HISTOPATH LEC (MODULE 1)  Can mutate into different form or strain

 Pathogenesis of COVID-19
IINTRODUCTION TO PATHOLOGY o Causative agent – SARS-CoV-2
Pathology o Transmitted through respiratory
droplets – can migrate to lungs esp.
 Refers to the study of disease alveoli
 Patho – disease, logy - study o Will then triggers the immune
 Devoted to the study of the structural, system of patient (cellular immunity
biochemical, and functional changes in and humoral immunity)
cells, tissues, and organs that underlie o Biochemical changes –
disease. chemoreceptors, secretions of
 This is made possible by application of chemicals
various techniques such as the use of o Molecular levels – translation etc.
molecular, microbiologic, immunologic, and
morphologic techniques.
 Because of these, Pathology, help
clinicians explain the whys and wherefores
of the signs and symptoms manifested by
patients while providing a rational basis for
clinical care and therapy.
 Structural changes – lesions, any form of
tissue damages
 Biochemical changes – in enzymes, proteins
that are present in a tissue sample
 Molecular technique – PCR is to amplify,
reproduce multiple copies of gene
(BNA/RNA)
 Microbiologic technique – culture is
cultivating bacteria that are
synthetically/manmade prepared using
culture media. Sensitivity – application of
antibiotic test, to check what antibiotic is Etiology
resistant to bacteria. Staining – gram
staining, acid fast, hot and cold method  Study of causes, the cause of diase
o Histopathology staining techniques –  Pertains to the cause or origin of a disease
hematoxylin, Papanicolaou stain process. According to Robins, etiology can
(pap smear) to diagnose cervical be classified as genetic or acquired.
cancer, alcian blue o Intrinsic/Genetic - (e.g., inherited
o Hematology staining techniques – mutations and disease-associated
wright stain, giemsa gene variants, or polymorphisms),
 Immunologic technique – special test, breast cancer, testicular cancer,
detecting antigen and antibody, rapid test diabetes mellitus
kit, ELISA, enzyme immunoassay o Extrinsic/Acquired – caused by
 Morphologic technique – aims in the infectious agents like bacteria,
identification of structural changes in a viruses, fungi, parasites, basically
particular sample you acquire from the environment
(e.g., infectious, nutritional,
Pathogenesis chemical, physical), lung cancer,
bacterial diseases, COVID-19,
 Refers to the development or evolution of
malaria
the disease
o Idiopathic – cause of disease is
 Refers to the sequence of cellular,
unknown
biochemical, and molecular events that
follow the exposure of cells or tissues to an
injurious agent.
 ** Please note that portal of entry, portal of exit
and mode of transmission also play a role in
WHAT IS DISEASE? disease to occur.
 Structural or functional change in the Manifestations of Disease
body that is harmful to humans.
 Signs - physical observations made by the
person who examines the patient
o i.e, fever, unexplained weight loss,
clubbed fingers, coughing
 Symptoms - evidence of disease perceived
by patients
o i.e, feeling of fullness in the
abdomen, pain, feeling of tightness
in the chest, lumps, fatigue, loss of
smell and taste
 Laboratory Findings - observations made by
Morphologic changes
the application of tests or special
 refer to the structural alterations in cells or procedures
tissues that are either characteristic of a o i.e, x-rays, blood counts, biopsies,
disease or diagnostic of an etiologic chemistry test, CT scan, MIR
process.    infection increase in WBC
count
DISEASE PARADIGM  differential count if viral
(increase in lymphocyte) or
bacterial (increase in
neutrophils)
 anemia – hematocrit and
hemoglobin
Diagnosis

