HISTOPATHOLOGY LAB
LECTURE 1: QUALITY IN HP LAB/INSTRUMENTATION
PROF. THYNEE TAGO, RMT
JUNE 11, 2021
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I.
A.
QUALITY IN HP LAB
Quality Control
C.
Quality management system
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Is a set of coordinated activities to regulate a laboratory in
order to continually improve its performance
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Quality control and quality assurance is part of quality
management systems
Elements of quality management systems:
1. Skilled histotechnologist/histotechnicians
2. Proper specimen collection & processing
3. Efficient processing of results and documentation
4. High quality of reagents & equipment
5. Preventive maintenance of equipment
6. Continuous professional education of staff
II.
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B.
A procedure or set of procedures intended to ensure that a
preformed service adheres to a defined set of quality
criteria or meets the requirements of the client or customer
The machines in the lab need to be checked before using
to ensure that they are able to process the specimen
without any problems.
Quality control:
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Checking the machines if they function normally
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Checking expiration dates on reagent bottles are
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When we run samples in the machines, we also
run positive control and negative controls to check
if the reagents present in the machines are
working
A part of quality management system which is focused on
fulfilling quality requirements
It’s the inspection aspect of quality management
Quality control is merely a part of quality management
system (they are not the same thing)
Quality Assurance
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A part of quality management which is focused on providing
confidence that quality requirement will be fulfilled
It is the planned and systematic activities that is
implemented in the laboratory or within the quality
management system that can demonstrate that we are able
to confidently provide products or services that are of
quality
It includes:
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Having the license to do the service being offered
(license to operate) = the laboratory has been
checked and served certified by government
agencies that the lab is capable to provide the
clientele with the services
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Certificates of the different medtech on the wall to
provide assurance to clientele that the people
working are licensed and capable of processing
them accurately & precisely
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Availability of reagents and supplies
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Perform preventive maintenance of the materials
and equipments
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Evaluate the serves by asking feedbacks from
clients
It is the means of:
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Getting the right test
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At the right time
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On the right specimen
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From the right patient
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With the right diagnosis
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At the right place
Everything is in order.
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A.
Pathologist & Associate Pathologist
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B.
HUMAN RESOURCES
Most important
Without this particular element, you’ll not be able to run
samples or process samples in the laboratory
Head of the laboratory
Associate pathologist assists the head pathologist
Both have the same function
Pinpoint problematic situations
Find solutions or preventive measures
Monitor staff performance
Section hollow tissues to allow fixation of fixative and
forestall autolysis
Most important function: To ensure that the samples
submitted in the laboratory will not undergo too much
change due to autolysis
Histotechnologist & Histotechnician
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In the Philippines, the medtech is usually assigned to play
the role of the histotechnologist
In other countries, it could be especially trained individuals,
(may be a graduate of mls or not (other allied courses))
Histotechnician
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slightly lower in rank than the histotechnologist
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May be a graduate of mls or not
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Graduate of other allied courses
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Should be trained in the work of histotechnologist
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Mls but did not pass the board (trained)
Ensures reagents are fresh
Regularly filters hematoxylin
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Important to filter every now and then because it
undergoes “flaking”; it produces a type of surface
skin which tells us that it’s ripe but it has to be
removed otherwise
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Not filtering it would leave unwanted structures on
your tissue
Maintains equipments in high quality condition (microtome,
cryostat)
Works systematically to minimize errors
Analyzes problems & corrects them
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Important to log problems encountered that have
been analyzed in the laboratory so that when they
encounter the same problem again, they would be
able to provide a solution & solve them.
Provide “GOOD” slides
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Properly labeled, processed, stained, mounted
and sequenced
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III.
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IV.
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SERVICES
Tissue processing
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Samples submitted will be preserve to perform
tissue processing
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Usually starts with dehydration up to infiltration
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We process them to prepare tissues for sectioning
or cutting into very thin slices
Cytology
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Examination of a single cell type often from a fluid
specimen
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It is basically used to diagnose or screen for
cancers
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It can also be used for pap smears now
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In the Philippines, the main purpose of cytology is
for processing and pap smears
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Could be used for diagnosis of infectious
organisms and in other screening diagnostic
procedures
Frozen sections
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Mainly used for rapid diagnosis especially for
intraoperative management (patient is still in the
OR)
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A small portion of organ or disease tissue has
been removed for evaluation and will be sent to
the laboratory for the pathologist to process it
within 15-30 mins and reported immediately to
OR.
