*HISTOTECHNOLOGY
stage,substage, and mechanical stage.
- is the art and science performed by the
histotechnologist to produce a tissue section of
good quality that will enable the pathologist to
diagnose the presence & absence of disease.
*INSTRUMENTATION IN HISTOTECHNOLOGY
- Microscope
- Microtone
- Cryostat
- Histotechnician
- Automated coverslipper
- Automated H & E stainer
*”In the operation of any piece of equipment,
-----> read the manual”
*CURRENT FILE FOR EVERY PIECE OF
EQUIPMENT IN THE LABORATORY SHOULD
CONTAIN THE FOLLOWING INFORMATION.
-Name, Manufacturer, Model Number and Serial
Number
-Record of preventive maintenance performed as
prescribed by the manufacturer.
-Record of service calls and repairs performed
-Copy of operating manual
*MICROSCOPE
-is an instrument that enlarges images and allows the
visualization of morpholic cellular details that are too
small to be seen by the unaided eyes.
*PARTS OF THE MICROSCOPE
MAIN FRAMEWORK consists of base,arm,
1. Base – provides support for the microscope
2. Arm – supports and holds the magnifying and
adjustment system.
3. Stage – is the flat platform where the slide is placed
for examation.
4. Substage – is located directly under the stage and
holds the condenser and diaphragm.
5. Mechanical stage – permits the movement of the
stage while holding the slide in the phase of focus.
*PART OF THE LENDS SYSTEM ARE THE
nosepiece, objective, body tube, eyepiece, and focal
length
1. Nosepiece – is located at the end of the body tube
for holding the objectives.
2. Objectives – consists of a system of lenses located
at the end of the body tube that are held in place by
the nosepiece.
3. Eyepiece – is also referred to as the ocular. This is
the lens system nearest the eye.
4. Focal Length – is the distance between outer
length of objectives and the cover glass of slide.
*MAGNIFICATION
- is the process that increases the size of the
structure under examination.
Is achieved by the use of the microscope`s
lens system.
TOTAL MAGNIFICATION OF A MICROSCOPE
- magnifying power x eyepiece
Of objective
160mm – constant tube length.
* ILLUMINATION SYSTEM consists of conduser, * Every chemical should be labeled with RMT
illuminates, objectives, and ocular. certain basic information, including :

* DAILY MAINTENANCE OF THE MICROSCOPE - Chemical name

-wipe with lens paper - Manufacturer`s name and address

-remove dust with an air bulb. - Date purchased or made

-covered when not in use - Expiration date

- always support the microscope when carrying. - Hazard warnings and safety procedures

* QUALITY CONTROL IN HISTOPATH

SECTION??? * Storage of hazardous chemicals….

* Standardized operating procedures (SOP`s)
- to ensure that the laboratory is safe
- includes detailed procedures for handling hazardous
substances and personal hygiene practices.
* HANDLING OF SPILLS
- If the amount of spilled material is limited to a few
grams or milliliters ---- simply: wipe off towels or
sponge.
- If the spill is large --- the area must be sealed off.

*HAZARDS * FIRST AID

Health Hazards - Emergency eyewash stations

- Biohazards - Emergency showers
- Irritants
- Corrosive chemicals *HANDLING OF POTENTIALLY INFECTIOUS
- Sensitizers SPECIMEN
- Carcinogens
- Toxic materiasl - Fresh tissue and body fluids must ALWAYS be
considered potentially infectious.
Physical Hazards
- Combustibles *HAZARDS AND HANDLING OF COMMON
- Flammables CHEMICALS
- Explosives
- Oxidizers - “As a general during the process of dilution
concentrated acid should be added to water. (NEVER
WATER TO ACID) in order to prevent splashing and
should be done under a chemical fume hood”.

1. Acetic acid
- 1-10 % dilution is relatively safe
2. Aniline
- a potential carcinogen.
- excessive exposure may cause
________________.
3. Chromic acid
- Toxic to kidneys
-enviromental toxin
4. Isopentane 3.C. Pull apart

- is extremely flammable and highly volatile - done by placing a drop of secretion or

sediment upon one side and facing it to another clean
5. Methanol slide.