 Refers to the identification of the nature

of an illness or other problems by
examination of the symptoms
Categories of Diseases
1. Structural Diseases – also called organic
disease, characterized by structural
1.  Susceptible Host – harbors the changes within the body, called lesions
infection or person harboring (skin lesions such as psoriasis) or tissue
infection damage (presence of tumors)
2.  Pathogen or Agent - cause of disease, o Genetic diseases – caused by
responsible for causing disease abnormalities in the genetic makeup at
3.  Environment – exogeneous and the chromosomal or genetic (gene)
endogenous factors level
o Albinism (lack pigments)
Mode of Transmission: o Down syndrome (excess in
chromosome)
o Any mucous membrane opening o Degenerative and Inflammatory
o Inhalation of chemicals diseases
o Degenerative disease – refer to
o Portal of entry and exit (mouth, open
a condition wherein the
wounds/skin, mucous membrane)
function or structure of the
o Airborne
affected tissue or organs
o Direct and indirect contact
changes for the worse over
o Vectors (needle prick) time.
o Ingestion  i.e, alzheimer’s,
osteoporosis,
osteoarthritis
  naoobserve during old age
o Inflammatory disease – include
a vast array of disorders and
conditions that are
characterized by
inflammation.
o Inflammation – body coping
mechanism, body’s response to
an infection
 i.e., allergies, asthma,
autoimmune diseases,
glomerulonephritis,
hepatitis, lesions, tissue
damage (inflammatory)
o Hyperplastic and Neoplastic diseases –
basic abnormality is an increase in cell
populations, certain line of cells keeps
on multiplying uncontrollably,
continuous proliferation
o Hyperplasia – proliferation
(division and reproduction) of
cells with exposure to a
prolonged stimulus. Regresses or
stops when stimulus is removed.
Uncontrollable like
multiplication of cells.
o Neoplasia – results from genetic
changes that favor the growth
of a single population of cells.
Cancer that is genetic in nature.
 Benign – not cancerous,
localized because
encapsulated, can still be
remove through surgery
 Malignant – cancerous, has
the ability to localize or
spread to other organs
2. Functional Diseases - characterized by no
visible lesions, at least not at the onset of
the disease
o Tension headache
o Functional bowel syndrome
o Hypertension
o Mental illnesses – depression,
anxiety

Causes of Disease

o Physical – heat, cold, electricity,

radiation
o Chemical – carcinogenic in nature,
poisons, drugs
o Microbiologic – bacteria, viruses, fungi,
parasites
o Immunologic – antigen, antibody,
autoimmune diseases
o Endogenous – genetic in nature, inherited
types of diseases
 Once cell is expose to stress or injury stimuli
there is only 2 outcomes:

 Adapt
 Development of cellular injury
Cell Adaptation - reversible functional and
structural responses to changes in physiologic
HISTOPATH LEC (MODULE 2) states (e.g., pregnancy) and some pathologic
stimuli, during which new but altered steady
Cellular Adaptations, Cellular Injury and Cell states are achieved, allowing the cell to survive
Death and continue to function.
Cell injury occurs for many reasons. Some  Cellular response to cell injury
represent spontaneous alterations in the ability
of a cell to proliferate and function normally, CELL INJURY:
and in other cases, disease results when external  Irreversible – if reversible injury progress
stimuli produce changes in the cell's this may lead to cell death, severe
environment that make it impossible for the cell progressive
to maintain homeostasis. In such situations, cells o Cell death – either by necrosis or
must adapt to the new environment. These apoptosis
adaptations include hyperplasia, hypertrophy,
atrophy, and metaplasia which can be classified
as physiologic or pathologic depending upon
whether the stimulus is normal or abnormal. A
cell can adapt to a certain point, but if the
stimulus continues beyond that point, failure of
the cell, and hence the organ, can result. If cells
cannot adapt to the pathologic stimulus, they
can die.

