Tissue Processing and Assessment in Pathology
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Questions and Answers

What is the primary purpose of using a fixative in tissue processing?

  • To enhance the natural color of the tissue
  • To increase tissue solubility for better staining
  • To accelerate the decomposition process
  • To prevent bacterial decomposition and autolysis (correct)

    • During gross examination of a solid organ, what is the maximum thickness allowed for slices?

  • 10 mm
  • 3 mm
  • 5 mm (correct)
  • 7 mm

    • Which of the following is NOT a requirement for fixative during the fixation process?

  • Should accelerate cellular decomposition (correct)
  • Should preserve tissue in its natural state
  • Should be non-toxic and non-allergic
  • Should be economically viable

    • How should tissue samples be labeled prior to processing to prevent smudging?

    <p>Using printed labels or graphite pencil</p> Signup and view all the answers

    What is the role of clearing processes in tissue processing?

    <p>To remove water and prepare tissue for embedding</p> Signup and view all the answers

    What is the primary purpose of fixation in tissue processing?

    <p>To preserve tissues in a life-like state</p> Signup and view all the answers

    Which fixative is known for its effectiveness in immuno-histochemistry techniques?

    <p>Formalin</p> Signup and view all the answers

    What is the fastest fixative among the options provided?

    <p>Ethanol</p> Signup and view all the answers

    Which group of fixatives is known for its penetration speed and nuclear detail?

    <p>Alcohols</p> Signup and view all the answers

    What is the first step in the paraffin tissue processing technique?

    <p>Dehydration</p> Signup and view all the answers

    Which of the following is a disadvantage of using mercurial fixatives?

    <p>Poor penetration and tissue hardness</p> Signup and view all the answers

    What is the purpose of the clearing process in tissue processing?

    <p>To prepare tissues for embedding in paraffin</p> Signup and view all the answers

    Which fixation method is considered the most perfect among the techniques discussed?

    <p>Celloidin technique</p> Signup and view all the answers

    Which of the following is not a component of the major groups of fixatives?

    <p>Clearing agents</p> Signup and view all the answers

    Which of the following statements is true regarding paraffin embedding?

    <p>It uses dehydrated and cleared tissues.</p> Signup and view all the answers

    What is the optimal pH range for fixation to achieve the best results?

    <p>6-8</p> Signup and view all the answers

    Which fixative is primarily associated with Bouin's solution?

    <p>Picric acid</p> Signup and view all the answers

    What is the recommended volume ratio of fixative to tissue for effective fixation?

    <p>10:1</p> Signup and view all the answers

    During gross examination, what is typically done with the specimen once it is received?

    <p>Parts of it are placed in a plastic cassette.</p> Signup and view all the answers

    What is the maximum thickness of sections that can typically be made from paraffin-embedded tissues?

    <p>5-8 microns</p> Signup and view all the answers

    Why might different colored inks be used during specimen examination?

    <p>To identify different areas when malignancy is suspected.</p> Signup and view all the answers

    Which process allows fixed tissue to be transformed into a suitable form for microscopic sectioning?

    <p>Embedding</p> Signup and view all the answers

    What effect does increasing the temperature have on the fixation process?

    <p>Increases the speed of fixation.</p> Signup and view all the answers

    What characteristic of the fixative is crucial for the effective penetration into tissues?

    <p>Diffusability</p> Signup and view all the answers

    What temperature range is typically maintained within a cryostat for freezing tissue sections?

    <p>-20 to -30 C</p> Signup and view all the answers

    What is the primary purpose of the clearing process in tissue preparation?

    <p>To prepare the tissue for embedding in paraffin.</p> Signup and view all the answers

    Which step must occur before staining paraffin-embedded tissue sections?

    <p>Deparaffinization of the slides</p> Signup and view all the answers

    What is the primary purpose of using strong mineral acids in the decalcification process?

    <p>To remove calcium rapidly</p> Signup and view all the answers

    When performing sectioning with a microtome, what is a crucial characteristic of the knife used?

    <p>It must be very sharp.</p> Signup and view all the answers

    Which solvent is used to transition slides from paraffin to alcohol during the deparaffinization process?

    <p>Xylene</p> Signup and view all the answers

    What is the main advantage of using EDTA for decalcification of tissues?

    <p>It safely removes calcium without damaging cells.</p> Signup and view all the answers

    What is the purpose of coverslipping stained tissue sections?

    <p>To protect the tissue and preserve the section.</p> Signup and view all the answers

    What is one disadvantage of using organic acids such as acetic acid in the decalcification process?

    <p>They have a slow action on dense cortical bone.</p> Signup and view all the answers

    Which method has been shown to reduce the time required for embedding fixed tissues significantly?

