Problems, causes and countermeasures in the process of immunohistochemical staining

Good immunohistochemical staining section is the basis and premise for correct judgment of staining results. Because there are many steps or links in the immunohistochemical staining process, each step or link may affect the final result of the staining. Therefore, it is not very easy to prepare a high-quality immunohistochemical section. Pathological technicians and pathologists need to cooperate closely, coordinate and work together to ensure that qualified immunohistochemical sections are made. Although immunohistochemical staining can have various problems, from the result of staining, it can be generally divided into two categories: colorless film (ie, no positive signal) and "murmur" stained film (with positive signal). 1. After staining of the colorless film, no positive signal can be seen in the film. This is a relatively common phenomenon in routine work. There are two possibilities for this phenomenon: 1. True negative results: There is no problem in the entire staining process, and the tissue or cell does not express antibody-related antigens. 2. False negative result: This negative result is not a true reflection. False negative results can be divided into two cases: (1), the tissue or cells that are expected to be examined are not included in the slice at all. In this case, either the pathologist chose the wrong slice or the wrong antibody, or the technician chose the wrong wax block. Obtaining correct sections for staining is a prerequisite for obtaining correct results. This shows that making qualified immunohistochemical sections is not only a technician's job, but also a pathologist plays an indispensable role. (2) A problem occurred in one or some links in the dyeing process. For example, if the tissue is not repaired by antigen, some tissues must undergo antigen repair to detect antigen expression; or an antibody that can only be used for frozen tissue but not for paraffin-embedded tissue is selected; or the primary antibody fails, although antibody failure is theoretical The above is a gradual process, but occasionally encounters a sudden failure. The main reason is that the antibody has not been used for a long time and / or has expired. It can also be seen that a certain link is missed during the staining process, such as forgetting to add a secondary or tertiary antibody, or using two secondary antibodies without a tertiary antibody, or missing hydrogen peroxide when formulating DAB. In order to avoid this simple error, there is a simple method: at the end of the third antibody incubation, the third antibody on the slice is thrown on a white paper, and the prepared DAB is dropped on the white paper. , Observe whether there is brown. If it appears, it proves that the preparation process of the third antibody and DAB is correct. If this DAB is dropped on the slice again without any positive signal, the problem must be before the third antibody. If there is no brown reaction on the paper, the problem must be in the preparation of the third antibody DAB or DAB. This simple method can quickly help us find the possible cause of the problem. Solving the problem of negative staining is very simple, that is, setting up a "positive control". If the positive control has expression, it means that the whole process of staining and all reagents are no problem. If the test piece is still negative at this time, it is a true negative, indicating that the tissue or cell does not have the corresponding antigen expression. Conversely, if the positive control is not stained, it indicates that there is a problem with one or some steps in the staining process or with a reagent. The reasons should be sought one by one. There are two types of positive controls, one is called "self control" or "internal control", which refers to the presence of known antigens in the test slice itself, such as the presence of T and B cell antigens in normal lymph nodes, CD20 or CD3 There should be expression. The self-control is an ideal control. The control and the test tissue or cells are in the same slice and are under the same test conditions. The results are more reliable and comparable. When choosing your own pair of photos, it is best to choose a part that has both diseased tissue and normal tissue, which is helpful for comparison. The other is called "external control", and sometimes there is no known antigen in the test section. For example, if it is suspected to be malignant melanoma in the gastric specimen, it needs to be detected with HMB45 or Mart-1. In normal gastric tissues There is no relevant antigen in itself. If the lesion shows a positive result, it can still be a black evil. However, if a negative result occurs, it is impossible to determine whether the tissue contains no melanoma antigen or a technical problem. Therefore, an additional known positive control should be established. This positive control outside the test tissue is called "external control". In actual work, there are many cases where an external control needs to be set up. If each antibody has to choose a different positive control, the workload will be very large. In order to solve this problem, at present, there are units at home and abroad that integrate multiple different tissues into multi-tissue slices, "salami" and "spring roll" slices, tissue chips, etc., and their continuous slices are reserved for use. Can be used as a positive control. In addition, the simpler method is to use the appendix as a positive control, because the appendix contains many types of tissues compared with other tissues and organs in the human body, such as epithelium, lymphoid tissue, smooth muscle, interstitial, nerve, blood vessel, mesothelium and so on. An appendix slice can detect most commonly used antibodies. Setting up a positive control is the pathologist's task or responsibility, not the technician's responsibility. The pathologist observes the HE slice to see if there is a self-control in the slice. If not, he should tell the technician to use a positive control. Therefore, the role of pathologists in immunohistochemistry cannot be ignored. Antibody does not cover the test tissue: when many scattered small tissues are stained, a certain tissue stain may be missed. 