Received patient

specimen with
LABORATORY
REQUISITION FORM

PATIENT
INFORMATION should
match requisition form

ACCESSIONING

HISTOPATHOLOGY
TESTS

Fixed Tissue Specimen Fresh Tissue Specimen

Other Specimens Large Tissue Specimen Small Specimens

Lymph Nodes Organ or Tissue Surface of Thick Mucoid Secretion Seroud Fluids, Concentrated Mucoid Secretion
(>3mm) Small pieces of Immunohistochemistry In-Situ Hybridization Surgical Tissue Specimens
Specimen Freshly Cut piece Fresh Septum and Septum, Enzymatic Lavage from Fresh Septum and
Tissue (<1mm)
of Tissue Bronchial Aspirates GIT, Urinary Sediments, Vaginal Bronchial Aspirates
Pool, and Breast Secretions
General Steps
Cut into smaller pieces Cut the specimen
(1cm thickness) (about 2 mm Bivalved Squash
Teasing Method Touch Smear Tissue or Cell Frozen Section Method
Brain Colon Eye Hollow thickness) (Crushing) Spread Smear Method Streaking Method
Cancers Structures Method Pull-apart Smear Method
Method
Specimen wrapped in a Affix to glass slide
Inject fixative Cut open filter paper Procedures Considerations Procedures Considerations
10% NBF for Procedures Considerations Procedures Considerations Procedures Considerations Procedure
Small Polyps Large Polyps longitudinally
2-3 weeks Digest Protein Specimen Considerations
Cold Knife Procedure
Watch Glass 1. Specimen should be
Bisected Sides are Clean slide Application Clean slide
Clean slide dispersed evenly on the Add antibody Add probe
trimmed Two slides Application stick
Tied at Circle Supravital stain slide
away from stick Procedures Considerations Dry Fresh without No sharp or Cutted to small Gentle stroke of new
of Willis & 2. Pulled in a single,
the stalk Isotonic Procedures Denature fixative hard structures pieces scalpel upon cutting
suspended uninterrupted motion
Solution

Procedures Incubate Hybridize

Procedures Procedures Uses CO2 Equipment: Any Temperatures Cryostat Procedure
Stalk Trimmings Procedures
1. Tissue is touched on the slide. Microtome Knife: -40 to -60C
Transfer from container 2. Specimen leaves behind its Tissue: -5 to -10C
to cassette imprint in the form of cells. Wash Environment: 0 to -10C
1. Tissue is placed on the slide. 1. Specimen is gently spread
2. Forcibly compressed with onto the slide. Procedures Considerations
1. Specimen is immersed in watch glass with
another slide. 2. Mucous strands are teased 1. Rapid, but gentle zigzag
isotonic salt solution such as Muller's
3. Stain with supravital stain. apart using an application application of the material
Fluid, 1% Normal Saline, 30% alcohol
TISSUE PROCESSING stick. throughout slide. Procedure
2. Spread off the specimen away from each
other using a teasing needle Embedding Medium:
Temperature: -18 to -20C Equipment: Cryostat
3. Stain Optimal Cutting
Temperature

