Veterinary Epidemiology and Zoonosis

Collection of sample for histopathological examination

COLLECTION OF SAMPLES FOR HISTOPATHOLOGICAL EXAMINATION

Container for sample collection

• The mouth of the container should be broad. It should be leak proof and air tight.
• Cotton should be placed at the bottom of tissue collection container.
• Formalin 10% (fixative) should be filled up to 2/3^rd of the container.
• The tissues should be completely immersed into the fixative.
• Precautions: While handling formaldehyde wear eye goggles. Open the formalin containers in fume hood.

Fixation of specimen

• Keeping the tissues in a fixative for 24-48 hours at room temperature
  • serves to harden the tissues by coagulating the cell protein,
  • prevents autolysis,
  • preserves the structure of the tissue and
  • prevents shrinkage.
• Collect and fix samples as soon after death as possible.
• Tissue-fixative ratio should be 1:10 for initial collection and fixation.
• Replacement of the fixative during fixation reduces the time required especially if the tissues are bloody.
• The tissues have to be fixed for 48 hours before used for processing.
• The first 24 hours of fixation is crucial for better fixation of tissues. Before dropping the tissues, wash the tissues in normal saline.
• While dropping the tissues into formalin see that the tissue pieces don’t adhere to the sides of the container.
• Floating tissues (example lungs) should be kept submerged by laying absorbent material like paper towel or cotton wool on top of the fixative to cover the sample.
• Thin biopsies are fixed in an alcohol containing fixative such as formal alcohol, Carnoy’s fixative and Clark’s fixative.
• Rapid fixation of very urgent biopsy specimens: 10% formalin heated to 60ºC.
• Rapid microwave fixation: Fix tissues in 10% formalin at a temperature of 45-55ºC.

Collection of tissues

Do’s

• Sterile sampling should have priority.
• While incising or cutting the tissues for material collection, hold the tissues gently with fingers or forceps.
• For all parenchymatous organs use sharp knife or BP blade for taking pieces. The tissue pieces from parenchymatous organs should be one cm³.
• For all luminal organs use sharp edged scissors. While collecting luminal organs do not scrap the mucosa with fingers, scissors or knife. Collect 1sq.cm of tissues.
• If the lumen of tubular organ is smaller, collect 1cm length of the tube.
• Different portions of the same organ if collected from different locational lesions, they may be collected separately in separate containers and labeled.
• The entire set of tissues should be collected in duplicate and one set should be stored at the source institution for future reference.
• The other set is used for transport to the diagnostic laboratory.
• Always sample liver, kidney, lung and all lesions. However, sampling a full range of tissues is recommended as they can be unexamined, discarded, once the case is completed.
• The brain should be sampled when the history indicates need and in all grossly normal necropsies.
• For taking pieces of tissues the carcass may be used as a cutting board.

Dont’s

• Do not press the tissues heavily with tissue forceps.
• Do not use rat toothed forceps for holding the tissues directly. It can be used only for holding the connective tissue of parenchymatous organs.
• Avoid thin slices as low as 1mm which will curl in the fixative.

Organs with lesion

• Sample lesions at their margins as in cyst, abscess, tubercle, nodular lesion and tumour growths along with adjacent normal tissue.
• Incise close to petechiae, small cyst and abscesses (without opening), miliary nodules and necrotic foci on both the side of the lesion.
• Include only 1mm of normal tissue on both the sides.
• Infuse 10% formalin into large cysts and abscesses with a syringe and needle.
• Allow to fix for a day. Next day examine the contents and collect tissues involving the wall and adjacent normal tissue.
• Tuberculous lesions can be fixed in 10% formalin at 100ºC.

Special fixatives for specific organs

• Soft tissues (Mucosal surfaces of endometrium, intestine, stomach) - for quicker fixation of tissues use Bouin’s fixative for first 24 hours.
• Then the tissue should be transferred to 70% alcohol for next 24 hours. Thereafter, the tissues can be stored in 50% alcohol.
• Brain - 10% formalin, 10% formal saline, 10% neutral buffered formalin, Zenker’s fluid and Baker’s solution may be used.
• Eye – fix in buffered formaldehyde for 48 hours. Zenker’s fixative and Davidson’s fixative give improved results.
• Bone samples should be fixed prior to decalcification or a combined solution (eg. Gooding and Stewart) can be used