Surgical Pathology Laboratory

SURGICAL PATHOLOGY LABORATORY

Surgical Pathology: is the study of lesions of living tissues and cells that are the result of a disease, a trauma , or a malformation 

Autopsy :  post-mortem examination to discover the cause of death or the extent of disease.

Most of the surgical specimens come from the operating rooms 

Others come from emergency rooms or clinics

There are two types of specimens: 

1. organs or parts of organs 
2. biopsies 

There are various Histotechnological Techniques 

1. Gross techniques 
2. fixation and fixatives 
3. Tissue processing
4. Embedding Microtomy 
5. Staining and mounting 
6. Frozen section 

Cytology: 

• Fine needle aspiration biopsy 
• Exfoliative cytology 
• Fixation of cytology material 
• Processing cytology specimen 
• Staining of cytology material 

Gross Examination :

The specimen is properly identified and labeled after its reception in the laboratory.

A properly completed surgical pathology request form contains :

• the age and sex of the patient 
• essential clinical data
• type of operation and surgical findings 
• tissues submitted

Steps to examine material in container 

1. General inspection of specimen with identification of all normal and abnormal parts
2. Gross description = type of specimen,structures involved, dimensions, weight, shape, color/ lesion, size, color, consistency, relation with adjacent structures

Inadequate gross dissection and sampling will invalidate the microscopic description
A crucial part of pathologic examination is dissection, gross description and selection of sections for microscopic study.
It cannot be remedied if done poorly at the time of the initial workup.
If microscopic description is inadequate, the slide can be reviewed.

Gross examination is crucial because the tissue will be sampled and submitted to histology accordingly.

The specimen is kept wet for 2-3 months 

Fixation 

Preserves tissues by stopping autolysis

•     preservation of morphology by inactivation of molecules (ex: enzymes) that change the morphology of a cell
•  preservation of the chemical integrity

Fixation is accomplished by immersion or perfusion
Mechanism of action: modification of spatial configuration of proteins (denaturing)

Groups of Fixatives 

1. Aldehydes 
   • Formaldehyde and glutaraldegyde   
2. Mercurials
   • B5 and Zenker's
3. Alcohol: cytology 
4. Oxidizing agents 
   • dichromate fixative and osmium tetroxide 
5. Picrates 
   • Buin's Fixatives 

Formalin 

is a 10% buffered neutral solution 

• is made up of 40% formaldehyde (100 ml)
•    distilled water (900ml)
• Sodium phosphate, monobasic 4.0 gm
• Sodium phosphate, dibasic 6.5 gm
• there is 4% formaldehyde in the final liquid
• Best general fixative universally used
• colorless liquid, or colorless inflammable gas with a sharp odor 
• Immediate irritant to eyes, nose and throat
• Skin and respiratory system particularly affected
• Safety precautions include proper ventilation, restricted exposure time and thorough washing if spilled on the skin
• Formalin fixes by forming methylene bonds between protein chains

Tissue Processing 

There are 3 sequential steps designed to remove extractable water from tissue specimens and replace it with a medium that solidifies to allow sectioning:

1. Dehydration
2. Clearing
3. Infiltration

Processing is done by automatic tissue processor as an overnight schedule that enhances the processing by using heat, vacuum, pressure and agitation

Embedding 

is the process of surrounding tissue with a firm substance to facilitate the cutting of the sections

• the mostly used embedding medium is parrafin which has a melting point of 56ºC
• Stainless steel embedding molds are used for shaping liquid paraffin into blocks
• Tissues manually removed from the cassette and put into the molds.
• Paraffin should solidify in around 15 minutes: ready for sectioning

Microtomy 

The parafin block is inserted in  a rotary Microtome 

the sections are separated and placed onto a clean pre-marked slide 

Staining and mounting 

Hematoxylin and eosin  (H&E) is a routine stain by which all tissues pass by 

The hematoxylin stains nuclei in blue

The Eosin stains cytoplasm in pink or red 

It shows  the morphology of the tissue 

If special biochemical structures are to be stained: special stains for carbohydrates, lipids, amyloid, connective tissue fibers, microorganisms, pigments and minerals

Mounting 

Final step in preparation of a slide
Cover the portion containing the tissue with a thin glass: coverslip
This makes the slide permanent and permits microscopic examination
Protects tissue from being scratched and preserves the slide for the years to come.
Mounting media: synthetic resinsFinal step in preparation of a slide
 

Frozen Section 

Sectioning frozen tissue, the tissue being hardened by freezing the water that it contains.
Fresh or fixed tissues are frozen and sectioned instead of being routinely processed and embedded.
Sections done in Cryostat: Microtome in a deep freeze at -20ºC.

Cytology 

Cytopathology = study of disease in cells
Exfoliative cytology
Aspiration cytology
Papanicolaou or Pap stain; Main stain for cytologic preparationsCytopathology = study of disease in cells