → Ever-changing science
▪ Goal of resinous media ▪ Not definite and imperfect
▪ As if embedding or impregnating sample not with parafilm but ▪ Results can change over years due to subjectivity of trained
with glass eyes
● Resinous Media → Criteria
→ With refractive index greater than or equal to 1.518 ▪ To increase objectivity
→ Brands ▪ Scoring: Number of cells, etc.
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 − Based on this scoring, you can determine if it is benign,
malignant of indeterminate
− Different types of biopsies have different criteria
▪ Cell morphology
▪ Capsule
▪ Part of normal tissue should be compared with grossly
abnormal
→ Request Form
▪ Importance is emphasized
▪ This is what the pathologist refers to to know what they are
looking for
▪ Clinical eye is involved
● Are specimens stored forever? Would there not be issues
regarding storage logistics?
→ Walang forever :))
→ Labels are important :))
→ Criteria
▪ Depends on ISO, US, DOH Standards
▪ Requirements are constantly updated
▪ Minimum: 5 years
▪ Autopsy or crime-related: 10 years
o Include in the References
VI. REFERENCES
● Ulanday, G. E. L. (2021). Histopathological Techniques.
● BSPH 2022 Trans (2021). Introduction to Histopathological
Techniques.

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