- may cause blindness or death if taken in 3.D Touch prep

excessive amounts.
6. Sodium hypochlorite
- a strong oxidant, do not
formaldehyde or diaminobenzidine
7. Toluene
- repeated exposure can cause impaired
memory poor coordination, mood swing and
permanent nerve damage.
* FRESH TISSUE EXAMINATION
METHOD OF FRESH TISSUE EXAMINATION
1. Teasing or dissocation
- a special method of smear prep whereby
the surface of a freshly cut piece of tissue is brought
into contact and pressed to the surface of a clean
mix with glass side.
3.E. Frozen Section
- normally utilized when a rapid diagnosis of
the tissue in question is required.
- commonly used for: 1. rapid pathologic
diagnosis during surgery, 2.
_________________________________________,
3. Diagnostic and research demonstration of soluble
substance such as lipids and carbohydrates, 4.
Immunofluorescent and immunohistochemical
staining, 5. Some specialized silver stains, particularly
in __________________________.

-selected tissue spx is immered in a watch *The more commonly used method of freezing
glass containing isotonic salt solution, carefully include:
dissected or separated, and examined under the
microscope. - Liquid nitrogen

2. Squash Prep - Isopentane cooled by kiquid nitrogen

- small pieces of tissue not more than one - Carbon dioxide gas
mm in diameter are placed in a microscopic slide and
forcibly compressed with another slide. - Aerosol sprays

3. Smear Prep *PROCESSING OF TISSUES

- cellular materials are spread lightly over a Fixation

slide by means a wire ________ or applicator.
Dehydration
3.B. Streaking
Clearing
- with an applicator stick or loop, the material
is rapidly and gently applied in a direct or zigzag line, Infiltration (Impregnation)
attempting to obtain a uniform distribution of
secretion. Embedding

3.B. Spreading Trimming

- a selected portion of the material is Section cutting

transferred to a clean slide and gently spread into a
moderately thick film by _________________ apart Staining
with an applicator stick.
Mounting

Labeling
* FIXATION
- is the first and most critical step in histotechnology.
- no process of histotechnology is more critical to slide
preparation than fixation.
- The primary aim is to preserve the _____________
and ______________________.
-Prevents degeneration, decomposition, putrefaction,
and distortion of tissues after removal from the body.
- the ______________ is to hardeal and protect the
tissue from trauma of further handling.
- inactivated the lysosomal enzymes.
* 2 Basic mechanisms involved in fixation:
1. Additive fixation
- whereby the chemical constituent
of the fixative is taken in and becomes part
of the tissue by ___________________.
2. Non-Additive fixation
- the fixing agent is not incorporated
into the tissue but alters the tissue
composition and stabilizes the tissue by
removing the bound water attached to H-
bonds. (_________ fixatives).
* MAIN FACTORS INVOLVED IN FIXATION
- Hydrogen ion concentration
- Temparature
- Thickness of section
- Osmolality
- Concentration
- Duration of fixation
* PRACTICAL CONSIDERATION OF FIXATION
1. Speed
2. Penetration
3. Volume
4. Duration of fixation
* Characteristics of a Good fixative:
“So far, no single fixative has yet been known to
possess al the qualities of an ideal solution. There are
numerous fixatives available, and each has its own
advantages and disadvantages”.
* TYPES FIXATIVES ACCORDING TO composition.
A. Simple fixatives – are made up of only one
component substance.
B. Compound fixatives – are those that are made up
of 2 or more fixatives.
* TYPES FIXATIVES ACCORDING TO action.
A. Microanatomical fixatives
B. Cytological Fixatives
1. Nuclear fixatives – preserve the nuclear structures;
usually contain glacial acetic acid as the primary
component; have a pH of 4.6 or less
2. Cytoplasmic fixatives – preserve cytoplasmic
structures in particular; must never contain glacial
acetic acid; have a pH of more than 4.6.
* ALDEHYDE FIXATIVES
A. Formaldehyde (Formalin)
- One of the most widely used fixatives
- Commercially available solxn contains 35-
40% gas by weight.
- A “tolerant” fixative used for mailing spx
- Prolonged storage of formaldehyde may
induce precipitation of white
paraformaldehyde deposits.
- bleaching of tissues may be prevented by changing
the fluid fixative every 3 months.
B. 10% Formol saline
- For fixation of CNS tissues
- Normally used in conjunction ____________
fixatives to from a compound solxn.
- Fixes and precipitates nucleoproteins.
- When combined with potassium dichromate,
the lipid-fixing property of the latter is
destroyed.