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Used to perform enzyme immunohistochemistry
or immunofluorescence studies or lipids and
certain carbohydrates in tissue (help preserve)
Special staining
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Alternative staining technique that are used when
the traditional hematoxylin and eosin method will
not be able to provide all the information that is
needed by the pathologist to provide a proper
diagnosis
Immunostaining
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Immunohistochemical staining
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Process where we use antibody based methods
to detect a specific protein in the sample
DOCUMENTS
1.
Request Form
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Patient;s data
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Lab accession #
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Specimen type
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Specimen
source
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Pertinent history
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Surgery
performed
on the same
organ
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Test requested
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Procedure
performed
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Date & time
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Requesting physician
2.
Patient’s Report
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Data in request form
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Diagnosis
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Gross
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Microscopic
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Images
(evidence)
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Name of
pathologist
who did the
diagnosis
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Telephone reports
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Frozen
sections
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OR
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Informations needed ASAP
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3.
Preliminary reports
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Temporary results (48-72hrs)
Final reports
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Histopath results are released after a
week
Corrected reports
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If there are problems in the initial
reports, corrected reports are released.
Incident Reports
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Documents problems
related to personnel
performance or other
complaints
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Important to
guide the lab
staff in other
future
problems
encountered
in the future
How long do we need to keep these documents?
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If there’s space
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Depends on the SOP of the laboratory (can be 5-10 years or
longer)
V.
ROUTINE PROCEDURES IN HP LABORATORY
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Different steps that tissues go through:
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Receiving
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Receive samples from patient
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Checking of labels and request form
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Where most errors begin
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Accessioning
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Serial numbers are provided
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Letter A - autopsy specimen
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Letter S - surgical/ biopsy
specimen
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Gross examination
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Done by pathologist
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Microscopic characteristics of the tissue
are noted in detail
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Images gathered
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Cut tissue sample into representative
sections
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2 cm2 and not more than 4mm
thick
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Fixation
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Process where tissue is preserved in a
state as close to living tissue as possible
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Both chemically and
structurally
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Use fixative to terminate any ongoing
biochemical reaction, protecting tissue
from further damage or autolysis
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Decalcification
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Teeth, bones, or athersclerotic blood
vessels are calcified, they need to
undergo decalcification after fixation and
before dehydration
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In between process performed to soften
tissues by removing calcium deposits
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For tissues that are not calcified, they
can skip this step.
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Dehydration
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Immersed in concentrations of
hydrophilic fluids (ex. Alcohol, xylene)
that replaces free water in the tissue
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Important because we need to remove
water, because it catalysts further
autolysis
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VI.
A.
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Micrometer reader
Cut sections at around 5-10 micrometer thickness
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With rotary microtome, it can reach 20
micrometers
Type of microtome used in SPC HP lab
Can be manual, semi automatic, or automatic
Most common
Sliding Microtome
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Rotary; tissue block moves, knife stationary, Sliding: tissue
block is stationary, knife moves horizontally
Cant produce ribbons of the tissue sections
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One section at a time
Rocking Microtome
INSTRUMENTS IN THE HP LABORATORY
Microscope
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B.
Clearing
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Subject tissues to a reagent that help
increase their refractive indices
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Similar to glass slide used
Infiltration
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Fill tissues with a medium that fills up
natural cavities, spaces or interstices
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Creates a firm tissue that does
not collapse when cut in
sectioning process
Embedding
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Encasing tissue section in a solid
structure
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Same medium used in infiltration
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Wax (usually paraffin or
paraplast)
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Why?
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Irregularly sized tissue is
difficult to cut using the
microtome
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Square shaped - easier to cut
in thin slices
Sectioning
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Tissues are cut into uniformly thin slices
with the use of microtome
Staining
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Adding colors to the tissues is much
better way of evaluating them
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Makes identification easier
Mounting
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Protects tissue sections that have been
stained from physical damage, torn from
glass slides, fading of colors
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Use transparent medium that will stick a
coverslip on to the samples
Microscope at the top: Laser
microdissection microscope
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State of the art type of
microscope
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Cellular level of tissues
Microscope below: Digital
pathology scanner
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Can enlarge
specimens in the glass
slide
Ressembles a compound
microscope
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A museum type of instrument
Now only used for research
Named as such because the arm has to be moved in
stocking motion while cutting the section
Knife is held by a microtome thread
Able to create tissue ribbons
Freezing Microtome
Microtome
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Machine used to cut tissue samples into very thin
uniformed slices.
