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*HISTOTECHNOLOGY MAIN FRAMEWORK consists of base,arm,

stage,substage, and mechanical stage.


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

* HANDLING OF SPILLS
* Standardized operating procedures (SOP`s)
- If the amount of spilled material is limited to a few
- to ensure that the laboratory is safe grams or milliliters ---- simply: wipe off towels or
sponge.
- includes detailed procedures for handling hazardous
substances and personal hygiene practices. - 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.
- a special method of smear prep whereby
6. Sodium hypochlorite the surface of a freshly cut piece of tissue is brought
into contact and pressed to the surface of a clean
- a strong oxidant, do not mix with glass side.
formaldehyde or diaminobenzidine
3.E. Frozen Section
7. Toluene
- normally utilized when a rapid diagnosis of
- repeated exposure can cause impaired the tissue in question is required.
memory poor coordination, mood swing and - commonly used for: 1. rapid pathologic
permanent nerve damage. diagnosis during surgery, 2.
_________________________________________,
* FRESH TISSUE EXAMINATION 3. Diagnostic and research demonstration of soluble
substance such as lipids and carbohydrates, 4.
METHOD OF FRESH TISSUE EXAMINATION Immunofluorescent and immunohistochemical
staining, 5. Some specialized silver stains, particularly
1. Teasing or dissocation 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 1. Speed

- is the first and most critical step in histotechnology.

- no process of histotechnology is more critical to slide 2. Penetration


preparation than fixation.

- The primary aim is to preserve the _____________


and ______________________. 3. Volume

-Prevents degeneration, decomposition, putrefaction,


and distortion of tissues after removal from the body.
4. Duration of fixation
- the ______________ is to hardeal and protect the
tissue from trauma of further handling.

- inactivated the lysosomal enzymes. * Characteristics of a Good fixative:

* 2 Basic mechanisms involved in fixation: “So far, no single fixative has yet been known to
possess al the qualities of an ideal solution. There are
1. Additive fixation numerous fixatives available, and each has its own
- whereby the chemical constituent advantages and disadvantages”.
of the fixative is taken in and becomes part
of the tissue by ___________________. * TYPES FIXATIVES ACCORDING TO composition.

2. Non-Additive fixation A. Simple fixatives – are made up of only one


- the fixing agent is not incorporated component substance.
into the tissue but alters the tissue
composition and stabilizes the tissue by B. Compound fixatives – are those that are made up
removing the bound water attached to H- of 2 or more fixatives.
bonds. (_________ fixatives).
* TYPES FIXATIVES ACCORDING TO action.
* MAIN FACTORS INVOLVED IN FIXATION
A. Microanatomical fixatives
- Hydrogen ion concentration B. Cytological Fixatives
- Temparature
- Thickness of section 1. Nuclear fixatives – preserve the nuclear structures;
- Osmolality usually contain glacial acetic acid as the primary
- Concentration component; have a pH of 4.6 or less
- Duration of fixation
2. Cytoplasmic fixatives – preserve cytoplasmic
* PRACTICAL CONSIDERATION OF FIXATION 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 - Normally used in conjunction ____________
the fluid fixative every 3 months. fixatives to from a compound solxn.
- Fixes and precipitates nucleoproteins.
B. 10% Formol saline - When combined with potassium dichromate,
the lipid-fixing property of the latter is
- For fixation of CNS tissues 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 MEASURING EXTEND of DECALCIFICATION
issues in a second fixative. * Physical or Mechanical test
* Post-chromarization
* X-ray or Radiological Method
- Is a form of secondary fixation whereby a - most sensitive
primarily fixed tissue is placed in aqueous
solxn of 2.5-3% potassium dichromate for 24 * Chemical method (Calcium oxalate test)
hrs.
* POST DECALCIFICATION
* WASHING OUT
- Immersing in either saturated lithium
- Is the process of removing excess fixative carbonate solxn or 5-10% aqueous sodium
from the tissue after fixation. bicarbonate solxn.
- Tap water, 50-70% alcohol, alcoholic iodine. - 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