Histopathology and Cytopathology (Practical) part 1st

THIS TOPIC IS FOR MEDICAL LABORATORY 3RD YEAR DIPLOMA STUDENTS

1. Histopathology and Cytopathology (Practical) Perform the following tasks: Handle microscope. Prepare fixatives and fixation of tissues. Collect , transport & fix samples for histological & cytological studies. Process the grossed tissues. Cut sections using rotary microtome to get ribbons of tissue sections. Prepare reagents & stains used for Hematoxylin & Eosin stain, PAS stain, Alcian Blue stain and Ziehl-Neelsen stain. Prepare reagents & stains used for Giemsa and Papanicolaou stains. Stain sections by H/E stain, PAS stain, Alcian Blue stain and Ziehl-Neelsen stain. Prepare cytological fixatives and fixation of cells. Prepare cytological smears and stain with pap method. Stain FNAC smears by Giemsa and Papanicolaou methods. Mount stained smears/section. Demonstrate Barr body by Aceto-Orcein staining method.

2. 1. Handle microscope

3. Introduction A compound microscope is  a high-magnification instrument that uses a combination of lenses, including an objective lens and an eyepiece, to view extremely small specimens that are invisible to the naked eye. Principal: Commonly, the specimen or object to be examined is mounted on a transparent glass slide and positioned on the specimen stage between the condenser and objective lenses . A beam of visible light from the base is focused on the specimen by a condenser lens. The light emitted by the specimen is captured by the objective lens, which magnifies it inside the body tube to create the primary image. This image is once more magnified by the ocular lens or eyepiece . If a higher magnification is required, the compound microscope’s nosepiece is rotated after low power focusing in order to align the higher magnification objective with the lighted portion of the slide.

4. Operating Procedure of Compound Microscope The following points are the steps for operating microscope: The lowest power objective lens (for example, 4x) should be snapped into place by rotating the revolving turret/nosepiece. Place the microscope slide on the stage and secure it using the stage clips. Turn the focus knob to raise the stage as you look at the objective lens and stage from the side. Without letting the objective hit the coverslip , raise it as high as it will go. Adjust the focus knob to focus the image while looking through the eyepiece . Adjust the light’s brightness and the condenser’s position for the high amount of light. When the sample is in the center of the field of vision, move the microscope slide. 

5. For the clearest image, adjust the condenser and light intensity after focusing the sample using the focus knob. You can switch to the next objective lenses once you obtain a clear image of your sample with the lowest power objective. The sample’s focus and/or the condenser and light intensity may need to be adjusted. Make sure the objective lens doesn’t touch the slide! After you’re done, lower the stage, click the low power lens into place, and then take out the slide.

6. Applications of Compound Microscope A compound microscope is very helpful in pathology labs while conducting blood analysis for disease diagnosis. It aids in the visualization and understanding of the microbiological realm of bacteria and viruses. Compound microscopes can be used to determine if minerals are present or absent in addition to the presence of metals . The usage of a microscope in academic experiments is advantageous for students in schools and universities which facilitate them to see bacteria and viruses, that are normally undetectable to the naked eye, In forensic laboratories, human cells are taken and studied under a microscope to help identify and solve various crimes. A compound microscope is used to inspect plant cells and identify the microorganisms living within.

7. C are and maintenance of compound microscope Handling and Transport: Use two hands:   When moving the microscope, support the arm with one hand and the base with the other to prevent damage.  Carry gently:   Avoid jarring or excessive force when moving the microscope or adjusting its components.  Cleaning Optics:   Use soft, lint-free lens paper or a clean cotton swab to gently wipe dust from lenses. For tougher spots, use a special cleaning solution with a mixture of ethanol, petroleum ether, and ether, or the manufacturer's recommended cleaning agent.  Immersion oil:   Routinely clean immersion oil from oil-immersion objectives with lens paper after each use to prevent it from becoming sticky and attracting dirt.  Stage and condenser:   Use a damp, lint-free cloth to wipe the stage and condenser clean of dirt and stains, ensuring not to get moisture on internal parts.  Eyepiece:   If dust specks appear to move when rotating the eyepiece, the dirt is on the ocular lens, which can be cleaned gently with lens paper. 