 Reversible – once the stimulus is remove

the cell may be back to normal or may
return to homeostasis, mild, transient

Cell Death - This refers to an end result of

Homeostasis - Characteristic of cell being in progressive cell injury.
a steady state. This may also refer to absence of
cell injury brought about by stress or injurious CAUSES OF CELL INJURY
agent.
1. Oxygen deprivation of cells/Hypoxia -
 Steady state – equilibrium, characteristic decreased partial pressure of oxygen in
of the cell being in balance, absence of blood, reduction in aerobic oxidative
cell injury, the cell is normal respiration of the cell
a. Ischemia – reduce blood flow to a
Cell injury- This refers to exposure of cells to particular tissue/organ
stress or injurious agent causing injury to cell b. Cardio respiratory failure
and disruption of cell's homeostasis. c. Carbon monoxide poisoning in
cases of anemia
2. Chemicals, drugs, toxins
 a. glucose, salt in hypertonic die activate enzymes that degrade
solution could result to cellular the cell’s own nuclear DNA and
injury nucleo-cytoplasmic protein.
b. air pollutants (asbestos, o Regulated cell death by the cells.
insecticides, herbicides, oxides) o Sometimes it can be pathologic
c. mercury, recreational drugs such but most of the time it is normal
as alcohol regulated type of cell death.
d. carbon monoxide  Programed destruction of
3. Microbiologic/Infectious agents cells during embryogenesis
a. bacteria, viruses, fungi, parasites  Hormone-dependent
4. Physical agents involution in aging
a. Mechanical trauma (nauntog,  Cell deletion in
nadulas) proliferating cell
b. Extreme temperature changes populations
(extreme heat and cold)  Death of host cells that
c. Sudden changes in atmospheric have served its purpose
pressure (radiation, electric  Elimination of potentially
shock) harmful self-reactive
5. Immunologic reactions lymphocytes
a. autoimmune diseases  Cell death induced by
b. immune reactions to external cytotoxic T cells
agents such as viruses,  Necrosis - “accidental” and unregulated
environmental substances form of cell death resulting from damage
6. Genetic defects/derangement to cell membranes and loss of ion
a. enzyme defects in cases of inborn homeostasis. Sum of all the morphologic
errors of metabolism changes that follow after cell death. The
b. deficiency in proteins morphologic appearance of necrosis is
c. defect in DNA sequence the result of denaturation of intercellular
d. accumulation of damage DNA proteins and enzyme digestion of injured
e. errors in chromosome cell. Happens on pathologic conditions.
7. Nutritional imbalance o Liquefaction necrosis – result
a. Severe vitamin deficiency from action of powerful hydrolytic
b. Nutritional problems (anorexia enzymes which is characterized
nervosa) generally by digestion of dead
c. Aging – normal process, paghina cells.
ng cells  best exemplified by brain
TARGETS OF INJURIOUS STIMULI infarction (kinulang ng
oxygen supply ang brain)
1. Aerobic respiration – once aerobic brought about by ischemic
respiration of the cell is disrupted or destruction of brain tissue.
targeted by particular stimuli expect  Common “pus”/nana or
hypoxia to occur and eventually cellular present of dead leukocyte
damage/injury (creamy yellow material),
2. Integrity of cell membranes abscess. Another example
3. Protein synthesis is suppurative appendicitis
4. Cytoskeletal system there is also presence of
5. Integrity of the genetic apparatus of the pus.
cell – targets DNA and RNA o Coagulative necrosis – the
architecture of dead tissues is
PATHWAYS/TYPES OF CELL DEATH
preserved for a span of at least
 Apoptosis - refers to "programmed cell few days. Results from total
death", usually regulated but may also be occlusion of supplying vessels
pathogenic. especially in solid organs resulting
o Tightly regulated intracellular to:
program whereby cells destined to
  conversion of the cell to Adaptations are reversible changes in the
acidophilic tombstone size, number, phenotype, metabolic
 loss of nucleus but cell activity, or functions of cells in response to
architecture preserved changes in their environment.
 protein denaturation
(precipitation) 1. Hypertrophy – increase in size of
 exemplified by myocardial cells, refers to an increase in the
infarction leading to AMI size of cells, that results in an
o Enzymatic fat necrosis – increase in the size of the
destruction of fat resulting from affected organ
abnormal release of enzymes o
