Basics of Histopathology/Tissue Processing

HISTOTECHNOLOGY 

Histological technique deals with the preparation of tissues for microscopic examination. The aim of good histological technique is to preserve the microscopic anatomy of the tissues and make them hard, so that very thin sections (5 microns) can be made. After staining, these sections should represent the anatomy of the tissue as closely as possible to their structure in life. This is achieved by passing the total or selected part of the tissue through a series of processes. These processes are:  

• Fixation  
• Dehydration  
• Clearing  
• Embedding Cutting  
• Staining  

FIXATION 

This is the foundation for the subsequent stages in the preparation of sections necessary for making a diagnosis. This is the process by which the constituents of the cells and tissues are fixed in a physical and also partly in a chemical state, so that they will withstand subsequent treatment with various reagents with a minimum loss of architecture. This is achieved by exposing the tissue to chemical compounds called fixatives. 

Mechanism of action:  

Most fixatives act by denaturing or precipitating proteins, which then form a sponge or meshwork, tending to hold the other cell constituents. A good fixation is the most important factor for the production of satisfactory results in histopathology. The following factors are important: 

• Fresh tissue specimen 
• Proper penetration of the tissue by the fixative  
• Correct choice of fixative  

The inadequate penetration of the fixative is one of the commonest causes of poor results. It is a rule that no fixative will penetrate a piece of tissue thicker than 1 cm.  

Processing Special Tissues  

Bone: Unless there is a need for an immediate diagnosis, all bone specimens must be fixed and, later on, de-calcified (carried out by acids), forming soluble calcium salts. For this, strong inorganic acids such as Nitric Acid (5–10%) or Hydrochloric Acid are used. These have a rapid action and should not be used for more than 24–48 hours. Weak acids like formic acid can be used as a component of Bouin’s or Carnoy’s fixatives, recommended for very small bone pieces or for trephine biopsies. 

Amount of fixing fluid 

This should be approximately 10–20 times the volume of the specimen..  

COMMON FIXATIVES:   

1. Buffered Formal Saline    
2. Buffered Glutaraldehyde   
3. Zenker‘s Formal Saline  
4. Ethanol  
5. Methanol 
6. Ether
7. Cold Acetone 
8. Picric  Acid
9. Chromic  Acid
10. Osmium  Tetra-oxide  (Osmic  Acid)

FACTORS AFFECTING FIXATION:  

1. Size and thickness of the piece of tissue 
2. Tissues covered by large amounts of mucus or blood, or organs containing very large amounts of blood, fix slowly.  
3. Fatty and lipomatous tissues, fix slowly.  
4. Fixation is accelerated by agitation.  
5. Fixation is accelerated by maintaining a temperature around 60C.  

TISSUE PROCESSING  

In order to cut thin sections of the tissue, the tissues must have a suitable hardness and consistency when presented to the knife-edge. These properties can be imparted by infiltrating and surrounding the tissues with paraffin wax, celloidin or low-viscosity nitrocellulose (LVN), various types of resins, or by freezing. The process is called tissue processing. It is done in stages. It can be subdivided into dehydration, clearing, impregnation, and embedding.

It is important that all specimens are properly labelled before the processing starts. For labelling, a pen containing ordinary ink should not be used. Printed, graphite pencil-written, typewritten, or India ink-written labels are satisfactory. The label should be clearly written and must contain, in block letters, all of the necessary information.

The representative sections of the entire biopsy specimen, when of small size, are put in cassettes with their labels and are then transported from reagent to reagent in metal containers that have perforated walls, so that the reagent enters into the tissues. Tissue processing is a long procedure and requires approximately 24 hours.

1. Manually: In this method, the tissue is moved from one container of reagent to another by hand. Agitation is also done manually.  
2. Automatically: In this method, the same steps are completed automatically by a mechanical device. In automatic tissue processors, there are different jars containing reagents arranged in a sequence. A mechanical device moves the tissue from one jar to another. Agitation is also done mechanically. Timings are controlled by a timer. 

The steps involved in processing, whether done manually or mechanically, remain the same and are as follows:

Dehydration: Using increasing strengths of alcohol, e.g., 70%, 90%, and absolute alcohol, dehydrates the tissues. The time duration the tissues are kept in each strength of alcohol depends on the size of the tissue, the fixative used, and the type of tissue. After fixation in aqueous fixatives, delicate tissues need to be dehydrated slowly, starting in 50% ethyl alcohol directly, whereas most tissue specimens may be placed into 70% alcohol. Delicate tissues will shrink too much when exposed to a high concentration of alcohol. 

