Fixation refers to soaking the pathological tissue in the appropriate chemical reagents, with the help of chemical reagents, so that the protein in the tissue or cell condenses, precipitates or denatures into insoluble substances, and maintains the original morphological structure of the tissue and cell. Fixation is the most important step in the section preparation process, and a good histological section is based on timely and appropriate fixation.





Part one

immobilization

The primary goal of pathological fixation is to maintain clear morphological features (Eltoum et al 2001a, 2001b; Grizzle et al 2001).

Fixation is to penetrate some chemical reagents into the organs or tissues and cells that need to be preserved or made into slices, so that the substances contained in them can maintain the morphological structure, position and biological characteristics of the living state as far as possible.



Fixed purpose

① The morphology and structure of the cells remain similar to that of survival. Prevent cells from dissolving and destroying under the action of their own lysosomal enzymes, and autolysis occurs; Or due to bacterial reproduction caused tissue corruption.

② Keep the special components in the cell similar to the survival state. Fixation can not only precipitate or coagulate various components and pathological metabolites in the tissue, such as proteins, fats, sugars and pigments, but also preserve other carbohydrates, inorganic components and microorganisms after fixation, so that they can be located in the original parts of the cell, which is conducive to the exact positioning after staining. Different fixing fluids and fixing methods should be used for different substances.

③ Easy to distinguish different tissue components. Different substances in tissue cells have different refractive ratios after fixation, and have different affinities to dyes, which are easy to distinguish after dyeing.

④ is good for slicing. The fixed liquid has a hardening effect, which can harden the texture of soft tissue (such as brain) and solidify the colloidal object, so that the normal semi-liquid (sol) of the cell becomes an irreversible solid (gel), which is easy to make the production operation.

It is particularly important to preserve antigens and deoxyribonucleic acid (Deoxyribonucleic acid acidDNA) and ribonucleic acid (Ribonucleic acid acidRNA), especially for samples prepared for immunohistochemical staining and nucleic acid detection.



A common fixative

1. Simple fixative

(1) Formaldehyde:

The common concentration is 4%≈10% formalin.

Advantages: strong penetration, small shrinkage to the tissue, can make the tissue sclerosis, fixed tissue nucleus coloring;

Disadvantages: Closed antigen determinant family, improper use can form formalin pigment.

(2) Ethanol:

The best concentration is 80% ~ 95%.

Advantages: fixed, hardening, dehydration;

Disadvantages: Make the tissue brittle, obvious contraction, easy to appear uneven tissue fixation.
(3) Acetic acid:

Strong penetration, can make tissue expansion, generally not used alone.



2. Mix the fixing solution

Mixed fixative is used more in practical work. Its fixing effect is obviously better than that of single component fixing solution.

(1) 10% neutral formalin fixing solution:

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(2) ethanol-formaldehyde-glacial acetic acid (AFA) fixing solution:

Formaldehyde (40%) 100ml, 95% ethanol 850ml, glacial acetic acid 50ml



Precautions for fixing

1, timely fixation, cold ischemia time <30min, the earlier the better.

2, formaldehyde permeate the tissue speed of about 2-5mm/24h, fixed time at least 6-8h, preferably 12-48h, more than 48 hours can cause adverse effects, such as FISH failure.

3, the fixed liquid should be sufficient, generally 5-10 times the volume of the tissue.

4, to prevent tissue deformation.



The adverse impact of improper or inadequate specimen fixation on subsequent diagnosis or research is irreparable! Therefore, specimen fixation is an important part of the whole tissue specimen processing process!







Factors affecting fixed quality

1. Buffer and pH

In the weakly acidic environment, the fixing effect and cross-linking effect of formaldehyde will be weakened, and the metabolites of hemoglobin will undergo chemical changes to form brown and black insoluble birefringent crystal pigments. Pigment formation occurs at pH <5.7 and increases when pH is in the 3.0 to 5.0 range. Formalin pigments are easily recognized and generally do not affect the diagnosis of large amounts of hemoglobin breakdown products secondary to hematopoietic diseases. This pigment is easily removed in picric acid alcohol solution. In order to avoid formalin pigment formation, neutral buffer formalin is the preferred formaldehyde base fixative.