8. Storage: Cover:   Keep the microscope covered with its dust cover when not in use to prevent dust from settling on lenses and mechanical parts.  Dry environment:   Store the microscope in a clean, dry, and well-ventilated place away from direct sunlight, extreme temperatures, and corrosive chemicals.  Humidity:   In humid conditions, consider storing the microscope in a waterproof container with a drying agent to prevent fungus growth and corrosion.  Maintenance Bulb care:  Turn off the light source when the microscope is not in use to extend the bulb's life.  Annual check:  Have the microscope professionally serviced annually for more complex maintenance.  Slides:  Do not leave a slide on the microscope when it is not in use. 

9. 2. Prepare fixatives and fixation of tissues Fixatives in histopathology are  chemicals used to preserve biological tissues by halting decomposition, hardening them, and maintaining their microscopic structure for examination. Common types of fixatives Formalin : A 10% neutral buffered formalin solution is the most common fixative due to its excellent preservation and compatibility with various stains. Formalin is a cross-linking fixative that forms chemical bonds between tissue molecules.  Glutaraldehyde : Another cross-linking fixative, often used for electron microscopy because it preserves cellular structure very well.  Osmium tetroxide : Used for electron microscopy, it fixes and stains tissues simultaneously.  Alcohols : Ethanol and methanol are coagulant fixatives that work by precipitating proteins. They are faster but can cause cell shrinkage.  Picric acid : A coagulant fixative sometimes used in mixtures. 

10. Formalin The most common is 10% Neutral Buffered Formalin (NBF ) 10% Neutral Buffered Formalin (NBF) preparation Ingredients: Commercial 40% formaldehyde ( formalin ) Sodium dihydrogen phosphate (monobasic) Disodium hydrogen phosphate (dibasic, anhydrous) Distilled or deionized water  Procedure for 1 liter of 10% NBF: 1. Dissolve the buffer salts: In a 1 or 2-liter beaker, combine 4 g of sodium dihydrogen phosphate and 6.5g of disodium hydrogen phosphate with 900 ml of water. Stir until the salts are fully dissolved.  2. Add the formaldehyde: Carefully measure 100 ml of commercial 40% formaldehyde and add it to the buffer solution. 

11. 3. Mix thoroughly:   Stir the solution for about a minute to ensure it is well-mixed.  4. Store:   Pour the finished NBF into a clean, tightly sealed, and properly labeled container.  Glutaraldehyde : Materials needed: Glutaraldehyde stock solution:  Typically a 25% or 50% solution, often sold in sealed ampoules. Buffer solution:  A pre-made or self-prepared buffer, such as 0.2 M sodium phosphate buffer at pH 7.2, is a common choice. Distilled water:  To adjust the final volume. Activated carbon:  To stabilize the solution (optional, but recommended). Graduated cylinders and beakers:  For precise measurements. 

12. Preparation steps 1. Prepare the buffer:   Ensure you have a sufficient volume of the correctly pH-buffered solution. For example, prepare 50 mL of 0.2 M pH 7.2 phosphate buffer.  2. Add glutaraldehyde :   In a beaker, add the required amount of the glutaraldehyde stock solution to the buffer. For a 2% final concentration from a 50% stock, you would add 4 mL to 96 mL of buffer to make 100 mL of fixative. For a 4% solution, add 8 mL of 50% glutaraldehyde to 92 mL of buffer . 3. Adjust the volume:   Add distilled water to reach the final volume (e.g., to 100 mL ).  4. Add activated carbon (optional):   Add a small amount of activated carbon to the solution to help stabilize it and remove impurities.  5. Stir and filter:   Stir the solution thoroughly for about an hour to dissolve any components. Then, filter the solution to remove the activated carbon and any other solids. 6. Check pH:   Confirm the final pH is in the optimal range of 7.2–7.4 using a calibrated pH meter. 