1. Place piece of filter paper soaked in gum syrup on microtome

stage.
2. Short bursts of CO2 are applied - freezing filter paper on stage. Procedure
3. Selected tissue block (approx. 3-5mm thick) is oriented on
Hard Tissues intermittent bursts of CO2
Soft Tissues - Each for 1-2 secs duration | Intervals of around 5 seconds.
4. Frozen to a point it will be firm enough to section. 1. Tissue embedding in the mold
5. Lift tissue to the knife (manually) 2. Tissue loading in the frozen section chamber
6. Trim tissue block until surface is flat. 3. Specimen holder is clamped firmly onto the object head mounted
Fixation (same 7. Surface is warmed w/ finger til hard frozen tissue starts to thaw & on the spindle of the microtome
FIXATION procedure with soft 4. Tissue trimming
becomes visible to naked eye.
tissue specimen) - This is the DEW LINE (point at which section may be cut at 10 5. Antiroll plate
Types
microns thickness) 6. Sectioning
8. Remove sections that stick to the knife blade w/ camel hair 7. Section Lifting
brush or finger moistened w/ water. 8. Slide Fixation
Physical Fixation Chemical Fixation Decalcification 9. Transfer sections to dish of distilled water to separate.
- Dish water usually placed on dark or black background to see
Categories Types sections bc. they are colorless or very light colored.
10. Pick up sections individually for mounting & staining.
Strong Weak Chelating
Mineral Acid Organic Acid Agents
Heat Fixation Microwave Freeze-Substitution
Freeze-Drying Simple Compound Categories Categories
Fixation
Slide Preparation
NItric Acid Hydrochloric Formic Acid Trichloroacetic Sulfurous Chromic Acid Neutral EDTA
Categories Citric
Acid Acid Acid (Flemming's Acid-Citrate
Fluid) Buffer Solution
(pH: 4.5)
Osmium 10% Formol-Nitric Perenyi's Phloroglucin-Nitric Von Ebner's Temporary Stains Permanent Stains
Aldehyde Metallic Picric Acid Glacial Acetic Acid Alcohol Trichloroacetic acid Acetone
Tetroxide Aqueous Acid Fluid Acid Fluid
Specifications S
Types of Osmium NItric Acid
Specifications Specifications Tetroxide Solution
Specification Types of Glacial Acetic Acid
Specifications Types of Metallic Types of Picric Acid Nuclear Rapid Eyedropper Permanent Stains
Types of Aldehyde Types of Alcohol Flemming's Staining Method
Embryo & Precipitates Brain tissues structures &
All kinds of Bouin's Nucleoproteins, Bouin's for rabies, Nissl Fat
CNS tissue & 10% Zenker's Pituitary Carnoy's
General Tissue biopsies & Chromatins granules & Fixative
Formol-Saline Cytoplasmic
Post-mortem Flemming's Polychrome Methylene Blue
granules Cytoplasmic Toluidine Blue
w/o acetic Alcoholic pinacyanol
Pituitary structures Thionine
General acid
Tissue, gland, Bone Glycogen Picric Acid Hematoxylin & Eosin
POST-DECALCIFICATION
Iron-containing 10% NBF Marrow, Zenker-formol Enzymes 70-100%
pigment, & Blood- or Helly's Ethanol
elastic fibers containing
organs 1. Immersion for several
Mucopoly- hours in lithium
Gastrointestinal saccharides, & Process
Newcomer's carbonate or 5-10%
Tissue, Skin tumor Heidenhain Nuclear
Prostate Hollande's biopsies Susa aqueous bicarbonate
CHONs 2. Wash out for 1-4 hours
biopsies &
Bone marrow
Bone Marrow B5 Fixative
Biopsies
TISSUE
Formaldehyde
SOFTENER
Proteins & Chromic acid
Carbohydrates
Formol-Sublimate Specifications

Chromatin,
Mitochondria, Perenyi's 4% Aqueous 2% HCl 1% NaCl in
Molliflex
Glutaraldehyde Mitotic Fluid Phenol ROH
Regaud's or
figures, Golgi Muller's
bodies, RBC,
& Colloid-
Parafolmaldehyde containing

Early DEHYDRATION
Karnovsky's
degeneratove
Paraformaldehyde Orth's
processes &
Necrosis &
40% Aqueous myelin
Glyoxal
Acid Dioxane Cellusolve /
4% Aqueous Alcohols Acetone
mucopoly-
saccharides Lead
(Diethylene
Dioxide)
Ethylene Glycol
Monoglycol Ether
Triethyl Phosphate Tetrahydrofuran
EXAMINATION BY THE PATHOLOGIST BILLING OF PATIENT
& mucin

Lipids,
Mitochondria, 3% Isopropanol
Cytoplasm, Potassium
Chromatin, & dichromaye
Chromosome

Lead fixatives

PROCESS

PROCESS

Soak in fixative for 24 hours

70% Alcohol - 6 hours | Agitate - 24 mins

95% Alcohol - 12 hours | Agitate - 48 mins
WASHING OUT 100% Alcohol - 2 hours | Agitate - 8 mins
100% Alcohol - 1 hour | Agitate - 4 mins
100% Alcohol - 1 hour | Agitate - 4 mins

Excess Formalin, Osmic Acid, & Chromates Excess Picric Acid Fixatives & Gendre's Excess Mercuric Chloride

CLEARING

Tap Water 50-70% ROH Alcoholic Iodine Specifications

Common Urgent CNS tissues, CNS tissues, Embryos & Delicate Skin & Smooth
Clearing biopsies & Lymph nodes, Smooth Muscle, tissues Muscles
Agent Routine Embryos Skin
purposes
DEHYDRATION Clearing Agents

Categories Xylene or Carbon Methyl benzoate &

Xylol Benzene Chloroform Cedarwood Oil Aniline Oil Clove Oil Tetrahydrofuran Tetrachloride Methyl salicylate

Dioxane Cellusolve /
Alcohols Acetone
(Diethylene Ethylene Glycol Triethyl Phosphate Tetrahydrofuran
Dioxide) Monoglycol Ether
PROCESS
Blood &
Methanol
tissue films

Soak in clearing agent for:

Plant & 1 hour | Agitate - 4 mins
Butanol 1 hour | Agitate - 4 mins
Animal

PROCESS

70% Concentration- 6 hours | Agitate - 24 mins

95% Concentration - 12 hours | Agitate - 48 mins
100% Concentration - 2 hours | Agitate - 8 mins
100% Concentration - 1 hour | Agitate - 4 mins
100% Concentration - 1 hour | Agitate - 4 mins

