- Collection, Transportation and Accessioning of histopathology specimens
- Macroscopic Description (Gross Examination)
- Lab Procedures
10. Microscopic Examination and Interpretation
1. Purpose:
- To render tissue diagnosis on surgical specimens / biopsies.
- To standardize the system of Histopathology lab and monitor the operation phases
- To control process through appropriate verification & through adequate documentation.
2. Scope:
This procedure is applicable to Histopathology Lab when histopath ological examination on surgical specimen is performed.
3. Materials and equipment:
For Gross Examination
- Cutting board
- Knives
- Scalpel blades
- Forceps
- Scissors
- Weighing balance
- Kidney trays, sieve
- Apron
- Formalin
- Scales
- Tissue baskets / Cassates
- Saw
- Gloves
- Gauze cloth
- India Ink
- Petri Dishes
- Magnifying lens
- Nitric Acid
For Reporting
- Microscope EOP #
- Computer EOP #
4. Procedure
Collection, Transportation and Accessioning of histopathology specimen
Macroscopic Description or (gross examination)
1. Facilities
- Light must be adequate and preferably natural daylight.
- There must be strong exhaust fan to remove formalin fumes extraction at bench level.
- Running water must be available to wash out and clean tissues, and to wash bench and instruments.
2. Safety considerations:
- Rubber gloves, protective water proof aprons should be used
- Face masks for infectious cases.
- Eye protection must be available and used to protect formalin splashes fumes.
- All other safety considerations according to Safety Manual.
3. Method
- Before gross examination, specimens to be grossed are kept in order and all items required for gross are kept ready in the area specified for the gross examination.
- Identify number (Reception #. & Lab #) on the specimen container / request slip.
- Label Lab # on the tissue baskets / cassettes with lead pencil.
- Record size / quantity / weight (if required) of the specimen.
- Fragmentary and tiny biopsies are grossly described and wrapped in gauze cloth / filter paper and put in tissue baskets.
- Representative sections of large specimens are kept in tissue baskets/cassettes.
- Decalcification of bony specimen is done before processing when required.
- Tissue baskets / cassettes are kept in a container filled with 10% Formalin before tissue processing.
4. Decalcification
- It is a processing which calcium removes salts from a bony tissue to facilitate its sectioning.
Aim of Decalcification:
Bony biopsies, teeth normally and sometimes pathological tissues are hard enough to be sectioned due to deposition of calcium salts in them which include calcium phosphate about 85% calcium carbonate, about 10% and the remaining 5% are chlorides and fluorides of calcium and magnesium. All such tissues need to be softened by removal of calcium salts prior to sectioning.
Decalcifying agent of choice:
In the department 5% Nitric Acid is preferably used to perform decalcification of bony biopsies. This decalcifying agent is prompt in action.
Formula:
- Conc. Nitric Acid - 5ml
- Distilled Water - up to 100 ml
Method:
- At the receipt of a bony biopsy or other pathologically calcified tissue, the following steps are taken:
- Immerse calcified biopsies in bulk of fresh fixative to ensure complete fixation prior to decalcification.
- Write down gross features of the biopsy on back of the request slip.
- Smaller bony biopsies are put to decalcifying agent, freshly prepared, as such.
- From large bony tissues 5 mm thick blocks are cut by a small saw and then these are decalcified.
- Status of decalcification is regularly checked with intervals till the process is completed.
- Decalcified biopsies at the end are washed in running water and are then routinely processed like other tissues.
5. Lab procedures (tissue processing, embedding, microtomy and staining).
5. Reporting:
The Histopathologist carries out microscopic examination on Histopathology/Cytopathology slides and records his/her findings on Test Ordering slip, which is sent to the typist who prepares the final Test Report. After verification reports are signed out by the Pathologist.
6. Tissue Processing:
After the gross examination, the tissue specimens need to undergo tissue processing to prepare them for microscopic examination. This involves a series of steps such as dehydration, clearing, and infiltration with a suitable embedding medium (e.g., paraffin wax). The tissues are placed in cassettes or containers and processed in an automated tissue processor.
7. Embedding:
Once the tissues are adequately processed, they are embedded in paraffin wax or another suitable embedding medium. The processed tissues are oriented appropriately and placed in embedding molds filled with molten wax. The molds are then cooled to solidify the wax, forming a solid block with embedded tissue.
8. Microtomy:
The embedded tissue blocks are trimmed to expose the desired area for sectioning. A microtome is used to cut thin sections (usually 4-6 micrometers thick) from the tissue blocks. These sections are carefully collected on glass slides and labeled.
9. Staining:
The tissue sections on the slides undergo staining to enhance cellular and tissue structures, making them visible under a microscope. The staining process typically involves hematoxylin and eosin (H&E) staining, which provides contrast between the cell nuclei (hematoxylin) and cytoplasm (eosin). Additional special stains may be employed to highlight specific cellular components or pathological features.
10. Microscopic Examination and Interpretation:
The stained slides are examined under a microscope by a histopathologist. They observe the cellular and tissue characteristics, identify abnormalities, and make a diagnosis based on their findings. The histopathologist records their observations, interpretations, and diagnostic conclusions.
11. Reporting:
The histopathologist prepares a detailed report summarizing the findings from the microscopic examination. The report includes the diagnosis, any relevant histopathological features, and additional comments or recommendations. The report is then reviewed, verified, and signed by the pathologist before being released to the referring physician or healthcare provider.
12. Archiving and Storage:
After the examination and reporting, the slides and blocks are typically retained for a certain period as per institutional protocols. These materials are archived and stored appropriately to ensure their long-term preservation for potential future reference or further investigations.
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