13. Storage and use: Freshness is critical:  Use the solution within 8 hours of preparation, as glutaraldehyde decomposes over time.  Storage:  Store the fixative in a sealed container in a cool, dark place.  Disposal:  Dispose of any unused solution properly after 8 hours.  Osmium tetroxide : Osmium tetroxide (𝑂𝑠𝑂4) is a heavy metal compound used in histology as both a fixative and a stain, primarily for electron microscopy. It works by reacting with and fixing lipids, which adds electron density to tissues for visualization. This reaction turns lipids a dark brown color under a light microscope and makes them highly visible in electron micrographs. 

14. Safety precautions: Work in a fume hood:  All preparation must be done in a certified chemical fume hood with good ventilation to contain the toxic vapors.  Wear PPE:  Wear chemical-protective gloves, eye protection (goggles), and a lab coat.  Protect surfaces:  Cover the hood's working surfaces with plastic-backed absorbent pads to contain spills.  Post warnings:  Place a warning sign on the fume hood to alert others.  Know emergency procedures:  Ensure the safety shower and eyewash stations are accessible and operational.  Handle ampoules carefully:  Wrap the glass ampoule in a paper towel before opening it to contain any glass fragments.  Preparation steps: 1. Obtain materials:   Use a liquid osmium tetroxide solution (2% or 4%) to avoid handling the powder. You will also need the buffer or distilled water, a graduated cylinder, and a tightly sealed glass or shatter-resistant container.

15. 2. Add the ampoule: Carefully break the osmium tetroxide ampoule and place the contents into the container. 3. Add the solvent: Use a Pasteur pipette to add the buffer or water to the container. This helps ensure the solution is prepared to the correct final concentration and rinses the ampoule. 4. Seal and mix: Seal the container tightly and wrap the cap with parafilm to prevent vapor escape. Gently swirl or roll the container to mix the solution. 5. Label clearly: Label the container with the date, concentration (e.g., 1% OsO ₄ in 100 mM cacodylate buffer"), and preparer's initials.  Post-preparation storage: Store securely:  Store the solution in a secure, designated location with no unauthorized access. Store properly:  Osmium tetroxide can penetrate some plastics, so store solutions in a sealed glass container and place that container inside a secondary container. Refrigerate:  Store the container in a refrigerator. Separate from incompatibles:  Keep osmium tetroxide away from acids, bases, strong reducing agents, and other organic materials. 

16. Alcohols Common alcohol-based fixative preparations EthMeth :   Mix 3 parts of 100% ethanol with 1 part of 100% methanol.  Methacarn :   Prepare by mixing 6 parts 100% methanol, 3 parts chloroform, and 1 part glacial acetic acid.  Ethanol + Formalin mix :   Combine 9 parts alcohol (such as ethanol) with 1 part 10% neutral buffered formalin (NBF).  Buffered Ethanol :   Combine 70% ethanol with 0.5× phosphate-buffered saline (PBS) to maintain pH.  Other components like glycerol or glacial acetic acid can be added to the buffer for specific purposes. 

17. Picric acid Picric acid is used as a fixative in histopathology by combining it with other agents, most famously in Bouin's solution (picric acid, formaldehyde, and glacial acetic acid). It can also be used as a saturated alcoholic solution for fixative mixtures, but picric acid can make paraffin infiltration difficult, so tissues must be thoroughly washed with 70% ethanol after fixation. Common picric acid fixative solutions: Bouin's Solution:  A widely used fixative containing aqueous picric acid, 40% formaldehyde, and glacial acetic acid. It is known for providing excellent nuclear detail and is particularly useful for certain tissues like those from the gastrointestinal tract, endocrine glands, or the testes.  Formalin Picric Trichloroacetic (FPT) Fixative :  A solution of picric acid, formalin, and trichloroacetic acid. 