C. 10% Neutral buffered formalin or phaspate * ALCOHOL FIXATIVES

buffered formalin.
A. Methyl alcohol
- For preservation and storage of surgical,
post-mortem and research spx. - For fixing dry and wet smears, blood smears
and bone marrow tissues.
D. Formol-Corrosive (Formol Sublimate) B. Isopropyl Alcohol

- Contains mercuric chloride - Less irritant

- For fixing touch preparations
E. Alcoholic Formalin (______________) C. Ethanol

- Contains glacial acetic and _______ acid. - Used at concentrations of 70% - 100%.
- Fixation is faster: use to fix sputum D. ________ Fluid

F. _______________ - For fixing chromosoes, lymph glands and

urgent biopsies.
- Made up to 2 formaldehyde residues, linked - Most rapid fixative
by 3 carbon chains.
- Buffered _____ dehyde+_____________ = * OSMIUM TETROXIDE (OSMIC ACID)
Satisfactory for electron microspy
A. Flemming`s solution
* METALLIC FIXATIVES
- Most common nucleos stain
A. Mercuric chloride
- most common Buffered _____ B. Flemming`s solxn without acetic acid.
dehyde+_____________ = Satisfactory for
electron microspy * TRICHLOROACETIC ACID
- tissues contain black precipitates of mercury
- recommended for renal tissues. - May be used as a weak decalcifying agent.
- Zenker`s fluidm Zenker`s formol,
Heidenhain`s susa solxn, B-5 fixative. * ACETONE

B. Chromate Fixatives (CROP) - For the study of water diffusible enzymes

eps. Phosphatases lipases.
b.1 Chromic acid
b.2 Potassium dichromate *HEAT FIXATION
b.3 Regard`s (Mullers) Fluid
b.4 Orth`s fluid - Usually for frozen sections and preparations
of bacteriologic smears.
C. ________ Fixatives
* SECONDARY FIXATION
- Recommended for acid
mucopolysaccharides
- Fixes connective tissue mucin

* GLACIAL ACETIC ACID

___________________________________
___________________________________
___________________________________
___________________________________
 - The process of placing an already fixed
issues in a second fixative.
* Post-chromarization
- Is a form of secondary fixation whereby a
primarily fixed tissue is placed in aqueous
solxn of 2.5-3% potassium dichromate for 24
hrs.
* WASHING OUT
- Is the process of removing excess fixative
from the tissue after fixation.
- Tap water, 50-70% alcohol, alcoholic iodine.
MEASURING EXTEND of DECALCIFICATION
* Physical or Mechanical test
* X-ray or Radiological Method
- most sensitive
* Chemical method (Calcium oxalate test)
* POST DECALCIFICATION
- Immersing in either saturated lithium
carbonate solxn or 5-10% aqueous sodium
bicarbonate solxn.
- Running tap water.

* FIXATION ARTIFACTS * TISSUE SOFTENERS

1. Formalin pigment – formed under acid - Perenyi`s fluid, Molliflex, 2% HCL, or 1%

condition. HCL in 70% alcohol.

2. Crush artifact – intense ecsinophilic staining, * DEHYDRATION

present in surgical liver biopsies.
- Process of removing intercellular and
extracellular water from tissue
* DECALFICATION - Many of these dehydrating agents are
alcohol,
- Is the process whereby calcium or lime salts - Starts by placing the fixed specimen in 70%
are removed from tissues following fixation. ethanol progressing tp 90% to 100%
- As a general rule, whatever dehydrating
1. ACID DECALCIFYING AGENTS agent is used, the amount in each stage
should not be less than 10 times the volume
- Are the most widely used agents for routine of the tissue.
decalcification.
2. CHELATING AGENTS *COMMONLY USED DEHYDRATING AGENTS
3. ION EXCHANGE RESIN ARE:
4. ELECTROPHORESIS
1. Alcohol
* FACTORS TO CONSIDER IN DECALCIFICATION
2. Acetone
* The recommended ratio of fluid of tissue volume is
20.1%. 3. Dioxane 4 cellosolve
- Most expensive
* The optimum temperature recommend is room 4. Cellosolve (Ethylene glycol monoethyl)
temp. - Cleaning and dehydrating
5. Triethyl phosphate
* The ideal time required is 24-48 hours. 6. Tetrahydrofuran
* CLEARING d. Tissue-Tek
e. Disposable Embedding mold
- Also called as dealcotiolization. - peel away
- Is the process whereby alcohol or - plastic ice trays
dehydrating agent is removed from the - paper boats
tissue.
* The process by which a tissue is arranged in
- COMMONLY CLEARING AGENTS USED ARE: precice positions in the mold during embedding is
known as orientation.
Xylene, Toluene, Benzene, Chloroform,
cedarwood, aniline oil, clove oil, carbon teirahloride, * MICROTOMY
Methyl benzoate and methyl salicylate.
Microtome is consists of three essential parts,
* IMPREGNATION AND EMBEDDING namely;

