Learning Outcome 17 MLDY HISTOLOGY

MLT 1027
PATHOLOGY

 The science of the causes and effects of diseases,

especially the branch of medicine that deals with the
laboratory examination of samples of body tissue for
diagnostic or forensic purposes.
HISTOLOGY

 Branch of anatomy that deals with the minute

structure of animal and plant tissues as discernible
microscopically
MICROANATOMY
 The branch of anatomy dealing with microscopic
structures (distinguished from gross anatomy).
HISTOTECHNOLOGY
 Technical histology concerned especially with
preparing and processing (as by sectioning,
fixing, and staining) histological specimens
LABORATORY PATHOLOGY
 Clinical Pathology
 the branch of pathology dealing with the study of
disease and disease processes by means of chemical,
microscopic, and serologic examinations.
 Biochemistry
 Hematology
 Transfusion Services
 Microbiology
LABORATORY PATHOLOGY
 Anatomical Pathology
 the branch of pathology dealing with the morphologic
changes in the tissues, both gross and microscopic;
pathological anatomy.
 Histology
 Cytopathology
 Genomics (Cytogenetics and Molecular Diagnostics)
 Flow Cytometry
 By Law, any tissue removed from a human body
MUST be histologically examined, with the
exception of a few considered relatively non-
patholological
ISCHEMIA, AUTOLYSIS,PUTREFACTION
 As soon as a tissue is excised from the body the
blood flow is interrupted- Ischemia (cold)
 Oxygen and nutrients no longer supplied
 Metabolic wastes no longer removed
 Autolysis- enzymatic breakdown
 Putrefaction- micro-organism breakdown
 Desquamation- shedding of outer layer of tissue
 We need the tissue to be as life-like as possible
 Preservation
 Stops Autolysis and Putrefaction

 Fixation
 Maintains structure by stopping Autolysis and
Putrefaction through stabilization of proteins to keep
the tissue as life like as possible and increases
mechanical strength and stability
QUALITY MANAGEMENT SYSTEM
 Aligned to
 ISO 15189:2012 Medical Laboratories -
Requirements for Quality and Competence
 ISO 9001:2015 Quality management systems-
Requirements
 Quality is the degree to which healthcare
services strive to provide accurate desired
outcomes for patients and are consistent with
current professional knowledge
QUALITY MANAGEMENT SYSTEM
 Safety
 Ensuring safety of working personnel and
environment is of paramount importance
  Safety issues that may come up in a histopathology
lab are primarily those related to
 potentially hazardous chemicals
 biohazardous materials
 accidents linked to the equipment and instrumentation
 general risks from electrical and fire hazards
QUALITY MANAGEMENT SYSTEM
 Quality management of a laboratory should
ensure that there are systems in place to monitor
and improve areas such as organization and
quality management systems
 liaison with users, human resources, premises,
the local environment, equipment management,
information systems and materials
QUALITY MANAGEMENT SYSTEM
 address the pre-examination process, the
examination process, and the post-examination
phase as well as evaluation and quality assurance 
 Regular audit of the various components of the
system will provide evidence of compliance with
standards for accreditation
QUALITY MANAGEMENT SYSTEM
 identify any trends and issues for concern, and
confirm quality systems are working.
 all of these measures should identify areas for
quality improvement and show whether any
improvements are working
 Improving Patient Safety is the #1 concern
QUALITY MANAGEMENT SYSTEM
3 PHASES
 pre-analytical
 analytical
 post-analytical
PRE-ANALYTICAL

 Sample collection, transport, accession

 Patient or specimen identification is one of the most
important aspects and it begins with specimen
labeling and accessioning

 70% of all errors occur in Pre-Analytical

phase
PRE-ANALYTICAL

 Specimen container must be labeled with

 3 unique demographic identifiers
 Specimen type
 Site
 Laterallity
 Requisition must also document relevant clinical
history
 Specimen label MUST match requisition
PRE-ANALYTICAL