Rotary Microtome
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Based on a movement that it has to go through to generate
tissue ribbons
Rotating
Handwheel/ Flywheel - moves block holder up and down
Coarse handwheel
Knife holder - razor type of knife
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With levers used to manipulate
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Already out of commission
Designed for the preparation of frozen section
When doing frozen sections, we don't embed and infiltrate
the tissue. Rather, it is frozen to cut the tissue
Usually uses a compressed carbondioxed to freeze
sections
The mechanism of operating this freezing microtome is
much similar to the sliding microtome, where the tissue is
kept immobile by the carbon dioxide
Knife is moved around to create sections of tissue
We use a cover to concentrate the carbon dioxide onto the
tissue section. Otherwise, it is going to spread out
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Tissue processors
Cryostat Microtome
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Makes our job easier and saves our time.
Machines that can perform the steps required to take a
human tissue from fixation to the state where it is
completely in concreted with a suitable histological wax
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Fixations → infiltration
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We can also do tissue processing manually
Carousel-Type processor
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Moves around to the next container
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Upgrade of the freezing microtome
Is actually a rotary microtome encased in a container
Can freeze and cut tissues at low temperature, typically
around -15 to -30C
The selected portion of tissue to freeze is placed on a
prepared metal mounting base called the CHUCK
The tissue is encased in a squirt of rapidly freezing material
that’s usually liquid at room temperature
OCT or Optimum cutting temperature compound is used for
freezing the tissue sections
Tissue can be flattened and the freezing can actually be
expedited with a use of a steel weight that would create a
flat surface making the cutting easier
Freezing the tissue in the cryostat can take about 3 MINS,
or depending on the size of the tissue
Tissu Tek Express
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Enclosed from fluid type
Ultra Microtome
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Designed for cutting by a mechanism similar to our rotary
microtome, but it is more sophisticated and precise. It is so
precise that it can change the section thickness with
differences of several nanometers
Special microtome used to cut sections very thinly that are
usually observed under the electron microscope
Sensitive to movement, air, and vibrations. Thus, it has to
be placed on top of a non vibrating surface in a special
room where air has to be regulated
Observe positioning or degrees to achieve the right
thickness of sections to be cut
Trough
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Where you collect your thin sections
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Filled with distilled water
Attached in a microscope to assist the technologist in
making sure that the sample and the knife is positioned
properly
Turning off the backlight would allow you to see clearly
Apparatus that you would need to cut sections very thinly
for electron microscopy.
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Embedding Consoles
Next machine that we used right after tissue processors
After infiltration we need to embed our tissue sections
Embedding- in cased our tissue sections in a medium that
would hold it in a firm position, usually wax
Has separately heated paraffin dispensing system with an
integrated filter
Refrigeration spot○
Enables to consistently produce an excellent
paraffin embedded tissues with precise specimen
orientation
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OTHERS
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Flotation Baths/ water
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intermediate step between cutting paraffin sections and
placing them on glass slides
Simply sticking paraffin ribbons on slides will not work. You
will need a warm water bath to allow your tissue to relax
and smooth out prior to mounting on a glass slides
Warmth makes the paraffin to stick into the glass slides
Filled with a distilled water that is heated with temperature
with about 5-10 degrees below the melting point of paraffin
wax
While waiting for it to be used, it has to be kept at 40-50
degree celsius
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Slide dryers
Used to warm slides to a species temperature for fixing,
drying, and preparation for staining
Slides must be placed to a dryer or over for at least 15-20
minutes to dry out the water before the parafinization
Slides can also be dried for overnight at room temperature
to allow tissue to adhere to your glass slides
Wax Dispenser
It’s okay not to have an embedding console as long as you
have a wax dispenser.
Will heat up your wax that is used for infiltration and
embedding and keep it in a liquid state
Digital temperature control
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Provides accurate temperature in melting your
wax.
Ultra fast heating system
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Rapidly melt pelletized wax
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Freshly used wax is in pellet form
Tissue cassettes and molds
Where we placed our tissue sections right after gross
examination
Used to create truncated prism that we need during
embedding
Alternatives
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Gauze- wrap tissue sections, tied in a string and
attach label to it
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Disposable paper molds- to create blocks.
Making paper boats
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Fold the paper in 3 parts (lengthwise)
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Fold again (CW) in 3 parts
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Fold 4 corner boxes; allow the perpendicular lines to align
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After making the flaps, fold them towards each other at the
shorter end
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Microwave
For fixation of large specimens: brain tissue, accelerate the
action of fixatives and reduce the various stages of tissue
processing.
You need a day to create tissue blocks and another half
day to completely process it.
It can cut down 75% of working time
You can do fixation and dehydration for 15 minutes.
Can speedup penetration on the chemicals that we used in
tissue processing
NOT all tissue types can be processed with this apparatus.
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