especially lipases o Physiologic - caused by
 Exemplified by acute increased functional
pancreatitis demand or by
 Saponification: action of stimulation by hormones
lipase on fat results in and growth factors (e.i.
dissolution together with enlargement of skeletal
hydroxyl ions (-OH) will muscle during vigorous
produce soap with addition exercise, enlargement
of calcium will result in
of uterus during
formation of “chalky”
pregnancy)
material
a. Hormonal –
o Caseous necrosis – combination of
estrogen
liquefaction and coagulative
stimulation of
necrosis. Caseous means cheese
uterus in
like.
pregnancy
 only seen in tuberculosis
b. Compensatory –
process/infection
increased
 characteristic “cheesy
functional
appearance”, white friable
demand
appearance on necrotic
1. If skeletal
area
muscle is
 structureless, only
overworked
composed of live cell and
like pag
amorphous granular debris
nag ggym
o Gangrenous necrosis – according
nalaki
to robins’ it is not a specific
muscle
pattern of cell death. However,
2. Liver
this is use in clinical practice.
regeneratio
Applied to describe a limb, lower
leg that has lost its blood supply.
n
Combination of liquefaction and
o Pathologic – due to
coagulative necrosis. disease process, occurs
 2 types: dry & wet due to an abnormal
 Depends on stressor (e.i. increase in
predominance the size of the heart due
necrosis to aortic stenosis).
 Gangrene of the foot due a. excessive
to diabetes mellitus is hormonal
classified as DRY. stimulation
 Gangrene of loose organs, b. viral-induced –
e.g., appendix is classified growth factors
as WET. produced by virus
like papilloma
PRINCIPLES AND CONCEPTS OR FOUR viruses
TYPES OF CELLULAR ADAPTATIONS
 o CAUSES OF ATROPHY:
a. Decreased workload –
also known as atrophy of
disuse
a. i.e., fractured
bones,
b. Loss of innervation –
loss of stimuli, also
known as denervation
atrophy
c. Diminished blood
supply – gradual
2. Hyperplasia - as an increase in the decrease in a blood
number of cells in an organ or supply particularly in the
tissue in response to a stimulus cases of ischemia this
o Physiologic- due to the could lead to a gradual
action of hormones or decrease of organ or
growth factors occurs in could also lead to
several circumstances atrophy – pag nagkaroon
(e.i. enlargement of ng decrease of blood
breast during puberty) supply sa tissue this
o Pathologic- most are could lead to arterial
caused by excessive or occlusive disease which
inappropriate actions of is common in late adult
hormones or growth life particularly in brain
factors acting on target d. Inadequate nutrition –
cells (e.i.  growth of skeletal muscles need
adrenal glands due to proteins as a source of
production of energy
adrenocorticotropic a. Can develop
hormone (ACTH) by a cachexia
pituitary adenoma). e. Loss of endocrine
3.  Atrophy – decrease in size cells, stimulation – concerned
defined as a reduction in the size with hormones, if wala
of an organ or tissue due to a ng hormones na mag
decrease in cell size and number. stimulate this could lead
o Results from decreased protein to gradual atrophy of
synthesis and increased protein organs
degradation in cells. Reduced a. Breast and
metabolic activity could lead to reproductive
decreased protein synthesis. organs
a. Ubiquitin-proteasome b. Loss of estrogen
pathway after menopause
a. Activates could result to
ubiquitin ligases atrophy of
b. Accelerated endometrium,
proteolysis vagina, vaginal
b. Accompanied by epithelium and
increased autophagy – breast
consumption of the f. Aging
body’s own tissue as g. Pressure/tissue
metabolic process compression
occurring in starvation a. Presence of
and certain disease, enlarged benign
more like cell eating
 tumor surrounding o Not an adaptive mechanism but
tissue a change for the worse
o Physiologic- common
during normal
development (e.i. 
atrophy of thyroglossal
duct during fetal
development)
o Pathologic - occurs due
to many causes (e.i. 
Decreased workload
(atrophy of disuse, Loss
of innervation
(denervation atrophy),
diminished blood
supply, inadequate
nutrition, aging, loss of
endocrine stimulation,
pressure)
4. Metaplasia – replace adult cell by
another type, refers to
a reversible change in which one
differentiated cell type (epithelial
or mesenchymal) is replaced by
another cell type (e.i. epithelial
metaplasia, change of normal
columnar epithelial cells to
squamous cells in the respiratory
tract)
o CAUSES OF METAPLASIA:
a. Persistent irritation
b. Infection
c. Malnutrition

