Histopathology Lab intro to CLS (1) (1).pptx
- 3. Introduction • Tissues from the body taken for diagnosis of disease processes must be processed in the histology laboratory to produce microscopic slides that are viewed under the microscope by pathologists. • The techniques for processing the tissues, whether biopsies, or larger specimens removed at surgery, are described in this lecture. • The persons who do the tissue processing and make the glass microscopic slides are histotechnologists.
- 4. Introduction • Histology: is the microscopic study of tissues and their structures of plants and animals, it's commonly performed by examining cells of tissues under alight microscope or electron microscope. • Pathology: is the study of diseases and of the changes that they causes changes in a person, an animal, or plant that are caused by diseases. • Histopathology: is the microscopic study of tissues affected by disease, it is branch of pathology which deals with the study of disease in a tissue section.
- 5. • Specimen Accessioning: • Tissue specimens received in the surgical pathology laboratory have a request form that lists • 1. The patient information • 2. history of the condition • 3. description of the site of origin. • The specimens are accessioned by giving them a number that will identify each specimen for each patient.
- 6. Lab Request
- 7. • Gross Examination: • Gross examination consists of • 1. describing the specimen • 2. 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.
- 8. • Fixation - types of fixatives: • The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues. • There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated.
- 9. • There are five major groups of fixatives, classified according to mechanism of action: • Aldehydes • Alcohols Mercurials Oxidizing agents Picrates
- 10. • Fixation - factors affecting fixation: • There are a number of factors that will affect the fixation process: • Buffering • Penetration • Volume • Temperature • Concentration • Time interval
- 13. • Step 1: Dehydration: • Since paraffin is hydrophobic (immiscible i.e. not mixable with water), water inside a specimen must be removed before it can be infiltrated with paraffin. • This process is carried out by immersing specimens in a series of alcohol. • Alcohol progressively replaces water in all the cells of the specimen. • A series of increasing (typically from 70% to 100%) alcohol concentrations are used to avoid excessive distortion of the tissue.
- 14. • Step 2: Clearing • The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The commonest clearing agent is xylene.
- 15. • Step 3: Infiltration • The specimen can now be infiltrated with paraffin. Molten paraffin infiltrates tissues and when cooled solidifies to a consistency that allows sectioning on a microtome.
- 16. • Tissue Embedding: • The technique of placing cells or tissue in a supporting medium so that thin sections can be cut using a microtome.
- 17. • Tissue Embedding: •Most laboratories use modular embedding centers which consist of a paraffin wax dispenser, cold plate and heated storage area for molds and •processed tissue cassettes. Paraffin wax is dispensed from a nozzle into a suitably sized, warm mold. This final tissue block is placed on a cold plate to allow the paraffin wax to solidify.
- 19. • Sectioning: • Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. • This is done with a microtome. • The microtome is nothing more than a knife with a mechanism for advancing a paraffin block standard distances across it.
- 21. • Frozen Sections: • At times during performance of surgical procedures, it is necessary to get a rapid diagnosis of a pathologic process. • The surgeon may want to know if the margins of his resection for a malignant neoplasm are clear before closing, or an unexpected disease process may be found and require diagnosis to decide what to do next, or it may be necessary to determine if the appropriate tissue has been obtained for further workup of a disease process.
- 22. • Frozen Sections: • This is accomplished through use of a frozen section. The piece(s) of tissue to be studied are snap frozen in a cold liquid or cold environment (-20 to -70 Celsius). • Freezing makes the tissue solid enough to section with a microtome. • Frozen sections are performed with an instrument called a cryostat. • The cryostat is just a refrigerated box containing a microtome. The temperature inside the cryostat is about -20 to -30 Celsius. The tissue sections are cut and picked up on a glass slide. The sections are then ready for staining.
- 24. • Staining • The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections. • Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylenes (or substitutes) to alcohols to water. • There are no stains that can be done on tissues containing paraffin.
- 25. • Staining • The staining process makes use of a variety of dyes that have been chosen for their ability to stain various cellular components of tissue. • The routine stain is that of hematoxylin and eosion (H and E). • Other stains are referred to as "special stains" because they are employed in specific situations according to the diagnostic need. • Frozen sections are stained by hand, because this is faster for one or a few individual sections.
- 26. • H and E staining: • Hematoxylin is the oxidized product of the logwood tree known as hematein. • In order to use it as a stain it must be "ripened" or oxidized. • This can be done naturally by putting the hematein solution on the shelf and waiting several months, • or by buying commercially ripened hematoxylin or by putting ripening agents in the hematein solution. • Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the tissues. This is provided by a metal cation such as iron, aluminum, or tungsten.
- 27. • Hematoxylin stains are either "regressive" or "progressive". With a regressive stain, the slides are left in the solution for a set period of time and then taken back through a solution such as acid-alcohol that removes part of the stain. This method works best for large batches of slides to be stained and is more predictable on a day to day basis. • With a progressive stain the slide is dipped in the hematoxylin until the desired intensity of staining is achieved, such as with a frozen section. This is simple for a single slide, but lends itself poorly to batch processing.
- 28. • Eosin is an acidic dye with an affinity for cytoplasmic components of the cell. • There are a variety of eosins that can be synthesized for use, varying in their hue, but they all work about the same. • Eosin is much more forgiving than hematoxylin and is less of a problem in the lab.
- 29. • Coverslipping: • The stained section on the slide must be covered with a thin piece plastic or glass to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come. • The stained slide must go through the reverse process that it went through from paraffin section to water.
- 30. • The stained slide is taken through a series of alcohol solutions to remove the water, then through clearing agents to a point at which a permanent resinous substance beneath the glass coverslip, or a plastic film, can be placed over the section.
- 31. • Decalcification: • Some tissues contain calcium deposits(e.g. Bone) which are extremely firm and which will not section properly with paraffin embedding owing to the difference in densities between calcium and paraffin. • A variety of agents or techniques have been used to decalcify tissue such as: EDTA
- 32. Cytology • The cell is the single structural unit of all tissues. The study of cell is called cytology. • A tissue is a group of cells specialized and differentiated to perform a specialized function. Collection of different type of cells forms an organ.
- 33. Cytology • All body fluids can be prepared for cytology. • As with histology specimens, Cytology specimens are spread on glass slides and fixed with some type of alcohol. (ethanol, isopropanol, methanol) or a waxy type of fixative (hairspray can be used). • The slide is then stained using a technique known as the PAPANICOLAU stain. • After staining the specimen is mounted using xylene, DPX and a coverslip- very similar to a tissue section. • The slide is then examined by a cytologist for the presence of malignant cells(Cancer)
- 34. Example of Papanicolaou stain: Malignant melanoma, fine needle aspiration biopsy of the liver, direct smear.
Editor's Notes
- #11: Fixation is best carried out close to neutral pH, in the range of 6-8. Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity. Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant. Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue. Concentration of fixative should be adjusted down to the lowest level possible, because you will expend less money for the fixative. Formalin is best at 10%;
- #33: CYTOLOGY IS THE STUDY OF CELLS TO DETECT CANCER.