Routine pathology HE production technology troubleshooting and precautions
First, the routine pathology HE production troubleshooting
1. Why does tissue dehydration start at a low concentration?
Answer: Ethanol is a colorless, transparent, flammable and hydrophilic liquid. It is easy to mix with other organic solutions. Dehydration is fast and reliable. Ethanol at all levels can be used for dehydration. If dehydration is carried out with ethanol, the initial concentration is preferably 70% aqueous ethanol, followed by 95% ethanol and 100% absolute ethanol. For particularly fragile tissue specimens, the initial concentration of ethanol used in the dehydration treatment is recommended to be 30% ethanol. High concentration of ethanol dehydration, easy to form hardening on the periphery of the tissue, commonly known as "polarization", hinder the penetration of the fixed solution, affecting the normal dehydration of the tissue. Therefore, the correct way to organize dehydration is to start from a low concentration of ethanol, step by step. Except for special needs.
2. How to prevent or reduce tissue slice holes?
A: This situation not only affects the aesthetics of the slice, but more importantly, it is easy to cause omissions to the pathologist's diagnosis. If the wax band is continuously deep-cut, the cavity becomes smaller and smaller, indicating that the cavity in the slice is artificially changed without being cut. When the force for cutting the wax block is too strong and too strong, small spots and small plaques in the tissue will fall off the wax block, leaving a small hollow in the slice. This situation is highly prone to occur in tissues that are dehydrated, transparent, or over-waxed. For example, specimens of the liver, spleen, kidney, brain, lymph nodes, and uterus, if taken too thin, are prone to over-treatment, and the wax blocks often appear hollow during roughing and slicing. Therefore, the thickness of the specimen should be moderate and not too thin. If you encounter such a wax block during slicing, you can cool the wax block on the ice tray for a while or wet it with wet cotton. The wax block should not be too strong or too heavy to contact the blade to reduce the occurrence of this artifact. If it is found that there is a hole in the tissue section on the wax band, as long as the tissue in the wax block is sufficient, it should be slowly sliced ​​as much as possible until the cavity disappears. In addition, the void may also be caused by the presence of residual air in the wax liquid when the tissue is immersed in wax. This condition is particularly common in lung tissue specimens. Even if you slice a hole in a row, it does not easily disappear.
3. What are the factors that affect the tissue slice?
Answer: 1) The nature of paraffin, such as the hardness of wax, the melting point of wax and the additives of wax, especially some additives with hardening properties. 2) The thickness of the slice. 3) The water temperature of the piece. For most paraffin waxes, the temperature of the bleaching oven is generally set at 40 ° C ~ 44 ° C, if the temperature is higher than 45 ° C, the wrinkles of the slice will disappear quickly, and the paraffin will dissolve. 4) For some slices that are prone to breakouts, you can make a second shot. To do this, place the tissue section on a glass plate with 30% ethanol and gently push it into the floater. Alternatively, place the tissue section in a container filled with cold water, then pick up the slides and gently move to the floater. In addition, the tissue section is erected vertically, so that the bottom edge first contacts the surface of the drifter, and then the scorpion is gently pushed to the right and outside, and the smooth surface of the tissue slice is smoothly touched by the water surface. This can reduce the occurrence of bubbles on the surface of the water due to pulling and rolling the sheet.
4. What is the cause of staining white spots in tissue sections?
A: After the dewaxing step is completed, a large white spot appears. If these spots are not found or removed in time, the stain will show irregular staining or uneven dyeing changes after staining. The reason is that the temperature of the baking (bake) sheet is too low, the slide is not fully baked (baked) before dewaxing; the slicing time in the xylene is insufficient, or the xylene is used too long, resulting in dewaxing. Treatment strategy: If the slide is not baked (baked), first remove the water from the slide with anhydrous ethanol, and then remove the paraffin with xylene. If the phenomenon of insufficient drying (bake) is serious, it may cause the slices to fall off, and it cannot be remedied. If the white spot is caused by insufficient retention time of the xylene in the slice, or if the xylene used is too long, the slice needs to be returned to the xylene for a relatively long residence time, or the xylene may be replaced, decolorized, bleached, and re-stained.
5. How to understand the concept of tissue dehydration?
A: Today's technicians are increasingly aware that organizational over-dehydration is not really excessive. Because some people have used repeated experiments to show that tissues with long dehydration are beneficial for slicing, such as autopsy specimens. Therefore, the excess here is not due to the excessive dehydration of the organization, but may be because the organization is either poorly fixed or insufficiently dehydrated, resulting in a certain amount of water inside the tissue. When it is transparent to xylene and paraffin is saturated, Hardening caused by the action of temperature.
6. Is the tissue wax block frozen in the refrigerator or soaked in cold water?
A: The optimum temperature for the tissue block is not static. Placing the tissue block in the freezer or cooling the tissue with ice helps to cut hard tissue because it maintains a uniform hardness between the tissue and the wax block. If the tissue is placed in cold water, the hardness can be uniformly lowered, and the humidity of the tissue block can be appropriately increased. The use of spray coolant to cool the surface of the tissue block is not ideal. If it is necessary to use it, try to spray only a small amount to prevent the surface of the tissue block from freezing or the entire tissue block from cracking.