 Histology specimens are considered non-

retrievable
 Any discrepancies must be corrected by submitting
area before acceptance for accessioning
 Any non-conformances MUST be documented
 Sufficient fixation, volume of fixative and time-
insufficient fixation cannot be fixed!
FIXATION
 Many different fixatives with different modes of
action
 Two main types of action
 Coagulation
 Forming additive compounds
FIXATION
 Main general fixative: Formalin
 Formaldehyde= poisonous, flammable gas
 Aqueous saturation 37-40%
 Concentrated or 100%
 At low pH contact with bloody tissues forms
pigment= acid formaldehyde hematin
 Prevent formation by buffering pH ~7
FIXATION
 Typical use is 10% Neutral Buffered Formalin
 10% solution of concentrate= 3.7-4.0%
formaldehyde
 10% Neutral Buffered Formalin (NBF)
 Penetrates at ~1 mm/hr
 Protein cross linking takes approximately 12 hr to
take place
 Optimum fixation time 24-72 hr
FIXATION
 Volume ratio of fixative to tissue must be at least
20:1
 Any large specimens must be opened or sectioned
to allow for adequate fixation
 Bread-loafing
ANALYTICAL
 Gross analysis
 Precise and systematic gross description
 Adequate sampling
 Careful handling of specimen
 Inking of relevant margins for microscopic
identification
 Orientation
GROSS ANALYSIS
 As opposed to microscopic
 Describe and dissect in order to document any
pathological processes
 Descriptive terminology not diagnostic
 Any inquiries regarding the specimen must be
clarified prior to specimen dissection by
consulting with colleagues, the responsible
pathologist and/or submitting physician
GROSS ANALYSIS
Specimens should be oriented in 3
dimensions.
Superior to Inferior
Anterior to Posterior

 Medial to Lateral

Appropriate margins must be marked in order

to be seen microscopically
GROSS ANALYSIS
 Margins of the
specimen are
marked with blue
ink.
 Ink will show
microscopically if
tumour is present
at margin
 Inked margins clearly visible microscpically
GROSS ANALYSIS
 Appropriate tissues will be taken for histologic
examination

These tissue sections should be chosen to
document the features in the description
 The location and description of the sections
should be documented in a summary fashion at
the end of the description
GROSS ANALYSIS
 Tissues should be trimmed to 2-3 mm thick and
should fit in the tissue cassette so that there is a
complete rim of empty space surrounding the
tissue
 Small specimens can be placed in tissue cassettes
between sponges, in a filter bag or in an
appropriate sized cassette to prevent loss of tissue
or cross-contamination during processing
GROSS ANALYSIS
 All fresh, fixed or embedded tissue should be
considered as a source of contagion and should
be handled only by appropriately trained and
competent personnel with appropriate safety
equipment.
 e.g. Nitrile gloves, eye protection, well ventilated
area or respirator, use of formalin
absorbent/neutralizing pads.
 Proper thickness/size of tissues. Smaller pieces placed between sponges
 Lung tumour, possible pleural extension (ink)
A large bowel specimen with anatomical complexity requiring a good
description and multiple blocks to be taken for full analysis.
 Lung tumour with possible extension to hilar margin (blue ink)
 Radical prostatectomy specimen, sectioned
 Thymectomy specimen with portion of lung
 Squamous cell carcinoma, nose
 Limb amputation
 Traumatic injury usually gross description only
 Ischemia or tumour needs further investigation/sectioning e.g. peripheral vascular disease or sarcoma
GROSS ANALYSIS
 Grossing area must always be kept clean and
orderly in order to prevent “carry-over” or cross
contamination of one specimen to another.
 Instruments and dissection area must always be
cleaned between cases. Special care should be
taken with any instruments with grooves or
recessed areas that could harbor carry-over tissue,
especially when dealing with smaller diagnostic
specimens.
 Hydronephrosis- a condition that typically occurs when a kidney swells due to urine failing
to properly drain from the kidney to the bladder. This swelling most commonly affects only
one kidney, but it can involve both kidneys. Hydronephrosis isn't a primary disease
 Uterus with IUD and
extensive scarring of
endometrial cavity
 Distal esophagus tumour and
partial gastrectomy
 Wider excision for Melanoma with sentinel node biopsy
GROSSING TUMOURS
 Tumour: Size, location, distance from margins or
landmarks configuration/descriptive
characteristics– exophytic,endophytic,flat,
circumferential (annular) ? Colour,consistency
presence of hemorrhage or necrosis
 Maximum depth of invasion: superficial, invasion
into underlying tissue?
 Invasion to other structures?
GROSSING TUMOURS
SKIN PUCH BIOPSY
 Skin biopsy is one of the most important
diagnostic tests for skin disorders
 Punch biopsy is considered the primary
technique for obtaining diagnostic full-thickness
skin specimens
 involves the use of a circular blade that is
rotated down through the epidermis and
dermis, and into the subcutaneous fat, yielding a
3- to 4-mm cylindrical core of tissue sample
SKIN PUNCH BIOSY
 Grossing
 In Toto if 5 mm or less
 bisect if 6 mm or larger, submit both halves
 marking the cut surface to be embedded down
 Describe:
 diameter and thickness
 appearance of skin surface
 Subcutaneous tissue included?
 The entire biopsy is submitted
 The specimen must be embedded on edge
SKIN PUNCH BIOPSY
SKIN PUNCH BIOPSY