5. (not a cell adaptation response,

only an addition) Dysplasia
o DERANGED DEVELOPMENT
a. Proliferation and
atypical cytologic
alterations
b. Change in size, shape
and organization
 Protective practices/measure:
1. Proper hand hygiene
2. PPE (gloves, lab gowns, eyewear)
3. Sharp containers for prevention of needle
prick
4. Maintain sterility of equipment
5. Clean and disinfect environmental
surfaces (Clorox – 1 to 10 dilution)
6. Proper segregation of waste

HISTOPATH LAB (MODULE 1)

Introduction to Histopathologic Tissue
Processing Techniques for Paraffin Sections
Histopathology
 a branch of pathology that deals with the
study of disease in a tissue section. In the
laboratory, the tissue is subject to a
series of procedures before it is
thoroughly examined microscopically to
arrive at a particular diagnosis.
Medical technologist/Histotechnician
 process the tissue sample, transform
tissue in a tissue slide According to the OSHA (Occupational Safety
Histo – tissue and Health Administration), the types of
hazards are:
Pathology – study of disease
1. Biological hazard – infectious agents,
Histopathology Laboratory including airborne or blood borne
 It should be consist of rooms, each with organisms such as bacteria and viruses
2. Physical hazard – wet floors, heavy lifting
proper ventilation, good light, proper
distribution of equipment and proper (e.g., boxes and patient transfers)
3. Electrical hazard – dangerous high voltage
storage of chemicals and reagents.
equipment
Laboratory Hazards and Safety in 4. Chemical hazard – toxic, flammable (e.g.,
Histopathology alcohol, HCL, nitric acid, benzene,
xylene)
 Hazards – something that has potential to 5. Fire/explosives
cause harm, injury, risk to people, 6. Radioactive
property and environment.
 Safety – condition of being protected (numbers 1-4 – most common to encounter in
from or unlikely to cause danger, risk or histopath especially chemical hazards)
injury
Nuclear laboratory/medicine – they uses radio
Standard precaution – treat sample as if it is isotopes
potentially infectious especially blood borne
Equipment and Apparatuses in a
diseases
Histopathology Laboratory
  Microtome – used to cut sections/tissues Not label by names but by accession
into tiny slices number. Accessioning – assigning a
 Hot air oven – use for impregnation of number into a particular specimen.
section (used when melting impregnating 3. Preparation/processing of the tissue
medium) specimen to facilitate gross and
 Tissue processing machine / tissue microscopic examination
processor – use for tissue processing 4. Record keeping
 Water bath – use to heat samples in the Receipt of the sample:
lab
 Wax dispenser – use for embedding the For histopath it is called surgical request
section form.
 Cryostat – a special type of microtome
 Clinical details
used to cut the frozen section
 Adequate specimen
 Centrifuge – a machine with rapidly
rotating container that applies
centrifugal force to its contents, typically
to separate fluids of different densities
(purpose is to separate contents of Histopathologic Techniques
sample by means of sedimentation)
 Microscope – an instrument used to  Deals with the preparation of tissue for
examine minute objects (most common is microscopic examination.
bright-field microscope – the object will  Its aim is to preserve the microscopic
appear dark while the background will anatomy of the tissue and make the
appear white) basahin daw diff. kinds of tissue hard so that very thin section can
microscope she might give it daw sa quiz be made.
 Safety cabinet – an enclosed, ventilated
laboratory workspace for safely working
with materials contaminated with
pathogens requiring a defined biosafety
level (whenever you cultivate bacteria
from a sample to a medium, ginagawa
dapat sa biosafety cabinet - yung
mismong plates)
 Electronic balance – use for weighting
powders
 Glassware/plasticware – includes tissue
box, mold, bottles, specimen containers, Types of Tissue Processing
volumetric glass, glass rods, panel, flask
1. Manual tissue processing – in this process
and cylinder
the tissue is changed from one container
 Chemicals and reagents – xylene, of reagent to another by hand.
benzene alcohol, etc. 2. Mechanical tissue processing – in this, the
 Dyes/stains – such as hematoxylin, eosin, tissue is removed from one jar to another
alcian blue, Papanicolaou for pap smear, by mechanical device, also called tissue
etc. processor.
Responsibilities of a Histotechnician/Medical
Technologist
1. Tissue preservation – keep the tissue as
fresh as possible to prevent tissue
decomposition/decay
2. Specimen receipt and accessioning
(logging, releasing, labeling,
identification) – make sure there is no Tissue Processing Techniques
discrepancy. Before they label tissue
samples by means of accession number.
A. Paraffin method: these steps are as c. The water is from the previous
follows: step (fixation) because most of
1. Gross examination (still part of tissue the time fixation uses aqueous
processing but this is the job of solution. We want to remove
