Histopathology Laboratory

Frozen Sections

Surgical Pathology Sub-Specialties

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Immunostains (contact Dr. David LeBrun)

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Histopathology Laboratory

Once a specimen is received in the Histopathology Laboratory, it is identified by its own unique accession number. The gross examination is important because once the specimen has been sectioned, all gross information is lost. At this time the gross specimen is weighed, measured, and may be photographed or X-rayed. If necessary, the margins are inked to determine the adequacy of surgical margins. Finally, a report of the macroscopic findings are dictated.

Tissue that has been cut and placed in cassettes.

Following protocols for the dissection of various organs, the pathologist or pathologist's assistant determines which areas to sample for microscopic examination. 3mm sections of tissue are cut from the specimen and placed into cassettes, then returned to formalin.

Specimens are fixed in 10% buffered formalin for several hours to denature the proteins and harden them. This prevents further decomposition of the tissue by arresting cell metabolism. Some specimens are fixed overnight, prior to being examined grossly, to make them easier to handle and section.

Tissue processor for dehydration

Tissues must be dehydrated before they can be infused with paraffin. In the automated tissue processor, sectioned tissue is submerged in a series of alcohol baths to remove water. The alcohol must then be cleared with a clearing agent, such as toluol, which is miscible with paraffin. This process is performed overnight so that the tissue is ready to be embedded with paraffin the next morning.

Tissue embedded in molten paraffin.

The tissue is placed in molten paraffin at 52 - 56°C for several minutes so that once the paraffin cools, the tissue and block will be hard enough to cut. Depending on the tissue, it will need to be embedded in the paraffin in a specific orientation. For example, a tubular structure is positioned so that when it is cross-sectioned, the entire lumen is visible.

The paraffin blocks containing the tissue are cut producing 5µm sections with a microtome. This piece of equipment has a set mechanism for advancing the block across a very sharp knife. The cut sections are floated on a water bath to remove wrinkles, then transferred by hand to glass slides.

Cutting sections with the microtome.	Sections in waterbath picked up on slides.

In order for the water-soluble dyes to penetrate the tissue, the sections must be rehydrated. Toluol is used to remove the paraffin, then a series of alcohol washings rehydrates the tissue.

The slides are stained to differentiate the nuclear material and connective tissue from the rest of the tissue. The KGH histopathology laboratory uses the hematoxylin-phloxine-saffron (HPS) stain which is more effective than the regular hematoxylin-eosin (H & E) stain used by most laboratories. HPS stains the nuclei blue; the cytoplasm, muscle, and myelin red; and the connective tissue yellow.

Slides of surgical specimens

The tissue is again dehydrated with alcohol and toluol to preserve it indefinitely. The glass coverslip is applied by an automated machine. The slides are now ready to be viewed by the pathologist.

When specimens contain calcium, such as samples of bone or bone marrow, they must be de-calcified prior to cutting. Once the specimen is dissected into small sections and placed in plastic cassettes, it is de-calcified in RDO for approximately 4 hours.

Sometimes the pathologist will order a special stain which highlights various features of the tissue or chemicals present. Examples of special stains are:

Chemicals for special stains

Carbohydrate Stains
ex: PAS (periodic acid schiff) stain


Pigment Stains
ex: Prussian blue stain for iron


Micro-organism Stains
ex: Giemsa stain for H.pylori bacterium


Connective Tissue Stains
ex: Gordon and Sweet stain for reticulin

Pathology resident examing a frozen section.

Frozen Sections

During a surgical operation, the pathologist may be required to perform an intraoperative pathology consultation ("frozen section"). Analysis of a frozen section requires quick decision making and good judgement by the pathologist. A detailed diagnosis is not necessary from a frozen section since time is of the essence. Instead, the pathologist will assist the surgeon in determing the course of the operation by:

1. establishing the presence and nature of a lesion
2. determining the adequacy of surgical margins
3. establishing whether the tissue obtained contains diagnosable material or whether additional sampling is required

The frozen section laboratory is located close to the operating room, so that tissue removed in surgery can be processed immediately. When the specimen is received, it is labelled with its own unique accession number. The specimen is examined grossly and a section of tissue is sampled. The tissue sample is frozen in a Histobath containing isopentane at a temperature of -50°C. It is then transferred to a cryostat; a refrigerated box containing a microtome which cuts the tissue into 5µm sections. These sections are then mounted on slides and stained to differentiate the nuclear material from the rest of the tissue. The section is fixed in acidified alcohol, stained with hematoxylin and eosin, dehydrated in ethanol, and cleared in toluol. The entire process takes approximately 10 minutes to complete, allowing the pathologist to microscopically examine the specimen and quickly provide a diagnosis to the surgeon.

Freezing the tissue in the Histobath.	The cryostat
Cutting the tissue in the cryostat. The tissue is the pink area in the middle of a white frozen gel substance on a block called a "chuck".	Dehydration and staining chemicals

Surgical Pathology Sub-Specialties

Frequently Asked Questions

The histopathology laboratory examines all material that is removed from the body during surgery. These specimens may be whole organs, sections of tissue, or biopsies.

Dissection of a biopsy specimen.

A biopsy is a procedure to remove tissue from the body to be examined microscopically for a precise diagnosis. It is often used to determine whether a lesion is benign or malignant. The types of biopsy include:

• Excisional - removal of the entire mass
  ex: lymph nodes or breast lump
  
  

  Incisional - removal of a portion of the mass

  ex: soft tissue tumours
  
  

  Endoscopic - removal of tissue using a fibreoptic endoscope

  ex: GI(endoscopy), abdominal(laparoscopy) or bronchial(bronchoscopy)
  
  

  Colposcopic - removal of tissue using a telescopic type of endoscope

  ex: cervical tissue due to abnormal Pap smear
  
  

  Fine needle aspiration (FNA) - cells or tissue removed through a needle

  ex: tumours in deep structures like lung or liver
  
  

  Punch biopsy - removal of small area of skin

  ex: skin lesions
  
  

  Bone marrow biopsy - removal of bone marrow cells or a core of bone

  ex: abnormal blood counts

In all cases, the pathologist receives the slides of the specimen for microscopic examination along with a report of the gross findings, which may contain photographs, and the clinical history. The pathologist may be asked to view the gross specimen to decide which areas to sample.

Gross specimens are retained in formalin for several weeks, so the pathologist may order more samples if necessary. The paraffin blocks and the slides are kept indefinitely so that they will be available for further diagnostic review, should that become necessary. They also serve as valuable educational resources for pathologists, residents, and medical students.

The KGH histopathology laboratory processes approximately 25,000 cases/year, producing over 88,000 slides. Of these, approximately 700 cases are frozen sections.