Histopathology a.
But for machine (autotechnicon) – 40’C is
Histopathology Techniques- Art and Science
performed by MTs to provide the Pathologist with
good quality tissue sections on which to base
patient’s diagnosis
1.2 GENERAL TYPES OF TISSUE SPECIMEN
1. Biopsy- living source
2. Autopsy- dead source
2.Tissue excluded from mandatory submission to the
laboratory. (ex: fats, foreskin, fingernails)
3.Specimen for gross description only. (ex: accessory
digits, varicose veins, prosthetic breast)
TISSUE PROCESSING- this is done to make the
tissue suitable for viewing under microscope.
1. FIXATION
2. DEHYDRATION
3. CLEARING
4. INFILTRATION
5. EMBEDDING
6. TRIMMING
7. SECTION-CUTTING
8. STAINING
9. MOUNTING
10. LABELING
Fixation-
most critical step, to fix or preserve the tissue for
examination.
Primary goal- to preserve the tissue and prevent
autolysis due to bacterial attack, putrefaction and
action of enzyme.
Secondary goal- to harden the tissue and protect
the tissue from trauma due to further handling.
*apoptosis- cell death
1.MAIN FACTORS INVOLVED IN FIXATION
1.1 hydrogen concentration – pH 6-8
1.2 Osmolality- slightly hypertonic – 400-450
miniosmoles
1.3 Temperature- room temperature
the temp. required
b. Histochemistry – 0-4’C
c. Tissues with tuberculosis- 100’C
d. Rapid fixation- 60’C
1.4 Thickness of tissue section
a. Electron microscopy – 1-2 mm2
b. Light microscopy – 2 cm2 wide and thin no
more than 0.4 cm
c. Brain- submerged 2-3 weeks in 10%
buffered formalin
d. Concentration-
a. Formaldehyde- 10%
b. Glutaraldehyde- 3%
*low concentrations of glutaraldehyde (0.25%)
ideal concentration for immunochemistry.
2.PRACTICAL CONSIDERATION in fixation
2.1 Speed
2.2 Penetration
Formalin: 1mm/hr slows down as it goes
deeper
2.3 Volume
Tissue volume- 10-25x
Museum prep- 50-100x
Osmium tetroxide- 5-10x of tissue volume
2.4 Duration
Fibrous organs takes longer- uterus or
intestinal tract or loosely textured tissues
such as biopsies or scrapings
3.FACTORS BEYOND CONTROL OF THE
LABORATORY
3.1 Warm Ischemia- the time a tissue,
organ, or body part remains at body
temperature after its blood supply has been
reduced or cut off but before it is cooled or
reconnected to a blood supply.
3.2 Cold ischemia- the time between the
chilling of a tissue, organ, or body part after
its blood supply has been reduced or cut off
 and the time it is warmed by having its
blood supply restored.

4. FACTORS THAT RETARD FIXATION

4.1 Thick tissue

4.2 Cold temperature
4.3 Presence of mucous

5. FACTORS THAT ACCELERATE

FIXATION
5.1 Thin tissue
5.2 Hot temperature
5.3 Agitation

6. FACTORS CONSIDERED WHEN

CHOOSING FIXATIVE
6.1 Need for immediate fixation
Rapid diagnosis- speed fixative
6.2 Type of tissue to be processed
Bone marrow: B5
Liver: Zenkers
6.3 Tissue structure to be studied
Glacial Acetic acid, Glycogen:
Brasil’s solution
6.4 Staining technique to be applied
Bouin’s : Fuelgen stains

7. EFFECTS OF FIXATIVE IN
GENERAL
-hardens the tissue and inhibits bacterial
decomposition and reduce the risk of
infection

8. CHARACTERISTIC OF A GOOD
FIXATIVE
-cheap and should be easy to use, safe to
handle, relatively stable, fast acting
And inhibits bacterial growth