IMPREGNATION (INFILTRATION) 1. Block holder – where the tissue is held in

position.
- Is the process whereby the clearing agent is 2. Knife Carrier and knife – for actual cutting
completely removed from the tissue and 3. Pawl, Ratchet feed wheel and adjustment
replaced by a medium that will completely fll screws
all the tissue cavities.
- To line up the tissue block in proper position
EMBEDDING (CASTING OR BLOCKING) with the knife, adjusting the proper thickness
of the tissue.
- Is the process by which the impregnated
tissue is placed into a precisely arranged * 5 KINDS of MICROTOMES:
position in a mold containing a medium.
1. Rocking Microtome - for cutting serial
* 4 types of t issue impregnation and embedding sections of large blocks - largest
media
2. Rotary microtome – for cutting paraffin
a. Paraffin wax embedded sections.
b. Celloidin
c. Gelatin 3. Sliding microtome – for cutting celloidin
d. Plastic embedded sections.

d.1 Epoxy Embedding plastics EPON 4. Freezing microtome – for cutting

unembedded frozen sections
d.2 Polyester plastics
5. Ultrathin microtome – for cutting sections for
d.3 Acrylic Plastics
electron microscope
* DOUBLE EMBEDDING METHOD
* MICROTOME KNIVES
- Is the process in which tissues are first
infiltrated with celloidin and subsequently
embedded in paraffin. 1. Plance concave knife
- 25 mm in length
* TYPES OF BLOCKING-OUT MOLDS USED: - One side is flat while the other is concave

a. Leuckhart`s Embedding mold

b. Coumpound Embedding unit
c. Plastic Embedding Rings and Base mold
 2. Biconcave knife - Brittle or hard tissue
- 120 mm in length - over fixation
- Both sides are concave
- Clearing agent turns milky
3. Plane-wedge knife - incomplete dehydration
- 100 mm in leghth
- Both sides are straight - Tissue is opaque
- incomplete clearing
* HONING
- Tissue is soft when block is trimmed
- Involves the removal of gross picks on the - incomplete fixation
knife edge to remove blemishes and grinding
the cutting edge of the knife on a stone to - Air holes found on tissue during trimming
acquire an even edge. - incomplete impregnation

- Types of hones: - On trimming, wax appears crystalline

- contaminated wax
 Belgium fellow
 __________________ - Paraffin blocks after c ooling is moist and
 __________________ crumbles
- incomplete impregnation
- “Edge First, with a heel to toe direction”
* STAINING
* STROPPING
Restraining of old sections
- Is the process whereby the “burr” formed
during honing is removed and the cutting - Immersed in Xylene for 24 hours, placed in a
edge of the knife is polished. 0.5 potassium permanganate solxn, rinsed in
- Edge Last, toe to heel direction tap water and immersed in 5% oxalic acid for
- * Disposable blades 5 mins, then wash it again in running tap
* Glass knives water.
* Diamond knives
Broken slides
* OTHER EQUIPMENT
- Mount it to another clean xylene moist slide,
- Waterbath coverslip removed by soaking in xylene and
10’C below melting point, 56’C paraffin place the slide in incubator until all mountant
melting point. has been removed.
- The whole slide is then covered with 6 parts
- Drying oven or hot plate of ______________ and 1 part
__________.
- Forceps * H & E STAINING TECHNIQUE

- Clean slides - most common method utilized for microanatomical

studies.
- Ice tray
Results:
- Pencil
Nuclei- blue to blue black
* FAULTS OCCURING DURING TISSUE
PROCESSING Karyosome – dark blue

Berel angle – 27-32 Cytoplasm, proteins in edema fluid- pale pink

Clearance angle – 5-15
Wedge – 14-15 RBCs, eosinophilic granules, keratin – bright
orange red.