 H&E of skin punch biopsy

PROSTATE BIOPSY

 procedure in which small samples of the prostate are removed and then looked at with a
microscope. A core needle biopsy is the main method used to diagnose prostate cancer. It
is usually done by a urologist
PROSTATE BIOPSY

 Usually multiple needle cores from different areas of prostate

 Need to embed flat in order to visualize entire specimen
PROSTATE BIOPSY
 Visualize
glandular
architecture
BIOPSY OF ANTRUM
 Biopsy sampling of gastric
mucosa at diagnostic
endoscopy provides
information that cannot be
obtained by other means. The
most common indication for
gastric biopsy is whether or
not the patient is infected
with Helicobacter pylori, and
whether the stomach is
gastritic or not.
 Endoscopic view of
antrum of stomach
 Antral biopsy. Approx 3mm. orientation
FINE NEEDLE ASPIRATION
 Fine needle aspiration is a type of biopsy
procedure. In fine needle aspiration, a thin needle
is inserted into an area of abnormal-appearing
tissue or body fluid. As with other types of
biopsies, the sample collected during fine needle
aspiration can help make a diagnosis or rule out
conditions such as cancer
FINE NEEDLE ASPIRATION
 Usually
examined by
Cytopathology
 If positive
biopsy is then
perfomed
GROSS ANALYSIS
 Remaining “wet” tissue should be kept for the
time period specified by your retaining policy
and mandated by regional regulating bodies,
unless there are special considerations to keep the
specimen for a prolonged period of time, in
which case the specimen should be removed to a
special area.
GROSS ANALYSIS
 At the end of the retention time (typically 60 after
sign out) the specimens may be discarded.
 Excess fixative is removed and either neutralized
or collected as chemical waste for disposal.
 Tissues are treated as anatomical waste and
usually disposed of by incineration.
ANALYTICAL
 Purpose of
Histotechnology
is to produce high
quality glass
microscope slide
from which a
diagnosis can be
made
ANALYTICAL
 Refractive Index
 measure of the bending of a ray of light when passing from
one medium into another
 air, 1.0003; water, 1.333; crown glass, 1.517; dense
flint glass, 1.655; diamond, 2.417
 If we are mounting tissues on glass slides they
need to be of a similar Refractive Index
 Tissues typically have RI of 1.35-1.55
ANALYTICAL
 Tissue Processing
 Fixation
 Dehydration
 Clearing
 Infiltration

 tissue samples requiring processing need to be

placed in fixative as soon as possible after excision
from the patient.
ANALYTICAL
 Tissue Processing
 When tissue is immersed in fluid, an interchange
occurs between the tissue’s internal fluid and the
surrounding solution
 =Diffusion
 rate of diffusion, is dependent upon the tissue size and
density as well as the physical properties and
concentration of the processing reagents
FACTORS AFFECTING TISSUE PROCESSING