2. Immunohistochemistry of "murmur" staining tablets In addition to normal true positive signals, we often encounter abnormal background coloring. These abnormal tinting are called "murmur" staining. There are many types of "murmur" dyeing, and the causes are also varied. For ease of explanation, the author summarizes them as the following. 1. Full-chip coloring Full-chip coloring means that the entire slice is stained with color, and the intensity of coloring can be deep or light. In short, it is not clear which tissues are positive and those tissues are negative. The reasons for this phenomenon are: (1), antibody concentration is too high: too high primary antibody concentration is one of the common reasons. The solution is to test the working concentration of each new antibody before using it, so that each antibody can be personalized and find the ideal working concentration for its own laboratory, even if it is a ready-to-use antibody, it ca n’t be simple According to the instructions for dyeing. (2) The antibody incubation time is too long or the temperature is high: The solution is to strictly implement the operating procedures. It is best to wear a timepiece or clock with you to remind you in time to avoid prolonging the time due to forgetting. The currently popular two-step method (Polymer) is highly sensitive, requiring the primary antibody to be incubated not for the traditional 1 hour, but for 30 minutes. Therefore, it should be adjusted according to the staining results. (3), DAB deterioration and color development time is too long: DAB is best used now, if there is sediment, it should be filtered before use. The prepared DAB should not be stored for too long, because in the absence of enzymes, hydrogen peroxide will also release oxygen atoms and react with DAB to reduce the effectiveness of DAB. Unused DAB is stored in the refrigerator for a few days. It is not advisable to use this seemingly economical method. The color development of DAB is best monitored under a microscope, and the reaction is terminated immediately when the desired degree of staining is reached. However, when there are too many stained sheets or when using a staining machine, this seems unrealistic, but at least some new or less used antibodies should be monitored for color development to avoid excessive color development time. (4) Dried tissues: Failure to replenish the fluid in a timely manner after the repair solution overflows, too many stained slices, too slow movements, forgetting to drip, and dripping of the drip are all reasons that cause the tissue to dry out. The solution is to operate carefully, using DAKO pen or PAP Pen to draw a circle around the tissue, which can effectively avoid the loss of liquid and increase the operation speed. (5). The immersion time of the slice in the buffer or repair solution is too long (greater than 24 hours): the reason is not clear, but the phenomenon exists. Some laboratories like to dewax the slices to repair the day before, and add antibodies for immunohistochemical staining the next day. If the container with slices and repair solution is placed in a 4oC refrigerator overnight, there is no obvious effect on the results. At room temperature, especially on hot summer days, background coloring will appear, so it should not be stored for too long. (6). Polyclonal antibodies with degraded primary quality and poor quality: pay attention to the expiration date of the antibody. The expired antibody is either not colored or the background is colored. When using a newly purchased antibody, it is best to set up a positive control and compare it with the used antibody. 2. Slice edge coloring Slice edge coloring is also a common phenomenon. This phenomenon is called the edge effect. Causes: (1). The edge of the tissue is not firmly attached to the slide, the edge tissue is loose and floats in the liquid, and it is not easy to wash the reagents under the tissue every time it is washed. Solution: Prepare high-quality film (APES or poly-lysine), cut out tissue slices as thin as possible, no thicker than 4 microns, tissue pre-treatment should be standardized, try to avoid the use of more necrotic tissue. (2) The reagents dropped on the slices do not fully cover the tissues, the reagents on the edges are easy to dry first, the concentration is higher than the central tissue and the staining is deep. Solution: The reagent should fully cover the tissue, which should be 2 mm beyond the edge of the tissue. When drawing a circle with DAKO pen, in order to avoid the influence of oil, the circle should be 3-4 mm away from the edge of the tissue. 3. The "yin and yang face" coloring refers to two staining results where the tissue is half-colored and half-uncolored, forming a clear or unclear junction. The reason is that the reagent only covers part of the tissue, but not all. For example, after adding the reagent, the flow of the reagent is not dispersed but concentrated on some tissues. Generally, after adding reagents, carefully check whether some tissues are not completely covered by the reagents. If this is the case, it is recommended to use a toothpick instead of a pipette tip or reagent bottle to drain the reagents to cover the tissues. . In addition, the staining cassette is not flat and the slices are inclined. Although the reagents have completely covered the tissues at the beginning, the reagents flowed to one side later, and some tissues were not covered by the reagents. For this kind of problem, as long as you pay attention or think of it, it is easy to find and easy to solve. Sometimes, when using a DAKO (or PAP) pen to draw a circle around the tissue, the scribe line is too close or drawn to the tissue. Due to the mechanical principle of the pen oil, the reagent cannot reach the tissue near the scribe line. There are also bubbles that can cause a distinct coloration of yin and yang, but the non-colored area is circular. Because the bubbles contain gas, the reagent is pushed to the surroundings. Therefore, the tissue in the center of the bubble is not colored. The solution is to add light reagents when dropping reagents, pierce with toothpicks when there are bubbles. 