pathologist) the water so we use alcohol.
a. cutting up specimens d. We need to dehydrate the
b. grossing is an art water from the tissue para
c. most common way of cutting ma-apply yung wax
specimen is bread loafing style e. Paraffin wax is immiscible in
water
f. During dehydration – it should
start in least concentrated. So
bakit daw? Tayo na daw ang
magbabasa ukinam.
g. For delicate
specimen/biopsies – usually
After ilagay sa filter paper yung
begin at 30% concentration of
specimen ilalagay na to sa tissue
alcohol, 50, 60, 70, 75 etc.
cassette
then eventually absolute
2. Fixation - preservation of biological
alcohol (100%)
tissues to prevent decay due to
h. Average or normal tissue
autolysis or putrefaction.
sample – can begin at 60%
a. Prevent decay and preserve
i. Alcohol replaces the water in
cells and tissues “life-like”
all cells
state.
5. Clearing - replacing dehydrating fluids
b. Tissue can be preserve by
like alcohols with a clearing agent
preventing autolysis and
(xylene)
putrefaction to occur
a. Also known as de-
c. Formaldehyde – most common
alcoholization
fixative
b. Purpose is to remove the
d. There should be a new set of
alcohol so that it can be
fixative when you start
replace by a medium such as
fixation after grossing
xylene which is now miscible
3. Decalcification*** - removal of
with paraffin wax
calcium salts by applying decalcifying
c. It allows or make the sample
agents (nitric acid). This is only done
transparent
if the specimen is rich in calcium.
d. Process of replacing alcohol by
Calcium is for bone strengthening. IF
a solvent which is miscible
THE SAMPLE IS NOT CALCIFIED,
with paraffin.
OMMITT THIS STEP. KLARO? KLARO.
e. Most common clearing agent –
a. To make the sample soft
xylene - which makes the
enough to facilitate easy
tissue to become transparent
cutting using the microtome
6. Infiltration/Impregnation - saturation
b. This is done only in bone,
of tissue cavities and cells by a
teeth containing specimens
supporting medium such as paraffin
after fixation
a. Process whereby the clearing
c. Most common decalcifying
agent is completely removed
agent - nitric acid or weak
from the tissue and replaced
acid
by a medium that will
4. Dehydration - involves removal of
completely fill all the cavities,
water from the tissues by alcohol
thereby giving a firm
a. Can do this by application of
consistency to the specimen.
alcohols
b. Infiltrating agent – will tend to
b. Most common dehydrating
fill all the cavities that is/are
agent – ethanol
present in the tissue sample
c. Tissue cavities – empty spaces
 d. Most common infiltrating
agent – paraffin wax – which
fill all those cavities in the
tissue
7. Embedding - enclosing the tissue in a
medium (e.g. paraffin wax) and then
allowing the medium to solidify
a. Also known as blocking-out or
casting
b. Most common embedding 9. Mounting - tissues are
medium – paraffin wax placed/mounted into the glass slide
c. Need to prepare freshly a. Also called mounting of the
melted paraffin wax slide
b. Clean microscopic glass slide
are taken and the section is
floated in warm water is taken
on the glass slide in such a
way that no air bubble is
trapped between them.
c. The purpose of putting tissue
slide into warm water using
DIFFERENT EMBEDDING MOLDS: floatation bath is to remove
wrinkles/straighten the tissue
1. Peel-away – disposable type section and to assist/aid the
2. Paper molds/boats tissue on the glass slide
3. Metal 10. Staining - process of applying a stain
4. Plastic or dye to a tissue
5. Leuckhart’s – letter L wooden a. A process whereby tissue
material, di na ginagamit, obsolete na components are made visible
in microscopic sections by
direct interaction with a dye
or staining solution.
b. Staining allows proper
differentiation of tissue
components
c. Manual process, dip dip lang
ganern. Dip it gently not
vigorously.
8. Sectioning/Cutting - process by which 11. Cover with coverslips – applying a thin
tissue can be sectioned/cut glass coverslips to protect tissue
a. Also known as microtomy section for possible dirt
because it uses microtome contamination and for long storage
b. A paraffin embedded tissue is a. Coverslip in histopath is same
trimmed and cut into size with glass slide
uniformly thin slices or
“sections” to facilitate studies Microscopic examination
under the microscope.
 Process of examining processed tissues
under the microscope
 Performed by pathologist
Job of histotech/medtech will start from fixation
to cover with coverslips
  Incisional biopsy specimens - where tissue
is removed for diagnosis from within an
affected area
 Punch biopsies - where punches are used
to remove a small piece of suspicious
tissue for examination (often from the
skin)
 Shave biopsies - where small fragments of
tissue are “shaved” from a surface
(usually skin)
 Curettings - where tissue is removed in
small pieces from the lining of the uterus
or cervix
 Core biopsies - where a small tissue
sample is removed using a special needle
sometimes through the skin
(percutaneously).
Specimen Receiving and Preparation
Tissue for study can be obtained from