18. Preparation instructions 1. Mix components:   For solutions like Bouin's , mix picric acid, formaldehyde, and glacial acetic acid.  2. Adjust concentration:   Picric acid can be used in various concentrations, but a typical range is 2% to 15% of a saturated solution when combined with other agents.  3. Fixation time:   The fixation time depends on the size of the tissue. For example, a 3-5 mm thick piece may take about 8 hours to fix. Do not leave tissues in the fixative longer than necessary.  Post-fixation procedures: Do not wash with water:   After fixation, do not wash the tissue in water, as this can affect the staining characteristics.  Wash with ethanol:   Place the tissue directly into 70% ethanol to remove residual picric acid. 

19. Deal with picric acid deposits:   If picric acid remains after processing, it can affect the tissue's staining characteristics and deteriorate over time.  Treat with carbonate:   A few minutes of treatment with a carbonate solution before staining can also help neutralize residual acid.  Wash thoroughly:   If the tissue is to be processed for paraffin embedding, it must be washed thoroughly to remove picric acid, as it makes paraffin infiltration difficult.  Remove picric acid deposits:   Picric acid can dissolve small deposits of calcium in the specimen, so thorough washing before processing is crucial. 

20. Fixation of tissues Step 1: Obtain and prepare the tissue sample Obtain a fresh tissue specimen from its source.  If the initial fixation of an entire organ during surgery was inadequate, a second fixation after gross dissection is crucial.  Ensure the tissue is placed in a suitably sized container to prevent squashing.  Step 2: Fix the tissue Place the tissue in a chemical fixative solution, such as 10% formalin, ensuring the volume of the fixative is about 15 to 20 times the volume of the tissue.  Allow sufficient time for fixation to occur, which can be at least 6 to 8 hours but up to 24 hours.  The fixative solution should completely cover the tissue. 

21. 3. Collect, transport & fix samples for histological & cytological studies. Steps to Specimen Collection and Transport: From patient to pathologist, preparing tissue specimens for histological examination requires care, skill and sound procedures.  Avoid Mechanical Trauma: Tissue is removed gently to avoid trauma to the specimen caused by crushing or tearing. This applies both during surgery and during any further dissection that may be required of a fresh specimen. Prevent Specimen Drying: Specimen is not allowed to dry out prior to fixation. If immediate fixation is not practicable, gauze moistened with saline can be used to prevent this. Avoid Heat Damage: As far as possible avoid local heat damage to specimens Avoid Chemical Damage: Avoid contaminating fresh specimens with foreign chemicals or substances such as disinfectants.

22. Label Specimens Properly: Each specimen should be properly  identified , and all details recorded as soon as possible. Ensure Prompt Fixation: Fixation is always carried out promptly. If it is necessary that a specimen remains unfixed for a short period of time, it should be refrigerated at 4 °C. Fixation is delayed (degeneration of tissue elements commences as soon as the specimen is deprived of a blood supply). Use Sufficient Fixative and a Suitable Container: An adequate volume of fixative (ratio of at least 20:1) is used in a container of an appropriate size. This avoids distortion of the fresh specimen and ensures good quality fixation. Check Fixative pH: The fixative is of high quality and at the optimal pH. ( pH of 6.8–7.0. )

23. Expedite Large Specimen Fixation: The specimen dimensions allow rapid penetration of the fixative. Large specimens should be rapidly transported to the lab to allow  grossing  (tissue slices can be prepared to allow proper fixation to occur). Handle Specimens Gently   Specimens handled gently – fragile specimens remain intact. Transport: Avoid inappropriate containers:  Do not use glass containers or sharps containers for transport.  Safety:  Handle formalin with care, as both the liquid and vapors are harmful to humans. Report any spills according to your institution's guidelines.  Shipping:  Follow all relevant transport regulations for biological specimens. If shipping internationally, ensure you have the correct customs documentation. 

