DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: GPHCT

HISTOPATHOLOGY QUALITY IN HISTOPATHOLOGY LABORATORY

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

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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

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 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

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 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
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 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
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 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
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 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
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