 Biopsy – sample of tissue taken from the

body in order to examine it more closely
using a microscope in the laboratory
o A doctor/surgeon will make a cut
or incision of the patient’s body
part in order to remove a sample
of tissue which we call biopsy.
 Autopsy – examination of the body of a
HISTOPATH LAB (MODULE 2)
dead person. It may be restricted to a
RECEIVING, ACCESSIONING AND GROSS specific organ or region of the body.
EXAMINATION OF TISSUE SAMPLES
Requirement to send specimen
Specimens received for histological examination
 Requisition form/submission form
may come from a number of different sources.
 Container
They range from very large specimens or whole
organs to tiny fragments of tissue. Specimens  Fixative
are usually received in fixative (preservative) Specimen handling and transport to laboratory
but sometimes arrive fresh and must be
immediately fixed. Before specimens are When sending large specimens or small biopsy
accepted by a laboratory the identification samples:
(labelling) and accompanying documentation will  The specimen should be collected into a
be carefully checked, all details recorded and wide-mouthed container with a well-
“specimen tracking” commenced. It is vital that fitting lid to prevent leakage of the
patient or research specimens are properly fixative containing an adequate amount
identified and the risk of inaccuracies of 10% formol saline (formalin) to
minimized.  completely submerge the specimen.
The following are some of the specimen-types  For large specimens, the container should
commonly received in a histopathology lab: be larger than the specimen, preferably a
bucket.
 Excision specimens (surgical biopsies) -  Specimens should be transported to the
where whole organs or affected areas are laboratory as soon as possible to prevent
removed at operation decay of samples for proper fixation
procedures to be carried out.
  Specimen should be submitted in the  Ward no., bed no.
laboratory with the properly filled-up  Site and side – where the tissue sample or
request form biopsy is taken
 Multiple specimens from same patient
For frozen sections:
mark as A, B, C, D etc.
 The specimen should be sent in the fresh  Signature of doctor with date
state, without any fixative.
Accessioning procedure
 Specimens should be submitted in the
laboratory with the properly filled up - The specimens are accessioned by giving
request form them a number that will identify each
specimen for each patient.
Specimen identification and labeling
- Most hospitals label specimen using
A properly filled-up surgical pathology request accession number instead of patient’s
form must accompany the tissue specimen name
received in the laboratory. - Unlabeled specimens are absolutely
unacceptable for accessioning procedure
 Patient information – middle name, - Inconsistencies between the specimen
birthday, hospital number and other and the request form must be returned to
demographic data the operating room for correction by the
 Patient’s history staff concerned.
 Physical, laboratory and imaging findings - Incomplete specimens are likewise
 Patient’s diagnosis – pre-operative or unacceptable
post-operative diagnosis - Accessioning procedure is done right
 Description of the site of origin after inspecting samples
- Once the sample is accepted then
proceed to accessioning procedure or
assign a number that will identify the
specimen for a particular patient
Gross examination
- Grossing is an art
- A knowledge of what needs be taken for
microscopic study is crucial or important
for the final diagnosis
- Grossing involves:
 Accurate naked eye description of
intact specimen (specimen
identification)
 Identify all the anatomical
structures present
 Orientation markers should be
identified
 Measurements: length, width,
height
 Weight especially parenchymatous
Specimen identification and labeling organ
 Examine the external surface
Specimen container:  Cut all the organs at intervals of 1
 Plastic or glass jar – with well fitting lid cm thickness (bread-loafing style)
or cover, make sure that the jar is bigger  Describe cut surface, identify
than the specimen pathologic process
 Label matching requisition slip  If suspected lesion is present,
 Registration no. measure, describe with color and
 Full name consistency
 Age, sex  Surgical margins
  Histological sections Common errors during receipt of samples and
gross examination
Grossing room
- During receipt of sample
 Large room, well illuminated and
 No tissue received in specimen
properly ventilated
container
 Cutting board places inside a metal box
 Specimen received without
designed in such a fashion that all the
formalin
fluids flow directly into the sink
 Tissue samples received in
 Shelves for specimen container
wrongly sized containers
 Ready access to a sink with hot and cold
 Number of samples not consistent
water
with the request form
 Ready access to formalin
 Incomplete or incorrectly labelled
 Box of instruments, box with cassettes,
request form or specimen
labels
 Labelling of sample and request
 Large formalin container, photographic
form not matching
facilities
 Large table with sink for large specimens
- During data entry
 Central table for multiple purpose
 Sample and request form
allocated wrong number
 Sample and request form given
different laboratory numbers
(accession numbers)
 Sample and request form
allocated to wrong patient on
LIMS (Laboratory Information
Management System)
 Data entered incorrectly in LIMS