The effect of acetic acid and other acids is primarily to destroy the tertiary structure of proteins by lowering pH. The buffer keeps the solution at an optimal pH. The choice of specific buffer depends on the type of fixative and analyte to be analyzed. Commonly used buffers are phosphate, cacodylate, bicarbonate, bicarbonate, and acetic acid.

Formalin penetration rate:

2-3 mm / 24 hours (dense tissue)



2. Fixed time and specimen size

Medawar carried out some studies on the factors of diffusion of fixatives into tissues (1941). He found that a fixed depth (d) is proportional to the square root of a fixed time (t).

The formula is: d =k√t.

The constant (K) is the diffusion capacity coefficient, and the diffusion capacity of each fixative is specific. The coefficient is 0.79 for 10% methylaldehyde, 1.00 for 100% ethanol, and 1.33 for 3% potassium dichromate (Hopwood 1969).

For 10mm spheres, the fixative will not penetrate into the center of the sphere until 52 or 25 hours later. It is important to note that the various components of each composite fixative penetrate at different rates, so the specimen should be as thin as possible.

Penetration rate of dense tissue formalin:

2-3 mm / 24 hours



3. Fixed temperature

The diffusion of molecules increases with temperature because molecular activity and vibration accelerate at high temperatures. That is, the tissue permeability of formaldehyde is faster at higher temperatures, so microwave ovens are used in formaldehyde fixation to increase the temperature and speed of movement of gas molecules, however safety concerns need to be taken into account (Grizzle & Fredenburgh 2001.2005). Most chemical reactions also occur more rapidly at higher temperatures, so formaldehyde can react more rapidly with proteins (Hopwood 1985). The temperature of the fixed fluid in the closed tissue processor can be slightly higher than the room temperature, which is conducive to improving the fixed effect.



4. Concentration of fixative

The concentration of fixative is determined by the fixing effect and solubility. Formaldehyde concentrations above 10% tend to lead to an increase in the degree of tissue hardening and shrinkage (Fox et al 1985), while alcohol concentrations below 70% do not dehydrate tissue effectively.



5. Osmotic pressure and ionic composition of fixative

Osmotic pressure of buffer and fixative is very important; Hypertonic and hypotonic environments can lead to tissue contraction or expansion, respectively. The best solution is slightly hypertonic (400-450 mOsm); However, the osmotic pressure of 10% NBF should be about 1500 mOsm. Similarly, various ions (Na+, K+, Ca2+, Mg2+) affect the shape and structure of the cell regardless of the osmotic effect. Therefore, the liquid containing these ions should be as isotonic as possible to the tissue.



6. Selection and application of fixative

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7. Fixed timeliness

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8, the organization of fixed ways and methods

The specimen must be cut/cut/spread out for fixation.

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The container for fixing the specimen should be properly sized and airtight.

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Xiuwei specimen bag series




Remote sample processing

If the surgically removed specimen is far from the laboratory to be examined, the pathologist and technician should assist the surgeon and other operating room members to ensure that the specimen is not degraded by fixation with 10% neutral buffer formalin (NBF).

Breast cancer tissue specimens should be bisected before being immersed in 10%NBF for fixation.

Gastric cancer tissue specimens should be fixed on the specimen plate before being immersed in 10%NBF for fixation.

All specimens should be fixed in 10%NBF prior to transport.

The fixed start and end times for immersion of the tissue to be tested in 10%NBF should be recorded by the operator on the standardized histopathology application form.



9, the amount of fixed liquid to be sufficient

Fixed fluid to tissue volume ratio (at least 10:1).



10, Organize the fixed time to be appropriate

Depending on the laboratory situation, the fixed time is generally 8-48 hours.



Correct recording is an effective measure to ensure the fixed effect

It is important to record the sample excision to a fixed time;

The submitted specimens should be accompanied by a completed histopathological examination request form;

Responsibilities of surgeons and operating room staff:

Record the date and time of sample collection;

Responsibilities of pathologists and technicians:

Record a fixed start date and time;

Communicate effectively with multidisciplinary teams to ensure that records comply with operational procedures;

The application form should include:

Type of specimen;

Specimen location;

Suture position (for marking);

Imaging data (if available)