Biopsy and tissue

processing
Daudi Mshana, MD
Method of Biopsy Taking:
• Incisional biopsy
• Excisional biopsy
• Punch biopsy
• Core needle biopsy
• Curettage biopsy
Incisional biopsy
• A sample of abnormal tissue/part of a lump is removed for
histopathological evaluation.
• It is performed when removal of entire lesion is impossible.
• Often performed prior to major surgical procedure.
• Is strictly a diagnostic nature.
Excisional Biopsy:
• In this technique, the entire lesion is removed, usually with a rim of
normal tissue.

• It is performed when the lesion is smaller in size.

• The procedure serves the diagnostic and therapeutic function

Punch Biopsy
• * It is done by biopsy forceps.
• * It is performed in the lesion of uterine cervix, oral cavity, esophagus,
stomach, intestine and bronchus
Core Needle Biopsy
• It is done with special type of wide bore biopsy needle.

• * It permits a percutaneous approach to internal

• structures
Curettage Biopsy
• Curetting are usually done for diagnosis of endometrial disease.
Some General Rules for the biopsy
Procedure:
• The larger the lesion, the numerous the biopsies that should be taken
from it because of the fact that the diagnostic areas may be present
only focally.
• In ulcerated tumour, Biopsies should be taken from the periphery that
includes normal and diseased tissue.
• Crushing or squeezing of the tissue with forceps should be carefully
avoided.
• Once the biopsy is obtained, it should be placed immediately into
container with adequate volume of fixative
Handling of Specimen
• Specimen should be transported in glass, plastic or metal container or
in a plastic bag in 10%formalin. If formalin is not available at hand,
place the specimen in refrigerator at 4oC to slow down autolysis.

• *The container should have an opening larger enough so that the

tissue can be removed easily after it has hardened by fixation.
• Proper identification and orientation of the specimen.
• Unlabelled specimen should never be processed.
• A properly completed histopathology requisition form containing patient’s name, age,sex, relevant
clinical data, surgical findings, nature of operation and name of tissue submitted.
• Careful search and examination of all the tissue submitted in order.
• Place the specimen on cutting board in an anatomic position and record the following information:
• a. Types of specimen
• b. Structure included.
• c. Dimensions
• d. Weight
• e. Shape
• f. Colour
• g. Consistency
• h. Surgical margin, whether included or not involved by tumour
• Measurements are usually given in centimeter unless the specimen is very
small in which mm can be used.
• Endometrial and prostatic tissue should be measured by aggregate pieces in
volume.
• Tissue submitted for histopathology must not be more than 3 mm thick and
not larger than the diameter of slides used. Most specimens from solid tissues
are cut in the form of
• pieces measuring 10 to 15 mm on the slides and 2 to 3 mm in thickness.
• Discrete areas of calcification or ossification should be taken out and should
be decalcified in nitric acid.
• Small fragments of tissue must be wrapped in thin paper.
• Histological technique deals with the preparation of tissue for microscopic examination.

• The aim of good histological technique to preserve microscopic anatomy of tissue.

• This is achieved by passing through a series of process.

• These processes are:
1. Fixation
2. Dehydration
[Link]
4. Embedding
5. Cutting
6. Staining
Fixation
Definition:
• This is the process by which the constituents of cells and tissue are
fixed in a physical and a chemical state so that they will withstand
subsequent treatment with various reagents with minimum loss of
architecture.
• This is achieved by exposing the tissue to chemical compounds, called
fixatives
• *Most fixatives act by precipitating proteins
• *No fixative will penetrate a piece of tissue thicker than 1 cm.
• *For dealing with specimen thicker than this, following methods are
recommended:
• 1. Solid organ: Cut slices as necessary as but not thicker than 5mm.
• 2. Hollow organ: Either open or fill with fixative or pack lightly with
wool soaked in fixative.
• 3. Large specimen, Inject fixative along the vessels or bronchi as in
case of lung so that it reaches all parts of the organ
Properties of an Ideal Fixative:
• Prevents autolysis and bacterial decomposition.
• Preserves tissue in their natural state and fix all components.
• Make the cellular components insoluble to reagent used in tissue
processing.
• Preserves tissue volume.
• Avoid excessive hardness of tissue.
• Allows enhanced staining of tissue.
• Should be non-toxic and non-allergic for user.
• Should not be very expensive.
• *Amount: should be approximately 10-20 times the volume of the
specimen.
• *Fixative should surround the specimen on all sides
• A. Tissue fixatives
a. Buffered formalin b. Buffered gluteraldehyde
c. Zenker’s formal saline d. Bowen’s fluid
• B. Cytological fixatives
a. Ethanol b. Methanol c. Ether
• C. Histochemical fixatives
a. Formal saline b. Cold acetone
c. Absolute alcohol
Tissue processing
• *Tissue processing is a long procedure and required 24 hours. Tissue
processing can be done by manually or automatically.
• *It is done in stages. It can be subdivided into; dehydration, clearing,
impregnating and embedding.
• *It is important that all specimens are properly labeled before
processing is started.
• *For labeling, pen containing ordinary ink should not be used.
Printed, or graphite pencil written, are satisfactory
Sequence of manual tissue
processing
A. Dehydration:
• *Tissues are dehydrated by using increasing strength of alcohol; e.g.
50%, 70%, 90% and 100%.
• *The duration for which tissues are kept in each strength of alcohol
depends upon the size of tissue,fixative used and type of tissue.
• *The volume of alcohol should be 50-100 times that of tissue.
B. Clearing
• *The next step alcohol should be replaced by paraffin wax.
• *As paraffin wax is not alcohol soluble, we replace alcohol with a substance in which wax is
soluble.
• This step is call clearing.

