Histopathology: Tissue slide preparation

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1. Organ sample

After post mortem, the organ sample is taken in put in the bottle with 10% buffered formalin. If the size of organ is too big (eg Bull), cut of approximately 1.5 cm square area. Cut only the area of the organs showing the abnormalities (eg haemorrhage, ulceration)

The organ sample in the formalin is left overnight (at least-to proceed next process) to allow full absorption of the formalin. The function of formalin is to preserve the sample and kills the bacteria causing autolysis. The pH must be 7.2 (thats why the formalin is added with buffered)

2. Trimming of tissues

Trimming of organ

Tissue cassette

When the organ is taken out from the formalin, it goes to the second step that is trimming of organ sample into small pieces. The organ is cut in various shapes, such as triangular or square shape (depends of pathologist's preference) and next is place in the tissue cassette. The best suggestion is to put abnormal piece of organ such as kidney together with the normal piece of kidney in the same tissue cassette to allow the pathologist to distinguish the normal cell and abnormal cell of the particular organ.

4. Tissue processing- Dehydration

Modern Histokinate

After the tissue is fixed into the cassette, next it must be place in the histokinate for dehydration process. The tissue is immersed automatically in the low concentration of alcohol to high concentration of alcohol (100%) Once the tissue is dehydrated, next it is immersed in the xylene or chloroform to clear the water and covered with wax to made the tissue easier to cut in the later process. The dehydration process takes about 24 hours.

5. Tissue processing- Embedding with paraffin

Embedding in the paraffin wax

Tissue block

When the tissues is taken out from the histokinate, it will be taken to the embedding machine (I forgot the name!) to start to make paraffin block. The block is made from the melting paraffin wax and it will freeze within about 2 minute within the cassette.

6. Tissue processing- Cutting the sample

Tissue block on the microtome

The tissue block is now can be trim and cut by using microtome. The microtome's function is to cut the tissue block into a very thin piece of film (about 5 micrometer). One must ensure to trim the tissue carefully before taking the film to put in the water bath.

7. Tissue processing- "Fishing" in the water bath

Tissue's film in the water bath

The tissue's film must be immersed in the water bath (45 degree celcius) to remove the wax for staining purposes later. A glass slide covered with 1 drop of albumin is used to "fish" the film from the water bath. The function of the albumin is to adhere the film into the center of the slide.

Fishing the film

8. Tissue processing- Rehydration, staining and dehydration

After the tissue is adhere to the slide, proceed to the rehydration process. Rehydration process is to allow to tissue to gain water and therefore able to pick up the stain (H&E) to be able to see under the light microscope. The process is begins with immersion of the slide into the xylene 1, xylene 2 and xylene 3, 100% alcohol ---> 75% alcohol ---> distilled water ---> hematoxyline--> distilled water ---> 0.5% alcohol acid ---> 0.1% lithium carbonate --> 0.5% eosine--> alcohol concentration from low to high (75%, 90%, 100%,100%,100%) ---> xylene

The staining materials

9. Tissue processing- Mounting

After the slide has been stained, a cover slip must be put on top of the sample. Before that, depex is applied on the sample and mounted with a cover slip. The function of Depex is to fixed the tissue slide so that it is become adhere and therefore it does not remove from the slide. Once the mounting is done, the tissue slide is now ready to be observe under the light microscope.

Label the slide, and histopath complete!

Read it for me please?

Sources: Pathology Unit: Veterinary Service Department Kubang Kerian, Kota Bharu,Kelantan