    <p>Applying fewer steps in the embedding process.</p> Signup and view all the answers

    Which of the following should be avoided in the sectioning of tissues with a microtome?

    <p>Using a dull knife.</p> Signup and view all the answers

    What is the primary purpose of dehydration in tissue processing?

    <p>To prevent cellular distortion during embedding</p> Signup and view all the answers

    Which factor does NOT influence the duration tissues are kept in each alcohol strength during dehydration?

    <p>Initial temperature of the tissue</p> Signup and view all the answers

    Which clearing agent is noted for its tolerance to small amounts of water in tissues, though it is more expensive than xylene?

    <p>Toluene</p> Signup and view all the answers

    What is a major disadvantage of using acetone as a dehydrant in tissue processing?

    <p>It is a health hazard if used in large quantities.</p> Signup and view all the answers

    Which statement correctly describes the clearing process in tissue processing?

    <p>It removes water from tissues using xylene.</p> Signup and view all the answers

    What is indicated by the term 'turbulence' during the dehydration process?

    <p>The disruption caused at the water-ethanol interface</p> Signup and view all the answers

    Which of the following conditions affects the number of steps during dehydration?

    <p>The delicacy of the tissue</p> Signup and view all the answers

    Why is extensive exposure to clearing agents considered harmful to tissue?

    <p>It leads to excessive shrinkage or hardness of the tissue.</p> Signup and view all the answers

    The freezing technique is regarded as the most common method of tissue processing.

    <p>False</p> Signup and view all the answers

    Formaldehyde is considered the best fixative for preservation due to its rapid penetration into tissue.

    <p>False</p> Signup and view all the answers

    Mercurial fixatives can cause hardness in tissue but facilitate excellent nuclear detail.

    <p>True</p> Signup and view all the answers

    The celloidin technique is primarily used for biopsy samples as it provides the fastest processing.

    <p>False</p> Signup and view all the answers

    Oxidizing agents are classified as one of the five major groups of fixatives used in histology.

    <p>True</p> Signup and view all the answers

    10% neutrally buffered formalin is the standard solution used for aldehyde fixation.

    <p>True</p> Signup and view all the answers

    Alcohols, such as methanol and ethanol, are unsuitable for cytologic smears due to their slow action.

    <p>False</p> Signup and view all the answers

    Dehydration involves the abrupt removal of water from tissue using a single grade of ethyl alcohol.

    <p>False</p> Signup and view all the answers

    During the dehydration process, turbulence can cause damage or distortion to cellular components.

    <p>True</p> Signup and view all the answers

    The volume of alcohol used for dehydration should be 10-20 times that of the tissue.

    <p>False</p> Signup and view all the answers

    Xylene is an alcohol-soluble substance commonly used in the clearing process.

    <p>False</p> Signup and view all the answers

    Chloroform is considered a rapid clearing agent despite being a health hazard.

    <p>True</p> Signup and view all the answers

    Toluene is three times less expensive than xylene and works well in clearing tissues.

    <p>False</p> Signup and view all the answers

    The duration for which tissues are kept in alcohol depends solely on the size of the tissue.

    <p>False</p> Signup and view all the answers

    Formalin is only used in the dehydration step and not in any other part of tissue preparation.

    <p>False</p> Signup and view all the answers

    Methyl salicylate is rarely used in clearing due to its low cost and pleasant aroma.

    <p>False</p> Signup and view all the answers

    The primary tool used for sectioning tissues is called a cryostat.

    <p>False</p> Signup and view all the answers

    Deparaffinization is necessary before applying water soluble dyes to the tissues.

    <p>True</p> Signup and view all the answers

    Frozen sections are generally processed using automated stainers for efficiency.

    <p>False</p> Signup and view all the answers

    EDTA is known for its quick action in decalcifying dense cortical bone.

    <p>False</p> Signup and view all the answers

    Strong mineral acids can quickly remove calcium but can damage cellular morphology.

    <p>True</p> Signup and view all the answers

    The usual temperature range maintained within a cryostat is about 0 to -10 degrees Celsius.

    <p>False</p> Signup and view all the answers

    Acetic and formic acids are classified as strong acids for decalcification processes.

    <p>False</p> Signup and view all the answers

    Sections of tissue need to be embedded in paraffin to be properly stained.

    <p>False</p> Signup and view all the answers

    The process of coverslipping a stained section involves applying a thin glass cover.

    <p>True</p> Signup and view all the answers

    Histological technique aims solely to preserve the structural integrity of the tissue in preparation for microscopic examination.

    <p>False</p> Signup and view all the answers

    Specimens thicker than 1 cm will have their fixation compromised and typically cannot be processed effectively.

