Читать книгу THE ABCS OF HUMAN HISTOLOGY - Javier Munoz - Страница 9

Processing Methods in Histology

Macroscopic study: The study can only be possible with a fresh sample, observation and palpation of the organ without opening and only then cutting.

The data that is always included in the description is the weight (small biopsies and hollow organs such as the stomach or the large intestine are not weighed), colour, consistency, and measures are referred to three dimensions. The selected sample is placed in numbered capsules and so continues the processing.

Fixation: All material to be studied in pathology must be properly fixed. The procedure for establishing the basis for the conservation of the morphology is the fixation with formalin at 10%. In small biopsies is sufficient 1 to 2 hours. The largest biopsies, seemingly cutted and sectioned with a scalpel require several hours (6-7 hours). The surgical samples once opened and extended or anchored with a cork and with pins, will be necessary to be fixed for a period of 24 hours. The ratio tissue/formaldehyde should be 1/ 20.

Cut in microtome after embedded in paraffin.

Body tissues are composed of a large quantity of water. Moreover, the paraffin is a wax which melts at 60-65 °C and is completely insoluble in water. The process is basically to replace the water of the tissue by paraffin to impregnate it, obtaining a block of uniform density, which allows a fine cut with the microtome.

This process requires:

Dehydration: after obtaining the portion of tissue to be processed, with a previous fixation, is inserted into a plastic capsule, and properly identified, placed on a device called “tissue processor”, through solutions of ethyl alcohol with increasing concentrations (80, 96, 100) dehydrating completely the tissue; the last step is the xylol, who just finalized the dehydration and converts into a transparent tissue.

Paraffin embedding: the device introduces the melted paraffin (liquid) in the tissue; pulling from the capsule the apparatus tissue, and placing it in a mold with liquid paraffin and allowing it to cool.

Cuts with microtome: once the block is cold the cuts are done, usually 5 microns. The cuts are extended in an histology slide.The coloration of the cuts requires a series of steps currently performed automatically for the most common coloration - haematoxylin - eosin (H&E); dewaxing and hydration of the cuts: so that the paraffin can be removed, is necessary to make the cuts with heat to melt the paraffin; then passed through xylol that has just dissolved the paraffin. Subsequently is followed the hydration procedure which is the reverse to what was discussed above.

Hydration: Once stained, the dehydration with alcohol and xylol is done again.

Assembling of the Preparation: The process ends covering the histological sections with a small glass called “coverslip” and sticking by means of a transparent resin that solidifies quickly, getting a permanent preparation to be observed under the microscope and being then archived.