 Viscosity- solution with low viscosity has a faster

penetration rate
 Agitation- increases the flow of solutions around the
tissue.
 Heat- increase in temperature improves the fluid exchange
and penetration rate, excessive exposure to heat can cause
shrinkage and hardening of the tissue
 Vacuum & pressure- increase fluid mobility, increasing
 the infiltration rate and decreasing the time necessary to
complete each processing step
TISSUE PROCESSING STEPS
 Fixation – prevents autolysis and stabilizes tissue to
maintain cellular structure.
 Dehydration – removes water and unbound fixative
from the tissue.
 Clearing – displaces dehydrating solutions, making the
tissue components receptive to the infiltrating medium.
 Infiltration – permeates tissue with a support medium.
 Embedding – orientation of the tissue sample in a
support medium to create a tissue “block” suitable for
sectioning.
DEHYDRATION
 displaces the residual fixative as well as cellular
water
 Water is found in the tissue in two forms, free and
bound.
 bound water molecule is an integral part of the
macromolecules of the cell
 Correct dehydration during tissue processing should
only remove the free water, leaving the bound water
intact.
DEHYDRATION
 Graded alcohols are typically used in dehydration
to remove free water and keep the bound water in
place.
 When tissues are exposed to heat or excessive time
in the higher grade alcohols (95% or 100%), bound
water is removed= dry, brittle tissues
 Higher grade alcohols also induce turbulence when
contacting free water, damaging cell membranes
and organelles
DEHYDRATION
 Typical graded series
 70%
 85%
 95%
 100% (Absolute)
 100% (Absolute)
 100% (Absolute)
CLEARING
 Clearing agents must be miscible with
 the preceding anhydrous alcohols/dehydrants to
effectively remove them
 the ensuing paraffin wax to allow complete infiltration
 Xylene is the most widely used clearing agent in
tissue processing
CLEARING
 Can be applied neat (no graded series) since free
water has already been removed (no turbulence)
 Xylene will remove some bound water over time
causing excessive drying
 Change in RI occurs at this stage. Tissues become
“transparent”
INFILTRATION
 After clearing, tissue sections are infiltrated
(impregnated) with paraffin wax to support the
tissue, allowing thin sections to be cut (3-5 µm)
 must be sufficient to displace the clearant from
the tissues otherwise the wax will not harden
properly interfering with microtomy (sectioning)
INFILTRATION
 Tissues not allowed to infiltrate for long enough
will be soft and crumbly making sectioning
difficult
 Too much time in high temperature wax can
cause excessive shrinkage and produce dry,
brittle tissues which are equally difficult to
section
EMBEDDING
 process of creating tissue blocks by using an
external support medium to enable microtomy
 embedding medium should be completely
compatible with the infiltrating medium in order
to prevent tissue section separation and to
facilitate sectioning.
EMBEDDING
 Paraffin wax is dispensed into a suitably sized,
warm mold
 The tissue is oriented in the mold, fixed to the
bottom of it using the cold plate
 A cassette is placed on top of the mold and
completely filled with wax
 tissue block is placed on a cold plate to allow the
paraffin wax to rapidly solidify
EMBEDDING

 Choose appropriate sized mold

EMBEDDING

 Molten paraffin dispensed into mold

EMBEDDING

 Tissue oriented and embedded in paraffin

EMBEDDING

 Light pressure applied to flatten tissue

EMBEDDING

 Cassette is placed on mold to act as identifying “lid”

EMBEDDING

 Blocks are placed on cold plate to rapidly solidify

EMBEDDING

 Tissue block ready for microtomy

EMBEDDING
 Orientation  -
During embedding the orientation of tissue is
crucial. Correct orientation of tissue in a mould is
the most important step in embedding. ... it is
important to orient the tissue in a way that will
provide the best information to the pathologist.
EMBEDDING
 Tubular structures such as vas deferens are
embedded ‘on end’
 walled structures (e.g. gallbladder) and skin ‘on
edge’ so that all layers are visible
 Multiple pieces of tissue are aligned through the
long axis of the mold, with the epithelium facing the
same direction
 Embed on an oblique angle to avoid parallel edges-
minimize compression
EMBEDDING
EMBEDDING
 Tissue carryover.
 It is important to clean instruments and heated
embedding area between cassettes, to avoid cross-
contamination
MICROTOMY
MICROTOMY
 All excess wax wax must be trimmed from
surface of the block (Facing or coarse trimming)
to expose the entire tissue (full face)
 Typically trimmed at 20-30µm to avoid damage
MICROTOMY
 Once trimmed full face blocks are placed face
down on ice bath to cool and harden the wax for
sectioning
MICROTOMY

 Tissue block is the sectioned on a microtome at 3-5 µm as a ribbon

MICROTOMY

 Ribbon is “floated out” on a water bath kept at 5-10 degrees below the melting
point of the wax
MICROTOMY

