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NOTES FOR PATHOLOGY/MICROBIOLOGY CLERKSHIP STUDENTS ON

HISTOLOGICAL PROCESSING OF SPECIMENS

FIXATION

Specimens are usually fixed in a10 % neutral buffered formaldehyde (10% formalin) as

soon as they are removed from patient in surgery. Other fixatives can be employed but

for histology formalin is best for the demonstration of basic cell histology and it is also

the most economical. It is important for proper fixation that the size of container be

adequate to house the specimen and ten (10) times its volume of fixative usually

controlled for any particular period of time before reaching the laboratory.

ACCESSIONING

Specimens received in the laboratory are promptly assessed for correct labeling and

completion of request forms. Each patient is given a unique identification (accession

number) called a surgical number which also identifies the specimen by year of

accession, eg. S05/2005 or XS05/1085.

TRIMMING

Specimen(s) is/are then examined grossly, described and sampled by resident

pathologists and placed in the tissue processing cassettes which are used to make the wax

(paraffin) molds and these become the permanent blocks which are stored for years in

pathology archives.

PROCESSING

Specimen cassettes are processed using an automatic tissue processor (Citadel 2000).

Processing has the following steps:

(a) Further fixation – two changes of formalin for varying times dependent on

whether it is overnight, weekday or weekend processing.

(b) Dehydration – the removal of water from the tissue using ascending grades of

alcohol, each solution for an average of forty five minutes to an hour.

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(c) Clearing in xylene – this is a step which clears the tissue of opaque dull look from

previous stage structures making them more transparent and makes the tissue

more receptive to the next stage.

(d) Impregnations in molten paraffin wax in two changes of wax forty five minutes to

an hour in each change.

EMBEDDING

Tissue is embedded in molten paraffin wax using the plastic molds mentioned in the

trimming section which forms the permanent specimen.

CUTTING

Using microtome blades (disposables) tissue sections of 2 – 5 microns in thickness are

cut from cervical biopsy blocks. For cervical resection blocks, blocks are cut using

microtome knives. We aim for similar thickness of sections as for the biopsy but this

may be harder to achieve in samples such as cervix due to the characteristic toughness of

the cervical tissue.

STAINING

The slides are labeled with the same number that was written on the blocks, this is the

permanent slide of the permanent block. Slides are placed in a drying oven to melt off

excessive wax before staining procedure.

Dewaxing: The slides are cooled and placed in two changes of xylene for two

minutes for each change.

Rehydration: Water is put back into the tissue by passing it through descending

grades of alcohol, for two minutes in each.

Staining: Slides are overstained in basic Erlichs Haemotoxylin to stain the acidic

portion of cells (for five minutes. Decolourize in 1 % acid alcohol to remove excess

staining. This is controlled so we use sharp dips and then rinse in water.

Bluing up: Nuclei is allowed to blue in tap water five minutes or in alkaline

sodium hydroxide solution – 1 dip/ 1% Ammonia water-1dip.

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Counter stain: Is done using 1 % Eosin yellowish for one minute, rinse in tap

water, dehydrate in ascending grades of alcohol - sharp dips in each solution as

necessary.

Clearing: Xylene - 3 dips each.

MOUNTING

Slides are mounted using a synthetic resin then labeled with laboratory numbers and the

kind of stain and is given to the pathologist in order for a diagnosis to be made.

Most diagnoses can be made from only routine H&E slides only. For confirmation

Histochemical special stains may be requestd by pathologists to confirm the presence or

absence of organisms, or the characteristic cellular changes that occur in various disease

states. Immunohistochemical stains may also be requested, where specific monoclonal

antibodies are reacted with suspected antigen in the tissue.

In the first world countries the technology allows for direct DNA analyses including

FISH and PCR.

Prepared by:

Mrs. Sharon Harrison (DMT, BSc, MPH)

Chief Medical Technologist

Histology