CLEARING

Specifications

1. The slide is taken from the last xylene station, & excess xylene is wiped off the back of the slide
(opposite the specimen) & the edges of the tissue section
Common Urgent CNS tissues, CNS tissues, Embryos & Delicate Skin & Smooth 2. The slide is placed horizontally on a table top, w/ the tissue section facing upwards
Clearing biopsies & Lymph nodes, Smooth Muscle, 3. A drop of mounting medium is placed onto the tissue section
tissues Muscles 4. A cover slip (size appropriate to the tissue section) is then placed onto the tissue section
Agent Routine Embryos Skin - Lower one side of the cover slip onto the surface of the section w/ the other side following
purposes 5. Lower one side of the cover slip onto the surface of the section with the other side following
- In case bubbles are formed; push aside using applicator sticks or a forcep
Clearing Agents 6. Excess mounting medium is wiped or blotted off using tissue paper or gauze
7. The slides are kept flat overnight to dry

Xylene or Carbon Methyl benzoate &

Xylol Benzene Chloroform Cedarwood Oil Aniline Oil Clove Oil Tetrahydrofuran Tetrachloride Methyl salicylate

PROCESS

Soak in clearing agent for:

1 hour | Agitate - 4 mins
1 hour | Agitate - 4 mins PROCESS

IMPREGNATION DPX
GLYCERIN JELLY FARRANT'S WATER GLYCERIN APATHY'S BRUN'S (Dibutyl Phthalate Canada Balsam XAM Clarite
(KAISER'S 1880) MEDIUM MEDIUM FLUID and Xylene)
Specifications

Common Large & Hollow Cavities,

Impregnating Hard & Dense tissues, &
Agent Neurologic tissues AQUEOUS MOUNTING RESINOUS MOUNTING
MEDIA MEDIA
Impregnating Agent Impregnating Agent Impregnating Agent

Paraffin Celloidin Gelatin

Methods
Specifications MOUNTING MEDIUM

Manual Automatic Vacuum Bones, brain, Whole eye

teeth section
MOUNTING
Method Method
Method
PROCESS
Wet celloidin Dry celloidin Nitrocellulose

4 changes of Use of Use of

paraffin every machines Embedding
15 minutes oven w/
negative
atmospheric
pressure

EMBEDDING

Types of Mold

Plastic Embedding Rings

Leuckhart's Embedding Mold Compound Embedding Unit Disposable Molds
and Base Molds

EMBEDDING MEDIUM

3 hours in

Paraffin Celloidin Gelatin Plastic

TRIMMING

MICROTOMY

Rocking Rotary Sliding Ultrathin

SLIDE PREPARATION

PROCESS

1. Maintain the temperature of the floatation bath at 45-50C.

2. Float the ribbon on the floatation bath.
3. Separate the sections with a curved forcep.
4. Mount the sections on the glass slide.
5. Place the slide rack in the drying oven to dry the slides and melt the paraffin.
6. Cool the slide to room temperature before proceeding to staining.

STAINING

ROUTINE STAIN CARBOHYDRATES LIPIDS NUCLEIC ACID AND PIGMENTS CONNECTIVE TISSUE NERVOUS TISSUE
PROTEINS
Stains Stains
Stains Stains Stains Stains Stains

Hematoxylin & Eosin Prussian Blue Reaction Reticulin or Nuclear Fast Bielschowsky Method
Periodic Acid Schiff Oil Red O Feulgan Stain (Peris' Reaction) For Ferric Red Stain
Iron

Periodic Acid Schiff with Sudan Black B Methyl Green-Pyronin Y

Fouchet's Stain Trichrome Stain Cajal stain for Astrocytes
DIastase Stain

Acian Blue Stain Masson-Fontana Method Van Gieson Stain

Mucicarmine Stain
Von Kossa Calcium Verhoeff-Van Gieson (VGG)
Stain

Colloidal Iron
Rhodanine Stain

Weigert's Resorcin-Fuchsin
Congo Red Stain

Phosphotungstic Acid
Hematoxylin (PTAH) Stain

PROCESS

1. Deparaffinization w/ xylene twice 2.5 mins each

2. Rehydration w/ decreasing alcohol and increasing water concentration
3. Wash with tap water until oily appearance in slides disappear
4. Drop the basic dye for 15 mins
5. Wash with tap water several times until sections on slides are visible
6. Differentiation using lithium carbonate or 0.2% Ammonia water of 1-3 rapid dips
7. Wash with running tap water for 15 dips
8. Counterstain for 1 min
9. Dehydration w/ increasing alcohol and decreasing water concentration
10. Clearing w/ xylene for three time 15-20 dips each