Plasma cells and osteoblast- purplish pink

Cartilage- pink or light blue to dark blue

Calcium and calcified bone – purplish blue

* ADHESIVE AND MOUNTING MEDIA 4. Clarite (1.544)

ADHESIVES * RINGING

- Are essential for methods that require - The process of sealing the margins of the
exposure of sections to acids and alkalis coverslip to prevent the escape of fluid or
semi-fluid mounts and evaporation of
COMMON ADHESIVES mountant

1. Mayer`s Egg Albumin - Kronig cement and Durofix

- most commonly used
* ENDOGENOUS PIGMENTS
2. Gelatin
3. Starch paste 1. Hemosiderin
4. Plasma - iron containing pigment of hemoglobin
5. Poly-L-Lysine 2. __________
6. APES ( 3-Arminopropylthriethoxysilane) - iron free pigment of hemoglobin
3. Hematin
- iron free pigment of hemoglobin
4. Hemozoin
* MOUNTING -black granule formed by malarial parasites
5. ________________
- preventing the movement of the coverslip - brownish yellow pigment that occurs with
hemosiderin in hemachroromatosis
- prevent the distortion of image during microscopic
examination. * DIAGNOSTIC CYTOLOGY

- refractive index of the mountant should be as near A. Exfoliative Cytology

as possible to that of the glass which is 1.518
B. FNAB
- divided into 2 groups SPECIMENS:
a. Aqueous
b. Resinous - Cervicovaginal smear (pap smear)
- Nipple discharge
AQUEOUS MEDIA - Gastric or bronchial scretions
- Pleural and peritoneal fluids
1. Water - Sputum
2. Glycerin - Urine sediment
3. Farrant`s Medium (R.I 1.43) - CSF
4. Apathy`s Medium (R.I 1.52)
5. Brun`s Fluid - when specimen is more than a few drops, the fluid
should be centrifuged first (2,000 rpm 2min)

RESINOUS MEDIA - common fixatives are 95% _____ and 95% ethanol

1. Canada Balsam (1.524)

2. DPX (1.532)
3. XAM (1.52)
 - If spray fixative is used, kept the distance - Small cells, slightly cylindrical, occurring in
approximately 12 inches away. tightly packed groups.

Gynecological Specimens g. Endocervical glandular cells

- T-zone (junction of endoceal/ectocervical - A honey comb appearance when viewed on

mucosa) end.
- Sample should include squammons,
columnar and metaplastic cells.
NON_GYNECOLOGICAL SPECIMENS
* Cytological Collection and preparation
a. Respiratory Tract Specimens
a. Endocervical Brush – samples of
endocervical canal. - At least 3 consecutive morning sputum
b. Vaginal scrape – patients with hysterectomy specimens (Saccommano fluid)
c. Lateral vaginal scrape – for hormonal - Induced sputum (“down-deep”)
evaluation
d. For quadrant vaginal scrape – for localization b. Bronchoscopy Specimens
of vaginal adenosis
e. Vulvar scrape – for detection of herpetic - Washing the brochi with 1-2 cc saline
lesions or carcinoma
- Should contain ciliated brochial cells
* CELLS FOUND IN CERVICO-VAGINAL SMEAR
c. Gastric secretion
a. Mature superficial cells
- Patient should fast for at least 8 hours
- Polygonal squamous cells, presence of pale
pink staining cytoplasm and dark pyknotic d. Peritoneal, Pleural, Pericardinal Fluids
nuclei, true acidophilia – presence of
estrogen - Presence of malignant cells usually indicate
- metastatic involvement
b. Intermediate cells - Jelly like clots may be prevented by adding
300 units ______ for every 100ml of
- Medium sized polyhedral or elongated with asiparate.
basophilic vacuolated cytoplasm
-
c. Parabasal cells

- Round to oval cells, smaller than

intermediate cells and have a larger
vesicular nucleus, normally found from two
weeks of age to puberty, after childbirth, with
abortion and after menopause.
-
d. Navicular cells

- “Boat shaped”, suggests a combined

estroge-progesterone effect, found in the
latter half of the menstrual cycle
-
e. Pregnancy Cells

- Observed greatest at the center of the cell

due to glycogen accumulation, pushing the
nucleus to the side or towards the cell
membrane.

f. Endometrial cells