For routine sections not thicker than 7µm,  the following scheme may be followed:  

1. 70% alcohol–Methylated spirit: 1 hour  
2. 90% alcohol–Rectified spirit: 2 changes for two hours each 
3. 100% alcohol–absolute alcohol: 2 changes for two hours each  

In the above process, dehydration is helped by agitation of the tissues, hence the duration is typically 2 hours. If not agitated, it may take much longer for the procedure. In the absolute alcohol chamber, a 0.5-1 inch thick layer of anhydrous copper sulfate, separated by filter paper, may be used. It absorbs the water derived from the tissues. The volume of alcohol should be 50–100 times that of the tissues. If this is not possible, then frequent changes of alcohol are used.

Clearing (To remove alcohol): During dehydration, the water in the tissue has been replaced by alcohol. In the next step, alcohol is to be replaced by wax. As wax is not alcohol-soluble, we replace the alcohol with a substance in which wax is soluble. This step is called clearing. The clearing of tissues is achieved by immersing the tissue in any of the following substances:  

1. Xylene 
2. Chloroform 
3. Benzene 
4. Carbon Tetrachloride 
5. Toluene  

Xylene is commonly used. Small pieces of tissue are cleared in 1/2–1 hour, whereas larger pieces (5 mm or more thick) are cleared in 2–4 hours. Cedarwood oil can also be used. It is an excellent clearing agent, and tissues may be kept for months in it without hardening. However, it is slow in action, and extra time is required in molten wax. 

Impregnation with Wax: This is allowed to occur at the melting temperature of wax, which is 54–60°C. The volume of wax should be about 25–30 times the tissue's volume. For better results, impregnation is done serially in 3–4 jars; however, 2 jars are sufficient. The duration of impregnation depends on the size and type of tissue and the clearing agent employed. Longer periods are required for larger pieces and also for harder tissues like bones and skin, as compared to softer tissues like liver, kidney, spleen, and lung. Xylene is the easiest to remove, and 1–2 changes of wax are sufficient. A total duration of 4 hours is sufficient in all the jars for routine processing.

The types of waxes employed for impregnation are: 

1. Paraffin Wax: It is routinely used. It has a hard consistency, so sections of 3-4 micron thickness can be cut.  
2. Water-Soluble Wax: It has the advantage that the tissue can be directly placed in it, without dehydration and clearing. However, the disadvantage is that fragmentation of the section may occur in the floating bath.   

Other materials used for impregnation are:  

1. Celloidin  
2. Gelatin
3. Paraplast
4. Paraplast Plus  

Casting or Blocking: Embedded tissues are placed in a mold (metal or plastic) with their labels, and then fresh molten wax is poured on them and allowed to settle and solidify. Automated embedding stations are now available. Care is taken not to allow any bubbles to form. Once the block has cooled sufficiently to form a surface skin, it should be immersed in cold water to cool it rapidly. Failure to do this will often cause crystallization of the wax. After the block has completely cooled, it is cut into individual blocks, and each is trimmed. The labels are made to adhere to the surface of the block by melting the wax with a metal strip that is sufficiently warmed.

STAINING 

Staining is a process by which color is imparted to a sectioned tissue. Specially manufactured dyes are used for this purpose.

THE PROCEDURE OF STAINING  

Like processing, staining can also be performed manually or mechanically.  

• Manual Staining: In a small laboratory where only a few slides are stained, this is the method of choice. It is time-consuming but economical. Reagent containers are placed in a sequence. Slides are placed in a carrier and then moved from one container to another at specified intervals until the process is complete.

• Automated Mechanical Staining: The above procedure is performed with the help of a mechanical device similar to the one described for processing. Automated stainers of various kinds are now available. In these, the reagent jars are arranged according to a desired sequence. The carrier containing the slides is rotated through these at intervals, which are set by the operator. These are usually microprocessor-controlled and are both controllable and programmable.

  The advantages are: 
• They reduce manpower requirements  
• Precise control of timings 
• Large  number  of  slides  stained simultaneously  
• Less reagent is consumed  

HAEMATOXYLIN AND EOSIN STAINING  

This is commonly used for routine histopathology and in diagnostic cytology. Its particular value lies in its ability to impart proper differentiation, allowing for the distinction between different types of connective tissue, fibers, and matrices by staining them with different shades of red and pink. 

SPECIAL STAINING TECHNIQUES 

Here are the names of special stains used in histopathology:

• Ziehl-Neelsen (ZN) stain
• Grocott’s Methenamine Silver (GMS) stain
• Periodic Acid-Schiff (PAS) stain
• Alcian Blue stain
• Mucicarmine stain
• Congo Red stain
• Masson’s Trichrome stain
• Reticulin stain (Gomori's silver stain)
• Oil Red O stain
• Sudan Black B stain
• Toluidine Blue stain
• Prussian Blue stain
• Jones Methenamine Silver (JMS) stain
• Giemsa stain
• Elastin Van Gieson (EVG) stain