• *Clearing of tissue is achieved by any of the following reagents:

• • Xylene Chloroform Benzene
• • Carbon tetrachloride Toluene

• *Xylene is commonly used.

• *Small piece of tissue are cleared in 0.5 – 1 hour;
• *whereas larger (5cm or more thick) are cleared in 2-4 hours.
C. Impregnation with Wax
• *This is allowed to occur at melting point temperature of paraffin wax, which is
54-60oC.
• * Volume of wax should be about 25-30 times the volume of tissues.
• *The duration of impregnation depends on size and types of tissues and the
clearing agents employed.
• *Total duration of 4 hours is sufficient for routine
• impregnation.
• *Types of Wax employed for Impregnation:
• 1. Paraffin wax 2. Water soluble wax
• *Paraffin wax is used routinely. It has hard consistency, so section of 3-4 micron
thickness can be cut.
Embedding
• is the process by which tissues are surrounded by a medium such as
agar, gelatin, or wax which when solidified will provide sufficient
external support during sectioning.
• *Impregnated tissues are placed in a mould with their labels and then
fresh melted wax is poured in it and allowed to settle and solidify.
Once the block has cooled sufficiently to form a surface skin it should
be immersed in cold water to cool it rapidly.
• *After the block has completely cooled it is cut into individual blocks
and each is trimmed.
• *Labels are made to adhere on the surface of the block
Microtomy
• Sectioning the tissue block to obtain thin section(‘ribbon’) 2-
5micrometer
• Different types
• Rotary microtomes commonly used in Histopathology
Staining
• *Staining is a process by which we give colour to a section.
•
• *There are hundreds of stains available.

• *Classification of Stains:
• Generally the stains are classified as:
• A. Acid stains B. Basic stains
• C. Neutral stains
Classification of Stains:
• Acid Dyes:
• *In an acid dye the basic component is coloured and the acid component is colourless.
• *Acid dyes stain basic components
• *e.g. eosin stains cytoplasm red.

• Basic Dyes:
• *In a basic dye the acid component is coloured and the basic component is colourless.
• *Basic dyes stain acidic components
• *e.g. basic fuchsin stains nucleus blue.

• Neutral Dyes:
• *When an acid dye is combined with a basic dye a neutral dye is formed.
• *As it contains both coloured radicals, it gives
• different colours to cytoplasm and nucleus
• simultaneously. This is the basis of Leishman
• *Every stain is to be used according to a specified method.

• *Staining can be done either manually or in an automatic stainer.

• *Haematoxylin and Eosin staining:

• It is the most common used routine stain in histopathology laboratory
Special stains
• [Link] (Periodic Acid Schiff) stain: This stain demonstrates glycogen

• [Link] for micro-organism:

• a. Gram-stain:
• b. Ziehl_Neelsen stain: This stain detect acid fast bacilli.
• c. PAS stain: It is used for fungi, amoeba and Tricomonas.
• d. Modified Giemsa (2% Giemsa in water):For Helicobacter pylori.

• [Link]-red: It is used for identification of amyloid.

• [Link]-Black: It is used for fat staining.

• [Link]’s Trichrome:It is used for differentiation of

• connective tissue
•