    <p>True</p> Signup and view all the answers

    Using far fewer steps can significantly reduce the time required for embedding fixed tissues.

    <p>True</p> Signup and view all the answers

    The maximum recommended thickness for tissue samples submitted for histopathology is 5 mm.

    <p>False</p> Signup and view all the answers

    Decalcification is a necessary process for all types of tissue specimens submitted for histopathological examination.

    <p>False</p> Signup and view all the answers

    The primary function of histological fixation is to preserve the architecture of the tissue after exposure to a variety of reagents.

    <p>True</p> Signup and view all the answers

    Dimethoxypropane (DMP) serves only as a dehydrating agent and does not react chemically with water.

    <p>False</p> Signup and view all the answers

    The critical shrinkage point of collagen is approximately 75°C.

    <p>False</p> Signup and view all the answers

    Paraffin shrinks 12% in volume upon solidifying, while Paraplast plus shrinks 14% by volume.

    <p>False</p> Signup and view all the answers

    Biopsy specimens should be transported in a metal container with 10% formalin to prevent autolysis.

    <p>True</p> Signup and view all the answers

    Once a biopsy is obtained, it should be immediately placed in a container without any fixative.

    <p>False</p> Signup and view all the answers

    Automation in tissue processing typically involves instruments that require manual timing adjustments.

    <p>False</p> Signup and view all the answers

    In ulcerated tumors, biopsies should only be taken from the diseased tissue for proper diagnosis.

    <p>False</p> Signup and view all the answers

    Crushing or squeezing tissue with forceps is recommended during biopsy procedures.

    <p>False</p> Signup and view all the answers

    The embedding process is critical because tissues must be oriented properly in the paraffin block.

    <p>True</p> Signup and view all the answers

    Acetone is preferred as the dehydrating agent due to its effectiveness in the processing of all types of tissues.

    <p>False</p> Signup and view all the answers

    Study Notes

    Tissue Processing

    • Tissues for disease diagnosis are processed in a histology lab to create microscope slides
    • Tissue samples come from biopsies or surgery or autopsies
    • Three main techniques for preparing tissue sections: paraffin, celloidin, and freezing
    • Specimens are accompanied by a request form including patient info and site of origin
    • Specimens are accessioned by assigning a unique number
    • Tissue processing involves fixing tissue in paraffin, via dehydration and clearing steps

    Specimen Assessment

    • Tissue samples sent to the pathology lab are accompanied by a request form
    • This form includes patient information, history, and site of origin
    • The specimens are accessioned, given a unique identifier for each individual patient

    Tissue Processing Steps

    • The process of fixing tissue in paraffin is called tissue processing
    • The main steps are dehydration and clearing procedures

    Fixation

    • Fixation is preserving tissues in a lifelike state as much as possible
    • Fixation should be carried out as soon as possible to prevent autolysis
    • No perfect fixative exists, but formaldehyde is a good option
    • Various fixatives are available, depending on the tissue type

    Types of Fixatives

    • Major groups: aldehydes, mercurials, alcohols, oxidizing agents, picrates
    • Aldehydes: Formaldehyde (formalin), glutaraldehyde; good for immuno-histochemistry; formalin penetrates tissue well but is relatively slow
    • Mercurials: Contain mercuric chloride (e.g., Zenker's); penetrate poorly but rapidly; causes tissue hardness
    • Alcohols: Methyl alcohol (methanol), ethyl alcohol (ethanol); good for cytologic smears, act quickly
    • Oxidizing Agents: Potassium permanganate, potassium dichromate, osmium tetroxide
    • Picrates: Picric acid (e.g., Bouin's solution)

    Factors Affecting Fixation

    • Buffering: Fixation at a neutral pH (range of 6-8) is ideal
    • Penetration: The fixative's ability to diffuse into tissue depends on its diffusability
    • Volume: Fixative volume should be approximately 10:1 in relation to tissue volume
    • Temperature: Higher temperature increases the speed of fixation
    • Concentration: The concentration needs to be adjusted to the lowest possible level
    • Time Interval: The time between tissue removal and fixation

    Gross Examination

    • Tissues for diagnosis are examined in the pathology department
    • Examination involves describing the specimen and placing it in a small plastic cassette
    • The cassettes are stored in a fixative for further processing into a paraffin block

    Tissue Processing Steps (Continued)

    • Tissues are held firmly to allow for cutting thin sections with a sharp knife
    • Firmness achieved through embedding or freezing
    • The tissue, after fixation, must be processed into a format that can be sectioned into microscopic slides

