د.زياد

Histopathology Techniques

Tissue Processing and Staining Method of Biopsy Taking: 1. Incisional biopsy 2. Excisional biopsy 3. Punch biopsy 4. Core needle biopsy 5. Curettage biopsy

Incisional biopsy:

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 & 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

Kidney : (excisinal biopsy)

Some General Rules for the biopsy Procedure
1. 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. 2. In ulcerated tumor, Biopsies should be taken from the periphery that includes normal and diseased tissue. 3. Crushing or squeezing of the tissue with forceps should be carefully avoided. 4. 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.

General Principle of Gross Examination:

1. Proper identification and orientation of the specimen.2. Unlabelled specimen should never be processed.3. 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.4. Careful search and examination of all the tissue submitted in order.5. 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. Weighte. Shape f. Colorg. Consistency h. Surgical margin, whether included or not involved by tumor


6. Measurements are usually given in centimeter unless the specimen is very small in which mm can be used. 7. Endometrial and prostatic tissue should be measured by aggregate pieces in volume.

Sampling for Histopathological Examination:

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

Specimen accessioning

Gross Examination Consists of describing the specimen and placing all or parts of it into a small plastic cassette which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed into a fixative. When a malignancy is suspected, then the specimen is often covered with ink in order to mark the margins of the specimen. Different colored inks can be used to identify different areas if needed.

Histological Technique:

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 3. Cleaning 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, call fixatives

Mechanism of action of 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: 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:

1. Prevents autolysis and bacterial decomposition. 2. Preserves tissue in their natural state and fix all components. 3. Make the cellular components insoluble to reagent used in tissue processing. 4. Preserves tissue volume. 5. Avoid excessive hardness of tissue. 6. Allows enhanced staining of tissue. 7. Should be non-toxic and non-allergic for user. 8. Should not be very expensive

Classification of Fixatives

Tissue fixativesBuffered formalin Buffered gluteraldehydec. Zenker’s formal saline d. Bowen’s fluidCytological fixativesa. Ethanol b. Methanol c. EtherHistochemical fixativesa. Formal saline b. Cold acetonec. Absolute alcohol

Tissue Processing:

Tissue processing is a long procedure and required 24 hours. Tissue processing can be done by manually or mechanically. 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 TolueneXylene is commonly used. Small piece of tissue are cleaned in 0.5 – 1 hour;

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. Paraffin wax is used routinely. It has hard consistency, so section of 3-4 micron thickness can be cut.

D. Blocking:

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.



Tissue processor used for biopsy processing through passing the biopsy into multiple steps

Embedding with wax

CUTTING 
using the microtome

Tissue blocks are trimmed into thin sections (4-5microns) using the microtom

Staining:
Staining is a process by which we give color to a section. There are hundreds of stains available. Classification of Stains: Generally the stains are classified as: Acid stains Basic stains Neutral stains

Hematoxylin and Eosin (H & E)

Hematoxylin and Eosin staining: It is the most common used routine stain in histopathology laboratory Staining Procedure 1- Deparaffinize and hydrate to water 3- Mayer's hematoxylin for 15 minutes 4- Wash in running tap water for 20 minutes 5- Counterstain with eosin from 15 seconds to 2 minutes depending on the age of the eosin 6- Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess eosin is removed. Check under microscope 7- Clear in xylene, two changes of 2 minutes each 8- Mount in Permount or Histoclad Results Nuclei - blue Cytoplasm - various shades of pink-identifying different tissue components

STAINING

Special Stains:
1.PAS (Periodic Acid Schiff) stain: This stain demonstrates glycogen 2.Stains 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.3.Congo-red: It is used for identification of amyloid.4.Sudan-Black: It is used for fat staining.5.Masson’s Trichrome: It is used for differentiation of connective tissue

H-pylori This small curved to spiral rod-shaped bacterium is found in the surface epithelium of most patients with active gastritis. The rods are seen here with a methylene blue stain.

Amyloid deposits stain orange-red with Congo Red stain

It allows rapid diagnosis of the nature of the lesion whether benign or malignant to decide the next step in surgery. All laboratory staff should be informed and all preparations should be completed before arrival of tissue.

Cytology: is the study of normal & abnormal morphologic characteristics of human cells. e.g. fluid cytology , Pap smear from the uterine cervix , FNAC.