Aquitania, Mary Christelle G. Date Submitted: UST Medical Technology Intern Histolopathology

Precautions in Handling, Acceptance and Fixation of Specimen

Handling & Acceptance of Specimen

There are three very important responsibilities a good medical technology should do: 1) Record pertinent patient data, 2) Evaluate and inspect request form, and 3) Evaluate and inspect specimen. The whole process starts when the specimen is submitted in the laboratory reception or receiving area.

Specimen which may come from biopsy or autopsy are always accompanied by a request form in which are found the following data:

1. Name of the patient, age, sex, room number, address, civil status2. Date received3. Type of specimen4. Examination desired5. Clinical diagnosis6. Requesting physician or surgeon

The medical technologist staff or intern receiving the specimen should check first whether the specimen he/she is receiving is the same as that found in the request form. After doing this, it is his/her duty to enter all data in the logbook or record book. Numbers corresponding to the previous patients are found in this logbook, number next to the last patient should be given to the incoming patient. This is called NUMBERING. The number is preceded by either S (Surgical) or A (Autopsy).

NUMBERING is the first and an important step in all histopathologic techniques, the purpose of which is to identify properly all the specimens without writing the name of the patient to the accompanying tag of the specimen to be processed.

The medical technology staff or intern should evaluate and inspect the request form. A correct request form should have all of the following data above. It should be written legibly so as to understand. Then, the specimen, it should have proper container, proper fixative and properly labeled. A proper container should be proportionate to the size and amount of the specimen. To be easily viewed and identify, a proper container should be transparent and tight fitting. In addition, correct type of fixative, concentration and tissue fixation should be used. When the specimen enters the laboratory, remember to always check if the specimen is properly labeled with the patient’s name, specimen source and specimen number.

Fixation of Specimen

FIXATION is the first and most crucial step in histotechnology involving preservation of fresh tissue for examination. The quality of the section on the slide is only as good as the quality of the fixed tissue specimen. It is considered as more critical process other than slide preparation. If fixation is not adequate, the other processes that follow will also be inadequate. Poor fixation results in a poorly processed tissue and will make it difficult for the pathologist to render a proper prognosis.

The primary aim of fixation is to preserve the morphologic and chemical integrity of the cell in as life-like manner as possible. The cell is composed of different organic and inorganic substances. These cellular substances are loosely held together in the cellular protoplasm by hydrogen bonds that are in a

constant state of chemical and biological equilibrium. With fixation, the shape, structure, intercellular relationship, and chemical constituents of tissues are preserved. As a result, fixation prevents degeneration, decomposition, putrefaction and distortion of tissues after removal from the body.

The secondary goal of fixation is to harden and protect the tissue from the trauma of further handling, so it may be easier to cut during gross examination. Fixation allows the tissue to be more properly oriented in the cassette in preparation for paraffin embedding and microtomy. The use of formalin (Neutral buffered formalin) is used in most cases. However, certain specialized studies such as electron microscopy, histochemistry, and immunocytochemistry require specific fixation procedure.

The most important reactions for maintaining morphology in the fization of tissue for routine histopathology are those that stabilize the proteins. Fixatives have property of forming cross-links between proteins. Soluble proteins are fixed to structural proteins and thus rendered insoluble. This gives some mechanical strength that allows preservation of the shape and architectural pattern of the tissue.

Fixation preserves the tissue by stopping all activities so that the cells can be viewed under microscope. The tissue must be placed in the appropriate fixative as soon as possible after removal from the body. Leaving the tissue specimen in the air for prolong period of time will cause it to dry out resulting to morphologic distortion. Leaving the tissue in water (a hypotonic solution) will cause the cell to swell, while a strong salt (hypertonic solution) will cause the cell to shrink.

Fixation also prevents autolysis or postmortem decompostition. Every cell in the body has suicidal sac within its cytoplasm called lysosome. It contains hydrolytic enzymes and is released whenever a tissue is removed from the body, such as in surgical removal. In fixation, lysosomal enzymes are inactivated, or may be altered, stabilized chemically causing it to be insoluble. In addition, it protects the tissue from bacterial or fungal colonization overgrowth.

Main factors involved in Fixation1. Hydrogen Ion Concentration

Satisfactory fixation occurs between pH 6 to 8. Outside this range, changes may occur that are detrimental to ultrastructural preservation of the tissue.

2. TemperatureFor surgical specimen, it is traditionally processed at room temperature. Many laboratories used tissue processors that work at 40°C. For electron microscopy and some histochemistry, ideal temperature is 0-4°C.

