histo

2.  Histo : Tissue
 Histology : Study of anatomy of normal cell or
tissue.
 Histopathology : Study of diseased tissue at
microscopic level.
 Histotechniques :
Processing of tissue in such a manner that it
enables the satisfactory study of tissue.

3.  Paraffin embedding technique
 Frozen section
 Celloidin embedding

5.  Received in the surgical pathology laboratory
 Specimens: Biopsy, whole organ, Radical
specimens
◦ Patient's identity must be verified at the time of specimen
collection.
◦ Requisition form :
 Patient information ( Name, Father’s name, Age, Gender,
DOA, Registration No.,
 Clinical history ,
 Clinical diagnosis
 Description of the specimen
 Operating Surgeon’s / Referring Doctor’s Name & Signature

7.  Specimen should be labeled properly.
 There should be Standardized format of
label . ie only accession no, or with Patient's
name and nature of specimen
 Specimens are accessioned by giving them a
specific number that will identify each
specimen for each patient.
◦ Like H-121/21 write in requisition form as well as
on specimen container
◦ Write A,B,C or 1,2,3 if more than one container for
specimens and do proper labelling

8.  Do not attach label to the container lid (in
whole or part)
 Do not overlap label resulting in patient data
being covered.
 The unique specimen accession no. or bar
code must be consistent across all
applications: specimen container, requisition
form, cassettes and paraffin blocks and
slides.
 Specimens must be placed in appropriate
fixative.
 Volume of fixative to tissue ratio must be
appropriate i.e. 10% neutral buffered formalin
volume should be 15- 20 times the volume of
the specimen.

9.  For specimens submitted to the laboratory from
remote sites, there is a documented tracking
system to ensure that all specimens are actually
received.
 All specimens must be placed in leak proof
container.
 Specimens should be transported to the
laboratory immediately after collection.
 Specimens transferred from distant referral site
to pathology lab should be shipped under
temperature-controlled conditions to avoid over
heating or freezing.
 Specimen must be placed in appropriate fixative
and in appropriate volume.

10.  Solid tissues need to be fixed and processed
to preserve their structures, and eventually
impregnated with an appropriate hardening
substance to permit making thin slices
suitable for staining and microscopic
evaluation.

11.  This can be accomplished by preserving and
carefully processing solid structures and tissues
in the following order:
◦ Fixation
◦ Decalcification (optional)
◦ Dehydration
◦ Clearing
◦ Impregnation (Infiltration)
◦ Embedding
◦ Trimming
◦ Section-Cutting (Microtomy)
◦ Staining
◦ Mounting
◦ Labeling

12.  Fixation is the first step of any histological and
cytological laboratory technique.
 Helps to maintain the tissue nearest to its
original state in the living system.
 It is the process by which the cells in the tissue
are fixed in a chemical and physical state, and all
the biochemical and proteolytic activities within
the cells are prevented so that the cells or tissues
can resist any morphological change or distortion
or decomposition after subsequent treatment
with various reagents.

13.  Process of preserving the shape, structure,
relationship and chemical constituents of cells and
tissue with it’s hardening and preventing
autolysis and putrefaction.
 Fixatives : are the various chemical substances
which are used in the process of fixation

14.  The basic aims of fixation are the following:
◦ To preserve the tissue nearest to its living state
◦ To prevent any change in shape and size of the
tissue at the time of processing
◦ To prevent any autolysis
◦ To make the tissue firm to hard
◦ To prevent any bacterial growth in the tissue
◦ To make it possible to have clear stain
◦ To have better optical quality of the cells

15.  Prevention of autolysis of the cells or tissue
 Prevention of decomposition of the tissue by
bacteria
 Maintaining the volume and shape of the cell as
far as possible.
 Consistently high-quality staining particularly
routine stain such as haematoxylin and eosin
stain and Papanicolaou’s stain
 Rapid action
 Cheap
 Non-toxic

16.  Volume changes
◦ Shrinkage of the volume by formalin (33%)
 Hardening of tissue
◦ Mild degree hardening may occur.
 Interference of staining
◦ Inhibits routine stain: Osmium tetroxide inhibits
haematoxylin and eosin staining.
 Changes of optical density by fixation
◦ Nuclei may look like hyperchromatic

17.  The fixative can be classified on the basis of
the following criteria:
◦ Nature of fixation
◦ Chemical properties
◦ Component present
◦ Action on tissue protein

19.  Immersion fixation:
◦ This is the commonest way of fixation in the
laboratories.
◦ In this technique the whole specimen is immersed
in the liquid fixative such as tissue samples are
immersed in 10% neutral buffered formalin or
cytology smear in 95% ethyl alcohol.
 Coating fixation: This is commonly used in
the cytology samples. The spray fixative is
used for easy transportation of the slide.