24. Fix samples for histological To fix histological samples, immerse the tissue in a fixative like 10% neutral buffered formalin, ensuring the volume of fixative is at least 10 times the tissue volume and the container is large enough to prevent squashing.  1. Choose the right fixative: Use a buffered fixative, such as 10% neutral buffered formalin, for most routine histology. Buffered formalin prevents the formation of formalin pigment, which can interfere with diagnosis.  2. Use adequate volume and containers: Fixative-to-tissue ratio:   Use a volume of fixative that is at least 10 times the volume of the tissue. Container size:   Place the sample in a container large enough to allow the fixative to penetrate all sides without squashing the tissue. 

25. 3. Time and temperature: Fixation time:   Fix the sample for a minimum of 24 hours, and up to 48 hours for larger or more complex tissues, at room temperature. Penetration rate:   The time required for fixation depends on the thickness of the tissue. The general rule is about 0.5 mm per hour.  4. Prepare the sample for processing: Rinse: After fixation, rinse the tissue with tap water for about 20 minutes to stop the fixation process. Store: Store the rinsed sample in 70% ethanol at 4∘𝐶 to prepare it for dehydration. Avoid long-term storage in 70% ethanol, as it can cause drying. 

26. Collect, transport & fix samples for cytological studies To collect and transport cytological samples, use appropriate containers and fixatives, properly label all specimens with patient information, and follow specific instructions for different sample types.  For fluid or tissue, collection into a solution like Cytolyt or formalin is often necessary, while fresh smears require immediate fixation in alcohol and drying.  Transport specimens promptly, keeping them refrigerated if fresh and delayed, and always package them with the correct requisition form.  Sample collection: Fluid specimens:   Collect fluid in a clean container. If transport will be delayed by over an hour, add a fixative like Cytolyt . For certain tests, a portion may be spread as a direct smear and air-dried rapidly. 

27. Tissue samples:   Use appropriate collection devices to scrape or obtain the sample. Transfer the sample into a container with at least 20 times the volume of 10% neutral, buffered formalin. Make sure the tissue is completely submerged.  Smears:   Gently scrape the material onto a labeled slide. Immediately submerge the slide in 95% alcohol for fixation.  Urine:   Follow specific instructions, which may include voiding directly into a container with preservative, especially if a delay in transport is expected.  Cervical (Pap) samples:   Use a broom-like device and follow the specific instructions for collection and rinsing into the vial containing preservative fluid. 

28. Labeling: Label the container with the patient's name, date of birth, date of service, and physician's name.  For multiple samples, use a separate vial and brush for each site and label each one to identify the source.  Ensure the patient information on the requisition form matches the information on the specimen container.  Transportation: For fresh specimens requiring refrigeration, place the sealed container in a plastic biohazard bag with the requisition form and store in a refrigerator until transport.  For smears, ensure they are completely air-dried before transport.  Place the labeled container(s) and requisition form into a transport bag.  If using a separate specimen bag, place the vial in the smaller bag, which is then placed in the larger bag with the requisition in the front pocket.  Transport samples to the laboratory as soon as possible, especially if they were not collected in a fixative. 

29. Fix samples for cytological studies To fix cytology samples, immediately place wet smears in a fixative like 95% alcohol or use a spray fixative, ensuring the sample is fully dry before sealing. For smear-based samples: Smear the sample:   Place a drop of fluid on a slide and spread it with a second slide, or use a collection device like a brush.  Fix immediately: Dip fixation:  Immediately place the wet slide into a Coplin jar containing 95% alcohol or other appropriate fixative.  Spray fixation:  Use a spray fixative, which is an alternative to alcohol immersion.  Dry:   Allow the slide to dry completely before any further processing or sealing. For spray-fixed slides, let them dry fully before sealing to prevent sticking.  Label:   Label the slide with patient information, preferably using a pencil, as ink can wash off. 