 Sections then “picked up” with glass slide. Tissue adheres due to electrostatic
differential ( tissue- neg charge, glass pos charge)
 Slides are then “dried” to melt excess wax and help adhere the
tissue to the glass
 At this point the tissue and glass have approximately
the same refractive index, detail cannot be seen.
 We need to do something to highlight and contrast
structures
 We do this by staining
 staining always involves the visual labeling of some
biological entity by attaching, or depositing in its
vicinity, a marker of characteristic color or form.
STAINING
 Routine Staining
 Hematoxylin and eosin (H&E) is the most widely
used histological stain. It is simple to use, easy to
automate and demonstrates different tissue structures
clearly.
 Hematoxylin stains the cell nuclei blue-black,
showing clear intranuclear detail, while eosin stains
cell cytoplasm and most connective tissue fibers in
varying shades and intensities of pink, orange and red
ROUTINE H&E
 Used as basis
for diagnosis.
 Further
procedures can
subesquently be
performed to
narrow down
diagnosis
OTHER PROCEDURES
 Special staining techniques
 Histochemical reactions
 Metallic impregnation
 Immunohistochemistry
 immunofluorescence
 in situ hybridization
 Molecular genomics
AUTOPSY PATHOLOGY
 Autopsy begins with two assumptions:
 The life of the individual is of the highest value
 The deceased is to be treated with reverence
 Autopsies do not benefit the dead, they benefit
the living
AUTOPSY PATHOLOGY
 Two types
 Hospital
 Medico-Legal

 Hospital case
 Consent can only come from next of kin
 Medico-Legal
 Ordered by Coroner ( or Medical Examiner depending on
jurisdiction)
AUTOPSY PATHOLOGY
 Purpose: Determine the cause and manner of death
 Cause of Death
 Chain of events that lead up to death
 Manner of Death
 Classification of death
 Natural
 Homicide
 Suicide
 Accidental
 Undetermined
AUTOPSY PATHOLOGY
 Coroners are medical doctors with specialized
death investigation training, who have been
appointed to investigate sudden deaths as
mandated by the Coroners Act.
AUTOPSY PATHOLOGY
 Pathologists are medical doctors who are experts
in disease and injury. Forensic pathologists have
further training and are experts in disease and
injury that result in sudden death. Pathologists
and forensic pathologists are the medical doctors
who perform autopsies, when required. Forensic
pathologists may also be appointed as coroners to
investigate cases of suspicious death.
AUTOPSY PATHOLOGY
 A death investigation is a process whereby a coroner or
forensic pathologist seeks to understand how and why a
person died. A coroner or forensic pathologist must
answer five questions when investigating a death:
 Who (identity of the deceased)
 When (date of death)
 Where (location of death)
 How (medical cause of death)
 By what means (natural causes, accident, homicide, suicide
or undetermined)
AUTOPSY PATHOLOGY
 A coroner is called to investigate deaths that
appear to be from unnatural causes or natural
deaths that occur suddenly or unexpectedly.
Additionally, a coroner may become involved
when concerns are raised regarding the care
provided to an individual prior to death.
AUTOPSY PATHOLOGY
 Certain types of deaths must be reported to a coroner.
These reportable deaths include, but are not limited to:
 deaths that occur suddenly and unexpectedly
 deaths at a construction or mining site
 deaths while in police custody or while a person is incarcerated
in a correctional facility
 deaths when the use of force by a police officer, special
constable, auxiliary member of a police force or First Nations
Constable is the cause of death
 deaths that appear to be the result of an accident, suicide or
homicide
AUTOPSY PATHOLOGY
 In some provinces, we have a Medical Examiners
system and in others a Coroners system. Medical
examiners must be medical doctors, but not
necessarily forensic pathologists. Only the Chief
and Deputy Medical Examiner are usually
forensic pathologists. In Ontario, coroners are
also doctors, but in the rest of Canada, coroners
are lay coroners and come from many
backgrounds.
AUTOPSY PATHOLOGY
 Steps in a post mortem examination
 Circumstantial and Medical History
 External Examination
 Internal Examination
 Organ and Tissue Removal
 Individual Organ Examination
 Examination of the Head, Skull, Brain, and Spinal Cord
 Microscopic Examination
 Postmortem Laboratory Analysis of Drugs, Chemicals, and
Microorganisms
POST ANALYTICAL
 Reporting
 Ensure valid report goes to appropriate health care
provider
 Ensure reports go to proper reporting agencies