20.  Vapour fixation:
◦ In this type of fixation, the vapour of chemical is
used to fix either a smear or tissue section.
◦ The commonly used chemicals are formaldehyde,
osmium tetroxide, glutaraldehyde and ethyl
alcohol.
◦ The vapour converts the soluble material to
insoluble material, and these materials are retained
when the smear comes in contact with liquid
solution.

21.  Perfusion fixation:
◦ This is mainly used in research purpose.
◦ In this technique the fixative solution is infused in the
arterial system of the animal, and the whole animal is
fixed.
◦ The organ such as the brain or spinal cord can also be
fixed by perfusion fixation.

22.  Freeze-drying:
◦ In this technique the tissue is cut into thin sections
and then rapidly frozen into −160 °C by immersing
it into liquid coolant. This is known as “quenching”.
◦ The commonly used fluids in the quenching bath
are liquid nitrogen, propane and isopentane.
◦ Alternatively the tissue section can be frozen by
keeping it in close contact with chilled metal.
◦ Subsequently the ice within the tissue is removed
with the help of vacuum chamber in higher
temperature (−30 °C).
 Advantages:
◦ Excellent for enzyme study
◦ No change of proteins •
◦ No shrinkage of tissue
◦ Preservation of glycogen

23.  Electromagnetic wave with frequencies between 300 MHz
and 300 GHz.
 Mechanism: Microwave creates electromagnetic field, and
the dipolar molecules rapidly oscillate generating heat by
kinetic motion. The generated heat accelerates the fixation
and also other steps of tissue processing.
 Advantages:
◦ Uniform heat production
◦ No volume change of tissue
◦ Good for electron microscopy after osmium tetroxide fixation
◦ Preservation of the tissue antigen
 Disadvantages:
◦ Tissue in the formalin for microwave fixation may produce toxic
gas and overhead hood is required.
◦ Heat injury may occur from microwave

24.  Simple Fixatives -are made up of only one
component substance.
◦ Aldehydes
 a. Formaldehyde
 b. Glutaraldehyde
◦ Metallic Fixatives
 a. Mercuric chloride
 b. Chromate fixatives
◦ Picric acid
◦ Acetic acid
◦ Acetone
◦ Alcohol
◦ Osmium Tetroxide

25.  Compound Fixatives : are those that are
made up of two or more fixatives which have
been added together to obtain the optimal
combined effect of their individual actions
upon the cells and tissue constituents.

28.  Precipitating (or denaturing) fixatives : act by
reducing the solubility of protein molecules and
(often) by disrupting the hydrophobic
interactions that give many proteins their
tertiary structure.
 The precipitation and aggregation of proteins.
◦ ethanol and ethanol
 Cross-linking of protein: They act by creating
covalent chemical bonds between proteins in
tissue. This anchors soluble proteins to the
cytoskeleton, and lends additional rigidity to the
tissue.
◦ Formaldehyde, Glutaraldehyde:

30.  Pure formaldehyde vapour dissolved in the
water is available as formaldehyde in 37–40%
concentration.
 This is also known as formalin and is
considered as 100% formaldehyde.
 10% of this formalin is used to make neutral
buffered formalin for routine laboratory
fixative.

31.  Mechanism: Cross linking of protein
 Rate of penetration: Formalin penetrates
approximately 1 mm/h.
 Volume of formalin: The amount of formalin
should be 20 times the volume of tissue.
 Removal of formalin from the tissue: As the
crosslinking of the amino acids and proteins
is a slow process, so if the tissue is washed
for 24 h in water, then 50% of formalin from
the tissue is removed.
 Precaution: Formaldehyde is irritant to the
eye and skin and toxic for inhalation. It is a
carcinogenic element.

32.  Advantages:
◦ High penetration rate
◦ Well-preserved cell morphology
◦ Cheap
◦ Stable
 Disadvantages:
◦ Slow fixation.
◦ Formalin reaction is reversible, and it can be
removed by washing.
◦ Fails to preserve acid mucopolysaccharides.
◦ Not good for staining of fat and enzymes.
◦ Highly vascular tissue may have dark-brown
granules.
◦ Exposure to the skin may cause dermatitis.
◦ Chronic inhalation may cause bronchitis.