- During tissue dissection/grossing

 Request form and specimen match
not performed
 Specimen containers wrongly
numbered
 Cassettes labelled with different
accession number to specimen
Inking
 Sub-numbering of cassettes
- The use of an assortment of ink colors is incorrect
useful in the following ways:  Tissue lost or contaminated during
 For resection margins section
 For orientation of a specimen  Tissue inadequately fixed or
 To reduce identification error decalcified
when multiple sampling is
ALWAYS REMEMBER!
required from the same tissue
 Embedding instructions  Histopathological examination is used to
 Identify cut surface provide diagnostic information that is
important for timely diagnosis to
Criteria for rejection of gross specimen
determine treatment plan
- Reject the specimen when:  Loss of specimen or specimen switching is
 Discrepancies between the tragic both for patient and pathologist
requisition and specimen labels  Handle all specimens with care and
 Specimen with no labels respect
 Mislabeled specimen  Fresh tissue is extremely fragile and
 Absent clinical data or history subject to autolysis. Therefore, handle
 Inappropriately identified all specimens quickly and correctly
specimens
Specimen reception (video) - If the patient wasn’t found in the system,
so new entry will be created. All the
In microbiology department
information came across and then put
Segregated by specimen type: technical details:
 Additional information
 Urine  Test which was requested
 Blood cultures  Specimen type
 Fluids  Time of collection
 Tissues  Received date
 Swabs - Saving these data means the quest is
Microbiology request form: booked and this request is new to specific
lab number which is used to record
 Patient hospital number sample request.
 Surname
 First name Providing result
 Date of birth  Results are only given to authorized
 Gender personnel (e.g, GP’s, ward staff)
 Specimen type
 Test requested **please take note that this video presented
 Consultant who requested test microbiologic samples but notice that protocols
 Location where sample was taken for specimen receipt are almost the same except
 Time that samples delivered in the histopathology
 Date laboratory are tissues that are soaked in
fixative. This video aims to give you an actual
Before opening the sample, check the integrity picture of what it is to be done in the reception
first. Like for example, if it’s not broken or area upon receiving the specimen.
anything.
Gross examination by pathologist (video 2)
Sample inspection
Gross Examination of Whole Prostatectomy
 Clean with disinfectant Specimens
 Minimum identification criteria (check if
it corresponds correctly)  Patient identification (verify patient’s
o Hospital number name on the container)
o Full name  Specimen orientation (e.g., seminal
o Date of birth vesicle)
 Check the sample (if it’s correct with o Seminal vesicles are posterior
what you receive) o Superior right side is inked green
 Sample mismatch (if the name doesn’t o Left side is inked blue
correspond with the name on the form or o Whole prostate is inked black
when you received a totally unlabeled o Black ink is more permanent when
specimen, then: adhering to tissues assuring better
o Reject easily repeatable samples identification of surgical margins
and request a retake and sealing the previous green
o If not repeatable, call medic to and blue inks.
confirm identity of sample o Fixing the ink by spraying the
specimen with acetic acid 20%
Booking in system  Begin sectioning specimen
 Lab number o The seminal vesicles are sectioned
 Hospital number at their intersection with the
 Patient’s surname prostate
 First name o Measuring
 Sex  Left and right
 Date of birth measurement
 Location  Height
 Length
 Weight
 Volume measurement by

doing water displacement
 Insert probe through
urethra helps to orient the
axis of the prostate
 Sectioning procedure at
4mm slices from apex to
base
 Embedding piece through
bread-loaf style (two little
sections)
 Make sections with wide
movements of the blade to
avoid crinkling of the cut
surface
 Section the seminal vesicle
 Labeling cassettes and submitting the
sections
o Crinoline bags

**This video shows how the grossing of a tissue

specimen is done from patient identification,
inking, cutting (bread-loafing style), describing,
measuring, labeling, and putting the tissue into
tissue cassettes in preparation for processing.