    Clearing

    • The alcohol is replaced with paraffin wax
    • Materials used includes xylene, benzene, acetone, chloroform, carbon tetrachloride, toluene.
    • Xylene is a routine clearing agent
    • Smaller tissue cleared in 0.5-1 hour, larger in 2-4 hours
    • Chloroform is no longer a preferred clearing agent due to potential health hazards
    • Removing the dehydrant with a substance, similar to paraffin (embedding medium), is called clearing

    Impregnation in Paraffin

    • Tissues are put in hot, soft paraffin at 50°C for a period varying from 6-24 hours
    • Tissues are transferred to hot hard paraffin at 55°C for further impregnation in an oven
    • Paraffin penetrates the tissue and hardens it, a necessary step prior to embedding

    Wax:

    • The volume of paraffin should be approximately 25-30 times the tissue volume
    • The impregnation duration depends on tissue size, type, and clearing agent usage
    • Impregnation takes about 4 hours for a routine process

    Embedding

    • Paraffin, a derivative of crude petroleum, is the typical embedding agent
    • Paraffin melts between 52° and 58°C, infiltrating the cells while hot
    • Impregnation should be performed few degrees above the melting point of paraffin
    • Firmness is achieved using a supporting medium

    Cutting (Sectioning)

    • Embedded tissues are cut into thin sections (4-6 microns) using a microtome with a very sharp knife
    • This method allows for the preparation of thinly sliced samples that can be examined under a microscope

    Frozen Sections

    • Frozen sections are performed using a cryostat
    • The cryostat is a refrigerated box containing a microtome
    • Frozen sections are used at temperatures ranging from -20 to -30°C
    • Tissue sections are cut and mounted on glass slides

    Staining

    • The embedding process is reversed to allow water-soluble dyes to penetrate
    • Slides are depraffinized using xylene, then alcohols, finally water
    • There are no stains applicable to tissues containing paraffin.

    Coverslipping

    • A thin piece of glass is placed over the stained section, protecting the tissue from scratches and preserving the tissue section for years

    Decalcification

    • Bone samples, as well as other calcified tissues, must be decalcified before embedding and sectioning
    • Mineral acids, organic acids, EDTA, and electrolysis are among the decalcifying reagents used
    • Strong mineral acids remove calcium rapidly, but may damage tissue morphology
    • Organic acids are slower but may be better for dense cortical bone
    • EDTA removes calcium safely, but is slow, penetrates poorly, and is costly

    Sampling for Histopathological Examination

    • Tissue specimens should not exceed 3mm in thickness nor be larger than the slides
    • Pieces from solid tissues are typically 10-15mm in surface area and 2-3mm in thickness
    • Discrete calcified areas should be removed and decalcified separately
    • Tissue samples should be wrapped in paper or gauze

    General Rules for Biopsy Procedure

    • The number of biopsies increases with the size of the lesion
    • Ulcerated tumors should be biopsied to include the periphery of the tumor, which includes normal tissue.
    • Tissues should not be crushed or squeezed too hard.

    Specimen Handling

    • Specimens should be transported in a container of suitable material, like glass, plastic or metal, along with 10% formalin
    • If formalin isn't immediately available, it can be placed in the refrigerator at 4°C to slow down autolysis
    • The container opening should have an adequate size for easy tissue removal after fixation

    Examination

    • Proper identification and orientation of the specimen
    • Unlabeled specimens should not be processed
    • Complete histopathology requisition forms include patient name, age, sex, relevant clinical data, surgical findings, operation type, and specific tissue name
    • The examination should be done in an orderly manner, searching all the submitted tissue and placing them in a proper anatomic position, recording specimen type, shape, dimensions, weight, color, surgical margins, consistency and pertinent information

    Automated Tissue Processor

    • The whole process is automated for efficiency
    • An instrument moves the tissue through the agents at controlled speeds

    Blocking

    • Impregnated tissues are placed in a mold and covered with fresh wax
    • After cooling, it is immersed in cold water for rapid cooling, then cut into individual blocks and each is trimmed.
    • Labels are adhered on the surface of the block.

    Histological Technique

    • Deals with tissue preparation for microscopic study
    • The aim is to retain microscopic tissue structures

    Fixation (Continued)

    • The process fixes the constituents of cells and tissues in a physical and chemical state to resist subsequent treatment
    • This is achieved by exposing tissues to chemical compounds known as fixatives
    • Solutions for dealing with thick specimens include cutting slices and soaking wool in fixative solution, or injecting fixatives along blood vessels.

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    Tissue Processing PDF

    Description

    Explore the essential techniques of tissue processing used in pathology labs for disease diagnosis. This quiz covers the steps involved, including fixation, dehydration, and accessioning specimens, as well as the importance of accompanying request forms. Test your knowledge on the preparation of microscope slides and the methods used for tissue sample processing.

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