33.  Preparation of Different Formalin Solution
 A. 10% neutral buffered formalin:
◦ 40% Formaldehyde : 100.0 ml
◦ Distilled water: 900.0 ml
◦ Sodium dihydrogen phosphate: 4.0 g
◦ Disodium hydrogen phosphate: 6.5 g
 B. 10% formal saline:
◦ 40%Formaldehyde : 100.0 ml
◦ Sodium chloride: 9 g
◦ Distilled water: 900.0 ml
 C. Formal ethanol fixative:
◦ 95% ethyl alcohol: 20 ml
◦ 40%Formaldehyde : 10 ml

34.  Glutaraldehyde is a larger molecule than
formaldehyde, and so its rate of diffusion across
membranes is slower than formaldehyde.
 Used as a fixative for electron microscopy because
it fixes and preserves the ultrastructure.
 For electron microscopy: 2.5% glutaraldehyde in
100 mM phosphate buffer at pH 7.0.
 Glutaraldehyde comes commercially as 25% or 50%
solutions in 10 ml
 Mechanism : Cross linking of protein

35.  It has a more stable effect on tissues, giving
a firmer texture with better tissue sections,
especially of central nervous tissues.
 It preserves plasma proteins better.
 It produces less tissue shrinkage.
 It preserves cellular structures better; hence,
is recommended for electron microscopy.
 It is more pleasant and less irritating to the
nose.
 It does not cause dermatitis.

36.  It is more expensive.
 It is less stable.
 It penetrates tissues more slowly.
 It tends to make tissue (i.e. renal biopsy)
more brittle.
 It reduces PAS positivity of reactive mucin.
This may be prevented by immersing
glutaraldehyde-fixed tissues in a mixture of
concentrated glacial acetic acid and aniline
oil.

37.  Not used routinely for tissues because they cause too
much brittleness and hardness.
 Very good fixative for cytologic smears because they
act quickly and give good nuclear detail. Spray cans
of alcohol used as fixatives for PAP smears.
 Ethanol (95%) is fast and cheap. Since smears are only
a cell or so thick, there is no great problem from
shrinkage, and since smears are not sectioned, there
is no problem from induced brittleness.
 Solid specimens taken from patients with gout are
usually fixed in 95% ethanol for subsequent
histochemical detection of sodium urate crystals,
which can be dissolved out of the tissue by water.

38.  Advantages:
◦ It is excellent for fixing dry and wet smears, blood
smears and bone marrow tissues.
◦ It fixes and dehydrates at the same time.
 Disadvantages:
◦ Penetration is slow.
◦ If left in fixative for more than 48 hours, t issues
may be over hardened and difficult to cut.

39. ◦ More frequently incorporated into compound fixatives
for better results.
◦ Fixation Time: 18-24 hours
◦ Advantages:
 It preserves but does not fix glycogen.
 It fixes blood, tissue films and smears.
 It preserves nucleoproteins and nucleic acids, hence, is
used for histochemistry, especially for enzyme studies.
 It fixes tissue pigments fairly well.
 It is ideal for small tissue fragments.
 It may be used both as a fixative and dehydrating agent.

40.  Disadvantages:
◦ Hemosiderin preservation is less than in buffered
formaldehyde.
◦ It is a strong reducing agent; hence, should not be
mixed with chromic acid, potassium dichromate
and osmium tetroxide which are strong oxidizing
agents.
◦ Lower concentrations (70-80%) will cause RBC
hemolysis and inadequately preserve leukocytes.
◦ It dissolves fats and lipids, as a general rule.
Alcohol-containing fixatives are contraindicated
when lipids are to be studied.
◦ It causes glycogen granules to move towards the
poles or ends of the cells (polarization).
◦ It produces considerable hardening and shrinkage
of tissues.

41.  pH of the fixative
◦ Neutral pH is preferable
◦ pH 6–8 is the best range.
◦ High acidity or alkalinity interferes fixation.
 Temperature
◦ Room temperature suitable for routine work
◦ High temperature facilitates fixation.
◦ Low temperature (0–4 °C) suitable for enzyme
histochemistry.

42.  Duration of fixation
◦ Depth of penetration of fixative is directly
proportional to the square root of time of fixation.
◦ Formalin fixes 1 mm/h
◦ Small tissue: 6 h in formalin is optimum.
◦ Large tissue: 24 h is the optimum time,
◦ Prolonged fixation in aldehyde: inhibition of
enzymatic activity,
 Osmolarity of the fixative solution
◦ Hypertonic: cell shrinkage
◦ Hypotonic: cell swelling
◦ Best: mild hypertonic (400–450 mOsm)

43.  Concentration
◦ Mild lower concentration with neutral pH is
preferable.
◦ Very low concentration prolongs the time of
fixation
◦ Higher concentration causes rapid fixation with
undesirable effect.
 Agitation
◦ Agitation increases rate of penetration.
◦ Rapid agitation: damages delicate tissue.
◦ Slow gentle agitation preferable.