4. The coloring areas in the sliced ​​slices of the stove are in the east and west, and are distributed in the shape of a slice. The reasons for this problem are: (1) The water is not exhausted during the mounting, and bubbles are formed in the local area to make the tissue protrude and stain. When the reagent penetrates, it is not easy to wash out, and the color is too dark. The solution is that the air bubbles in the bleaching box should be exhausted, the film heating platform cannot be laid flat, and it should have a slope of about 45 degrees, which is conducive to water flow and evaporation. (2). Necrotic tissue foci, after tissue necrosis, cell destruction, enzyme release, protein free, decomposition, complex peptide chain residues (such as Fc segment) may be combined with the primary antibody and / or secondary antibody, resulting in final coloration. The solution is to avoid selecting sections with more necrotic tissue when selecting stained sections. (3) When making APES film, the concentration of the glue is too high. After drying, white dots are left on the slide, and the white dots are colored when the color is developed. The solution is to follow the standard preparation method, that is, 5% hydrochloric acid alcohol (5ml hydrochloric acid + 95% alcohol 95ml) soak the slide for 4 hours, rinse the slide with hot water for 1 hour, wash the slide for 1 minute with distilled water, and soak the slide in acetone 5 After 2 seconds, air dry (room temperature), 2% APES (2 ml APES + 98 ml acetone), soak the slide for 5 minutes, slide the slide through acetone (1-2 seconds), slide the slide through distilled water (1-2 seconds Clock), dry overnight at 37 ° C, and store at room temperature for later use. If in the process of making tablets, when acetone gradually volatilizes and the glue becomes thick, some acetone can be added appropriately. 5. The interstitial staining is mainly in the interstitial. There are many reasons for the interstitial staining. For example, the antibody and the protein in the tissue are colored due to the interaction of the hydrophobic group of the protein to form a non-specific connection. The serum before the primary antibody is added to block this step It is to avoid non-specific combination. Another example is that the immunoglobulin in the serum often leaks into the interstitial tissue, and it is easy to bind to the antibody, causing interstitial staining, especially when lambda and kappa staining. When the thyroid gland overflows into the interstitial tissue, interstitial staining will also occur when doing thyroglobulin staining. Interstitial staining can also occur when the antibody is impure or the antibody is contaminated. We have encountered CD20 antibody that is impure, in addition to B cells, but also infected with interstitial. 6. Cytoplasmic coloring Cytoplasmic coloring is the most deceptive coloring of all "murmur" staining. The coloring area is limited to the cell and the interstitium has no coloring. It looks almost the same as the real immune reaction coloring and is difficult to distinguish. The cytoplasm contains more protein, so many non-specific stains can also appear in the cytoplasm in addition to the interstitium. The coloring caused by this reason can be solved by blocking the serum. There are also stains caused by endogenous enzymes, such as hemoglobin (erythrocytes), myoglobin (myocytes), cytochromes (granulocytes, monocytes), catalase (liver, kidney), these available hydrogen peroxide Closed. Macrophages engulf various antigenic substances or Fc fragments and appear cytoplasmic staining, which is not easy to avoid, but macrophages can be recognized by morphology and attract attention. The coloring of endogenous biotin is the most deceptive, because it is widely present in tissue cells. Our results show that: endogenous biotin is present in frozen tissues, and biotin is blocked after formalin-fixed paraffin embedding , The endogenous biotin exposure caused by the heating antigen repair, the intensity of endogenous biotin exposure varies in different tissues, from weak positive (+) to strong positive (+++), endogenous biotin in The distribution form in the tissue is both scattered and diffuse. It is mainly present in the cytoplasm in granular form. Endogenous biotin is widely present in epithelial tissues, especially glandular epithelial tissues, and some non-epithelial tissues. , Endogenous biotin exists not only in human tissue but also in rat tissue. The intensity of endogenous biotin exposure is related to the repair fluid, and its strength increases in the order of: citric acid, EDTA, EGTA, heat antigen repair exposure endogenous Sex biotin can be blocked by egg whites, and the non-biotin detection system Polymer two-step method (EliVision, EnVision) can avoid biotin interference. 7. Inappropriate tissue treatment of nuclear nucleus can cause nuclear staining. For example, if the tissue is immersed in xylene for too long (such as from Friday to next Monday), the immersion time in the buffer is too long, the tissue becomes dry, and microwave repair solution The pH value and repair time are improper or the repair fluid is left too little during the repair process, and the tissue has not been passed. The solution is to work strictly in accordance with the operating routine. Source: Biotechnology Forum

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