General Outline of Procedures in the Examination of Tissues in histopathology

General Outline of Procedures in the Examination of Tissues

There are several ways and techniques used to process tissues for laboratory examination with the intent of making accurate and proper diagnosis. Whichever technique is employed, there are factors which govern the whole procedure: the structures to be studied, the size and nature of the tissue, whether the specimen is fresh or preserved. and the urgency of the examination. Details of each stage of the investigation may differ from laboratory to laboratory but the basic principles remain the same.

RECEPTION

On arrival in the departmental reception, the specimen is checked at the earliest opportunity for the following: 

1. That the specimen is for histological examination. 

2. That the container is clearly labeled and accompanied by a completed request form. 

3. That sufficient fixative is in the container, or if the specimen is not in fixative or is in a wrong fluid.

The request form is dated and stamped; the specimen is given an identification serial number which remains with the specimen until all the investigations have been carried out. The pathologist, usually at a set time, will examine the specimen, where necessary describe the macroscopic appearance and select the pieces from which he wants the section to be prepared. A sink and running water are essential in this area usually referred to as the 'cut-up room'. 

The pathologist requires rubber gloves, sponge, scalpel and scalpel blades, large ham knife, plain and rat-tooth forceps, probes, scissors, bowel scissors, small bone saw, steel rule and some means of weighing the specimen. It is very important for the laboratory scientist assigned to the cut-up room to write the description of the specimen and record how many pieces are taken for processing.

FRESH SPECIMEN

Fresh specimens sent to the laboratory for histological examination are prepared as either squash or teased preparation, touch or frozen sections.

Squash preparation Small pieces of tissues not more than 1 mm in diameter are placed in the centre of a microscope slide; a cover slip is forcibly pressed down on them. Vital staining can be done by placing a drop of the stain at the junction of the slide and the cover slip. The stain is drawn in by capillary action and absorbed by the tissues. The purpose of squash preparation is to study the cellular contents of the tissue.

Teased preparation The fresh specimen of tissue, immersed in saline or Ringer's solution, is dissected with mounted needles. Pieces of the tissue are picked onto a microscope slide and mounted as a wet preparation under a coverslip. The slide is then examined by the ordinary light microscope or better still by phase contrast microscope. Stains like methylene blue can be applied to enhance the recognition of cellular structures. This method allows cells to be examined in living state. A big drawback is that it is not a permanent preparation. 

Smear Smearing a piece of fresh specimen of tissue evenly on the surface of microscope slide is an acceptable practice in histopathology. The making of such smears however, depends on the type of tissue to be examined. The smear can be examined fresh in which case it is stained as for teased preparation or by using supravital stain in conjunction with a warm stage. The preparation is never permanent. 

Impression smears these are prepared by touching a freshly cut piece of tissue with the surface of clean microscope slide. This way, cells are transferred and adhere to the slide. The smear can be examined with the phase contrast microscope or by using vital stain. It is also possible to fix the smear and stain accordingly.

Frozen sections Fresh tissue frozen on a microtome with CO, can be cut into sections of about 10-15 um in thickness. The sections are transferred to a dish and attached onto the slide before staining or from the dish carried on a glass rod through staining solutions.

FIXED TISSUES

To study normal or diseased tissues microscopically, thin sections of the tissue have to be cut. Sections are normally prepared from fixed tissues. Tissues are fixed to prevent putrefaction and other post mortem changes which occur soon after removal from the body. The choice of fixative depends to a large extent on the type of investigation,nature of the specimen, urgency of the examination and whether or not serial sections are required.

PARAFFIN SECTIONS

The most popular embedding medium for histological preparations is the paraffin wax. The embedding medium confers rigidity to the piece of tissue so that sections can be cut. The selected fixed pieces of tissue are first passed through a series of baths of alcohol to remove water from the tissues (dehydration). 

This is done because the fixative is not miscible with paraffin wax. The tissues are passed further to a bath or two of a medium that is miscible with alcohol and paraffin. This medium is the clearing agent. The clearing medium also raises the refractive index of the tissue, making it look transparent. It is this clearing medium that is eventually replaced by the molten paraffin wax. When the tissues have been sufficiently impregnated, they are embedded in fresh wax which solidifies on cooling. It should be noted that workers refer to clearing agents as ante-media because some of them don't actually clear the tissue. Using paraffin wax allows thin and serial sections to be cut easily, allows many staining techniques and facilitates handling and storage.

Celloidin Next to paraffin wax, celloidin is a commonly used embedding medium. Celloidin is a purified form of nitro-cellulose which is soluble in many solvents. Celloidin allows thicker sections to be cut and that makes it very useful in the study of central nervous system. It is a better medium for very hard tissues or tissues with varying consistency. It causes little or no shrinkage. However, it is a very slow method and serial sections are very difficult to produce. A variety of celloidin, the low viscosity nitro-cellulose (LVN) is also very popular. The LVN dissolves more easily and permits thinner sections to be cut.

Resin embedding This is a new method adopted for light microscopy from electron microscopy techniques. The major advantage of using resins is that much thinner sections can be cut though specially adapted knives and microtomes are required. The various stages of dehydration, clearing and embedding are referred to as processing and are usually done manually or by automatic processing machine.

CUTTING AND STAINING

Sections are cut with the microtome from the paraffin wax blocks. The microtome is a most useful piece of equipment in the histopathology laboratory and the microtome is as good as its knife. The different types of microtome are discussed later in this section. The cut sections are floated out in warm water, mounted on clean microscope slides, dried and then stained. The stained sections are mounted in a good mountant under a coverglass, then labelled and cross-checked against the information on the request form.

REPORT AND FILING

After examining the slides, the pathologist writes his report which is passed on to the secretary for typing and dispatch. In most cases the pathologist may require additional sections to be cut or special staining techniques to be carried out before making a final report. The slides are dried at 37°C for about two weeks and then filed away. The gross specimen is kept for at least one month before it is finally disposed off.

This is a general outline of the procedures of processing a histopathological specimen. It should always be clearly borne in mind to handle surgical specimens with utmost care because it is not always possible to repeat the sample.