44.  It is the process of placing an already fixed
tissue in a second fixative in order:
◦ To facilitate and improve the demonstration of
particular substances.
◦ To make special staining techniques possible (with
secondary fixative acting as a mordant).
◦ To ensure further and complete hardening and
preservation of tissues.
 Secondary fixation may be done before
dehydration and on deparaffinized sections
before staining, usually with 10% formalin or
10% formol saline as a primary fixative.

45.  For example, tissue fixed in 10% buffered
neutral formalin may require secondary
fixation with Zenker's solution (that acts as a
mordant) prior to staining with Masson's
trichrome (for connective tissue), Mallory's
aniline blue stain (for collagen), or
phosphotungstic acid-hematoxylin (PTAH)
stain (for striated muscle).

46.  Form of secondary fixation whereby a
primarily fixed tissue is placed in aqueous
solution of 2.5-3% potassium dichromate for
24 hours to act as mordant for better staining
effects and to aid in cytologic preservation of
tissues.
 .

47.  Process of removing excess fixative from the
tissue after fixation in order to improve staining
and remove artefacts from the tissues.
 Several solutions may be used :
 1. Tap water is used to remove:
◦ excess chromates from tissues fixed in Kelly's, Zenker's,
and Flemming's solutions
◦ excess formalin
◦ excess osmic acid
 2. 50-70% alcohol is used to wash out excess
amount of picric acid (Bouin's solution).
 3.Alcoholic iodine is used to remove excessive
mercuric fixative

49.  This is a pale yellow powder which dissolves
in water (up to 6% at 20°C) to form a strong
oxidizing solution.
 Used for fixation in electron microscopy.
 It reacts with unsaturated bonds in the lipid
molecules and fixes them.
 The penetration of the osmium tetroxide in
the tissue is poor, and if it is used alone, then
a good amount of protein and carbohydrate
may be lost during fixation.

50.  Advantages:
◦ This is a very good fixative for lipid.
◦ It preserves cytoplasmic organelles such as Golgi bodies
and mitochondria.
◦ Does not make the tissue hard.
 Disadvantages:
◦ It does not fix the proteins and carbohydrates and
therefore it should be used in combination with other
fixative.
◦ React with ribose group and may cause clumping of
DNA. This can be prevented by pretreatment with
potassium permanganate or post fixation with uranyl
acetate.
◦ Poor penetration in the tissue.
◦ Tissue swelling may occur.
◦ Toxic and volatizes at room temperature producing
harmful vapour. This vapour is toxic to the eye and
respiratory tract.
◦ Expensive

51.  Laboratory use:
◦ It is commercially available in sealed vial 0.1–1 g.
◦ Aqueous solution of 4% OsO4 is made. This should
be stored in clean glass vial away from sunlight.
◦ In laboratory 2–4% OsO4 in buffer solution of
pH 7.2 is used.

52.  It contains picric acid.
 This is an excellent fixative for glycogen.
 It reacts with protein and forms protein picrate.
 The tissue penetration rate of picric acid is high,
and it fixes small tissue biopsy within 3–4 h.
 Bouin’s fixative is not suitable for DNA
quantitative study as it damages the cell
membrane and causes hydrolysis of nuclei acid.
 Advantages:
◦ It is a good fixative for connective tissue and glycogen.
◦ Rapid penetration rate

53.  Disadvantages:
◦ It produces yellow stain to the tissue.
 Removal of yellow colour:
◦ The tissue should be washed thoroughly in 70%
ethanol.
◦ This yellow colour can be removed by dipping the
tissue in lithium carbonate in 70% alcohol.
 Bouin’s solution preparation:
◦ Picric acid solution (1% in distilled water): 15 ml
◦ 40% Formaldehyde stock solution : 5 ml
◦ Glacial acetic acid: 1 ml

54.  Rapidly acting fixative.
 Mercury is a poisonous substance and should
be used carefully.
 Mercury containing fixatives may corrode the
metal so the fixative should be kept in glass
container
 Examples:
◦ Zenker’s Fluid
◦ Helly’s Fluid
◦ B5 Fixatives

55.  It is a good fixative for nuclear chromatin and
collagen.
 Preparation:
◦ Mercuric chloride: 50 g
◦ Glacial acetic acid: 50 g
◦ Potassium dichromate: 25 g
◦ Distilled water: 950 ml

56.  Stock A solution
◦ Mercuric chloride: 12 g
◦ Sodium acetate: 2.5 g
◦ Distilled water: 200 ml
 Stock B solution
◦ 37% formaldehyde solution
 Before use mix 20 ml stock solution A with
2 ml stock