Histology Notes - Tissue Processing (PDF)

# HISTOLOGY. (PAPER - 502) ## Unit: 01 ### Tissue Processing Describes the steps required to take animal or human tissues from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome. #### Tissue Pro...

# HISTOLOGY. (PAPER - 502) ## Unit: 01 ### Tissue Processing Describes the steps required to take animal or human tissues from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome. #### Tissue Processing Steps Involves 1. FIXATION 2. DEHYDRATION 3. CLEARING (After Infiltration) 4. EMBEDDING 5. SECTIONING ##### 1. FIXATION: Fixation is the first processing and one of the primary purposes are to: * Preserve tissue morphology and structure * Prevent autolysis and putrefaction * Stabilize proteins and nucleic acids * Maintain the distribution of cellular components. ##### Mechanism of Action: Fixative act by denaturing proteins, either by cross linking them or coagulating them. And rendering them insoluble. These prevents enzymatic activity & Bacterial degradation. #### Types of fixatives: Fixatives can be classified into Additive & Non-Additive fixatives of coagulant, Non-coagulant fixatives * **i) Additive Fixatives:** *Chemically combines with the tissues to stabilize them.* * **eg)** Formaldehyde (Formalin) - Most widely used fixative, typically used as 10% Neutral - Buffered formalin (NBF). * **eg)** Glutaraldehyde - Used for electron microscopy due to its rapid fixation and ability to preserve ultrastructure. * **eg)** Osmium tetroxide - Used mainly in Electron Microscopy for lipid preservation. * **eg)** Mercuric chloride- An additive fixative used in compound fixatives like Zenker’s & B-5 Fixatives. It offers excellent Nuclear detail but is highly toxic. * **ii) Non-Additive Fixatives:** *These do not chemically bound to tissues but cause protein denaturation and coagulation by removing water.* * **eg)** Alcohol (Ethanol, Methanol): Used as a coagulative fixation in cytology for quick fixation. Ethanol is excellent for preserving nucleic acids but can lead to tissue shrinkage. * **eg)** Acetone: Commonly used for rapid fixation of frozen sections. It’s an effective fixative for enzymes but can cause dehydrate to tissues. * **iii) Coagulant Fixatives:** *These act by coagulating proteins, creating meshwork that allows better penetration of other processing solutions.* * **eg)** Alcohols: Mercuric chloride, staticacid * **iv) Non-Coagulant Fixatives:** *Cause cross-linking of proteins but don't form porous network that cause slower penetration and fixation.* * **eg)** Formaldehyde, glutaraldehyde ### Factors Affecting Fixation: 1. **Temperature** - Most tissues are fixed at room temp (20-25°c) or slightly below. ↑ Temp can affect Fixation & may cause tissue distortion. 2. **Fixative Concentration** - The conc” of fixative should be optimized for best results. 3. **Time of Fixation** - Typically fixation lasts between 6 to 48 hours depending on tissue size and fixative type. 4. **PH and Osmolarity** - Fixatives should ideally have a neutral PH (PH: 7.0). Osmolarity - Isotonic or slightly hypertonic solution are preferred to prevent tissue swelling / shrinkage. 5. **Volume of fixative** - Ideally tissue should be submerged in 10-20 times its volume of fixative to ensure adequate fixation. ### 2. DEHYDRATION Dehydration is a crucial step in tissue processing particularly in histology and pathology. It involves removing water from biological tissues to prepare them for embedding in a medium (usually paraffin). Alcohols are most commonly used in laboratory for tissue dehydration, since they are miscible with aqueous fixatives like 10% formalin. In this step alcohol penetrates and the water is replaced with alcohol. Since, alcohols act rapidly and may shrink and harden the tissue too much. One must be taken care when calculating the amount of time needed in the dehydration step. This step is performed at room temperature. Ethyl Alcohol and Iso-propyl alcohol are used most often, with methanol and luctand being used in some degree (temp) in special techniques. Since most alcohols (except for isopropyl) & paraffin are not miscible, 80 another step which is known as clearing is introduced. ### 3. CLEARING Clearing step is performed to remove the alcohol before infiltration with embedding media. This step is used to make tissue transparent or clear by using “Clearants” (cleaning agent). The most widely used clearing agent is Xylene. Clearants or dealcoholization agents must be miscible with both paraffin and dehydrants. Clearing is most often completed at R-T. As with the alcohols, clearing agents can act rapidly. On tissue, which cause the tissue to be hard and brittle. Xylene is miscible with alcohols as well as paraffin, but NOT with aqueous fixatives Xylene is intolerant of any water left in tissue during dehydration step, therefore. It will not prepare the tissue for paraffin infiltration if dehydration was not completed properly. ### INFILTRATION After clearing, tissues are transferred to a molten paraffin wax for filtration and impregnation. During this process clearing agent diffuses out and molten wax get infiltrated. The wax which has infiltrated in the tissues gets deposited. This process is called impregnation. Impregnation step is depends on: 1. **Size of tissue and type of tissue** - Longer time is required for thicker tissues 2. **Clearing agent employed** 3. **Use of Vacuum embedding** Tissue processing may be performed manually or with the help of tissue processor. ### Embedding Process The Embedding Process in tissue processing refers to the method used to prepare biological tissue samples for microscopic examination. Embedding is the process in which the tissues or the specimens are enclosed in a mass of the embedding medium using a mould. Since the tissues block are very thin in thickness they need a supporting medium in which the tissue blocks are embedded. The supporting medium is called embedding medium. #### The choice of Embedding Medium depends upon: 1. Type of Microscope 2. Type of Microtome 3. Type of Tissue (Eg: Hard tissue like bone or soft tissue like liver biopsy) #### Types of Embedding Medium: 1. Paraffin Wax 2. Celloidin 3. Resin 4. Agar 5. Gelatin 6. Carbowax 7. Methacrylate ##### Paraffin Wax Paraffin wax with higher melting point (56-62°c) is used for embedding. The molten wax is filtered inside the oven through a course filter paper into another container. This will protect the knife edge. ##### Agar Embedding Mainly used in double embedding. Multiple fragments and friable tissues may be impregnated in one block when sectioning on cryostat. ##### Celloidin Media *Is purified form of nitrocellulose. It’s used for cutting hard tissues.* #### Types of Moulds A variety of moulds are used for embedding. 1. **Moulds (LEUKHARD)** * Paper Blocks * Plastic moulds ##### 1- Moulds: * Most Laboratory uses L- Moulds. * L- Moulds are made up of metal. * Easy to procure. * Reusable. * Adjusted to make different size of blocks. ##### L-Moulds ##### Plastic Moulds: Nowadays most of the laboratory uses plastic embedding rings. These are relatively in-expensive, Convenient & Support the block during sectioning. And are designed to fit in microtome ### Microtomes: *A microtome is a device that slices organic or in-organic sample into strips called sections.* *Microtome blades generally consist of diamond rock, glass or metal, allowing for thin section of uniform size. It section the biological sample (tissues, biopsy) into thin form.* #### Types of Microtomes: 1. **Rotary Microtome: ** *Most common option for labs. These devices section paraffin-embedded tissue samples within the range of (0.5 to 60μm) * *These machines have a simple design, consisting of a weighted handwheel, a blade assembly, sample chuck and* 2. **Sliding Microtome: ** *Laboratory technicians. The knife or blade is stationary and the specimen slides under it during sectioning.* *This microtome was developed for use with celloidin-embedded tissue blocks.*