3. Thickness of sectionStandard tissue size is 3 cm length, 2.5 cm width and 0.5 cm thick.

4. OsmolalityHypertonic solutions give rise to cell shrinkage. Isotonic as well as hypotonic fixatives cause the cell to swell and poor fixation. The best results are usually obtained using slightly hypertonic solutions (400-450mOsm; isotonic solutions

5. ConcentrationFormaldehyde is normally used as a 10% solution and glutaraldehyde is normally used as a 3% solution. The presence of a buffer causes polymerization of aldehyde, with consequent decrease in its effective concentration.

6. Duration of fixationPrimary fixation in buffered formalin is usually carried out for 2-6 hours during the day the specimen is obtained, but they may remain in fixative over the weekend without much adverse effect. Most of the formalin can be washed out after fixation for 24 hours. Prolonged fixation may cause shrinkage and hardening of tissues, and may severely inhibit enzyme activity and immunological reactions.

Effects of Fixatives in General1. They harden soft and friable tissues and make the handling and cutting of sections easier. This is

usually accelerated by the action of alcohol during dehydration process. 2. They make the cells resistant to damage and distortion caused by hypotonic and hypertonic

solutions used during tissue processing.3. They inhibit bacterial decomposition.4. They increase the optical differentiation of cells and tissue components thereby rendering a more

readily visible specimen during examination.5. They act as mordents or accentuators to promote and hasten staining, or they may inhibit certain

dyes in favor of another.6. They reduce the risk of infections during handling and actual processing of tissues.

Characteristics of a Good Fixative1. It must be cheap.2. It must be stable.3. It must be safe to handle.4. It must kill the cell quickly thereby producing minimum distortion of cell constituents.5. It must inhibit bacterial decomposition and autolysis.6. It must produce minimum shrinkage of tissue.7. It must permit rapid and even penetration of tissues.8. It must harden tissues thereby making the cutting of sections easier. 9. It must be isotonic, causing minimal physical and chemical alteration of the cells and their

constituents. (This is not, however, a strict rule since there are some hypotonic solutions, such as glacial acetic acid, producing tissue swelling, which are being used in conjunction with hypertonic solutions, such as picric acid, causing shrinkage of cells, to produce a compound which would give an optimal effect on the tissue structure.

10. It must make cellular components insoluble to hypotonic solutions and render them insensitive to subsequent processing.

11. It must permit the subsequent application of many staining procedures to facilitate easier and more profitable examination.

So far, no single fixative has yet been known to posses all the qualities of an ideal fixative solution. There are numerous fixatives available, and each has its own advantages and disadvantages.

Types of Fixative according to Action and Composition

According to COMPOSITION1. Simple Fixatives – are composed of only one component

a. AldehydesFormaldehydeGlutaraldehyde

b. Metallic FixativesMercuric chlorideChromate fixativesPotassium dichromateChromic acid

c. Lead fixativesPicric acidAcetic acid

AlcoholOsmium tetroxide (Osmic acid)Heat

2. Compound Fixatives – are those that are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon cells and tissue constituents.

According to ACTION1. Microanatomical Fixatives – are those that permit the general microscopic study of tissue

structures without altering the structural pattern and normal intercellular relationship of the tissues in question

10% Formol saline10% Neutral buffered formalinHeidenhain’s SusaFormol sublimate (Formol corrosive)Zenker’s solutionZenker-formol (Kelly’s solution)Bouin’s solutionBrasil’s solution

2. Cytological Fixatives – are those that preserve specific parts and particular microscopic elements of the cell itself.

a. Nucler Fixatives – are those that preserve the nuclear structures (e.g., chromosomes) in particular. They usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin. They have a pH 4.6 or less.

Flemming’s fluidCarnoy’s fluidBouin’s fluidNewcomer’s fluidHeidenhain’s Susa

b. Cytoplasmic Fixatives – are those that preserve cytoplasmic structures in particular. They must never contain glacial acetic acid which destroy mitochondria and Golgi bodies of the cytoplasm. They have a pH of more than 4.6

Flemming’s fluid without acetic acidKelly’s fluidFormalin with “post-chroming”Regaud’s fluid (Muller’s fluid)Orth’s fluid

3. Histochemical Fixatives – are those that preserves the chemical constituents of cells and tissues10% Formal SalineAbsolute Ethyl AlcoholAcetoneNewcomer’s fluid

FixativesSIMPLE FIXATIVES