30. Dip Fixation Spray Fixation

31. For fluid-based samples: Collect the sample:   Collect the fluid in a sterile container or a vial with the appropriate preservative fluid, such as ThinPrep or CytoLyt .  Rinse device:   If using a collection device like a brush, rinse it thoroughly in the preservative fluid, cap the vial tightly, and shake gently to dislodge the cells.  Label the vial:   Label the vial with patient identifiers and place it in a biohazard bag with a completed requisition.  General tips: Avoid formalin:  Do not use formalin for most cytologic specimens, as it can affect staining quality and should be kept separate from cytology samples.  Air-dry for stains:  Create one air-dried slide for immediate evaluation with rapid stains and one alcohol-fixed slide for stains like the Pap stain.  Submit slides:  Always submit unstained slides in addition to any pre-stained ones.  Label tubes:  Use ink for labeling tubes and avoid adhesive labels on slides. 

32. ThinPrep CytoLyt

33. 4. Process the grossed tissues Processing grossed tissues involves preparing them for microscopic analysis by first grossing (inspecting, measuring, and trimming) the specimen, then processing it to remove water and infiltrate it with paraffin, and finally embedding the tissue in a wax block for sectioning.  This is a multi-step process performed by a pathologist or histotechnologist in a pathology laboratory.   1. Grossing Inspection and logging:   The specimen is received, logged into the system, and inspected visually for identification and diagnostic information.  Trimming:   The pathologist or histotechnologist measures and trims the tissue into uniform slices, typically no more than 3–4 mm thick, to ensure proper fixation and processing. 

34. Sampling and marking:   Representative areas of the tissue are sampled, and the margins are inked to be clearly visible in later steps.  Cassette preparation:   The tissue slices are placed into a labeled tissue cassette, which is a small, perforated plastic container.  2. Processing (Paraffin Technique): Dehydration:   The tissue in the cassette is placed in a series of alcohol baths to remove all water.  Clearing:   The alcohol is removed by a clearing agent, such as xylene , which makes the tissue clear and permeable to paraffin.  Infiltration:   The tissue is infiltrated with hot paraffin wax to fill the spaces left by the clearing agent, hardening the tissue for sectioning. 

35. 3. Embedding: Molding:  The paraffin-infiltrated tissue is placed into a mold.  Orientation:  The tissue is carefully oriented within the mold, and the labeled cassette is placed on top.  Hardening:  Molten paraffin is added to the mold and allowed to harden, creating a solid block containing the tissue. This block is then ready to be sectioned into extremely thin slices for staining and microscopic examination. 

36. Grossing specimen

37. 5. Cut sections using rotary microtome to get ribbons of tissue sections. To cut sections with a rotary microtome, mount a paraffin-embedded tissue block, set the desired thickness, and use a steady, rhythmic motion on the handwheel to rotate the block against a stationary blade.  As sections are cut, a "ribbon" of sections will form on the knife edge, which can then be carefully separated with a brush and transferred to a warm water bath for flattening before being mounted onto a slide.  Before cutting: Secure the blade:   Install a fresh, sharp blade into the microtome and ensure it is properly tightened and aligned with the specimen.  Trim the block:   Mount the tissue block on the specimen clamp. Use a trimming function or a razor blade to remove excess wax until the tissue is fully exposed, creating an even, flat surface. 

38. Rotary microtome

39. Set section thickness:   Adjust the microtome to the desired section thickness, typically between 1 and 60 micrometers.  Adjust knife holder:   Set the knife holder to a steep angle for optimal cutting.  Cutting the sections: Start the rotation:   Lock the block holder and use the handwheel to rotate the block toward the blade at a moderate, consistent speed to begin the cutting process.  Form the ribbon:   As you continue to turn the handwheel , the first few sections may be held with a brush. Once a "ribbon" of several sections forms, support the ribbon from underneath with the brush.  Control the cut:   A slow, uniform rotation is essential for cutting clean, consistent sections. Cutting too quickly can cause "chatter" or other distortions in the section. 

40. After cutting: Collect the ribbon:  Once a sufficient number of sections are cut, lock the handwheel . Use two brushes to detach the ribbon from the blade and carefully transfer it to a warm water bath.  Flatten and mount:  Place the slide into the water bath to flatten and smooth the sections. When the sections are flat, pick them up with a slide and place them in a drying oven.  Final drying:  Dry the slide in an oven to adhere the sections to the slide. 

41. Cutting section by Microtome