7. How to choose the paper used to wrap small tissues?
A: Try to choose a good quality paper or tea bag wrapping paper, and choose less paper or gauze with poor texture and adhesion to small tissues to affect the quality of the slices. In addition, the paper bag should not be too fair, so that when the wax is buried, the technician can hardly open the paper bag because he cannot find the paper corner. It is best to use the "package chaos" method when wrapping small tissue specimens, and it is easy to open the package.
8. What is the reason for the “terraced†changes in tissue sections?
A: “Tallfields†actually refers to the corrugated changes in tissue sections. Most of these cases are due to the fact that the wax block is not clamped or the blade is not tightened on the microtome. In addition to this, there may be other reasons, such as the looseness of the aging machine of the slicer. In addition, the ripple may also be due to the excessive inclination of the knife. Ripple often occurs in hard tissue wax blocks, such as uterine fibroids. The corrugated tremor visible to the naked eye can be seen in the float sink. For hard or tough tissue wax blocks, the surface of the wax block can be softened with 5N hydrochloric acid to improve the quality of the slices. Methods as below:
(1) Place the tissue wax block with the cracks in the slice face down, place it in a glass container or plastic container with 5 N hydrochloric acid, soak for 5 min to 10 min, remove the hydrochloric acid on the surface of the wax block with a dry gauze after taking out.
(2) The wax block was not frozen, and it was directly sliced ​​in a microtome at room temperature (25 ° C), and the thickness of the slice was 4 μm.
(3) When smashing, first place the sliced ​​face down in cold water, then use a clean glass slide to move it into the sink of the floater. The temperature is about 45 °C ~ 47 °C; slightly for a while, After the slices have been fully flattened, they are quickly attached to the slides.
(4) Conventional baking/baked slices, dyeing, and sealing. Most of the chatter or microvibration is caused by excessive dehydration during tissue processing. But it may also be because the knife is blunt, the angle is too large, or the slicing speed is too fast. If the tissue is indeed caused by excessive dehydration, it can help to alleviate this phenomenon by wiping the surface of the wax block with wet gauze or cotton with some warm water. For example, the lymph node tissue itself lacks water. If the material is cut too thin, the tissue will be over-treated and hardened when it is dehydrated. It will cause chattering or micro-vibration when slicing, and destroy the complete structure of the lymph nodes, making it difficult for pathologists. Make a correct pathological diagnosis and delay the patient.
9. Is there a remedy for decalcification when the embedded tissue is incomplete?
Answer: The method is as follows: (1) Place the tissue wax block with the crack in the slice face down, place it in a glass container or plastic container with 5 N hydrochloric acid, soak it for 5 min to 10 min, remove it and wipe off the wax with dry gauze. Hydrochloric acid on the surface of the block. (2) The wax block was not frozen, and it was directly sliced ​​in a microtome at room temperature (25 ° C), and the thickness of the slice was 4 μm. (3) When smashing, first place the sliced ​​face down in cold water, then use a clean glass slide to move it into the sink of the floater. The temperature is about 45 °C ~ 47 °C; slightly for a while, After the slices have been fully flattened, they are quickly attached to the slides. (4) Conventional baking/baked slices, dyeing, and sealing.
Second, the precautions in the operation process
1. Pay attention to the "secondary waxing" when the tissue is embedded.
Specific precautions for embedding operation: first take a stainless steel model of appropriate size, inject wax liquid on the hot stage, the liquid surface is just flush with the upper edge of the model, and should not be lower or higher than this limit. Remove the tissue in the embedding box. After correcting the correctness, put all the tissues into the bottom of the model with clean hot tweezers. After the tissue is fully integrated with the wax in the model, arrange the model and then quickly move the model to the cold station. The bottom paraffin wax has just begun to solidify, and the tissue is gently pressed with tweezers for a few seconds. After the tissue is buried, the waxing solution is quickly covered with an embedding box for about 10 s to 15 s. After the embedding box and the wax liquid around the model are solidified, Refilling a little wax solution, that is, performing "secondary waxing" to increase the stability of the embedding box, and preventing the slice tremor caused by loosening of the embedding box during slicing. Note that the liquid level should not be higher than the upper edge of the embedding box. When the embedding cassette is covered, it must be kept parallel to the plane of the bottom of the model, without tilting from front to back. When embedding the tissue in a plastic embedding box, it should be noted that after the paraffin wax at the bottom of the embedding model begins to solidify, the embedding box is covered, and after it is solidified around, the "secondary waxing" is performed. The purpose of this is to make the embedding box more securely in contact with the tissue wax block, reducing and preventing chattering during slicing or detachment of the embedding cassette from the tissue wax block. In addition, it should be noted that the secondary waxing can not be overfilled, otherwise the wax block can not completely match the microtome fixture, causing loose tremor during slicing and affecting the quality of the slice.
2, pay attention to "rough repair" when organizing slices
First, the slicing work process is divided into two steps: preparatory slicing and formal slicing, ie roughing and fine cutting. Secondly, the angle of the microtome should be checked and fixed every day before slicing, that is, the angle of the knife seat is about 10°, the upper and lower sides of the clamp are parallel to the cutting edge, and the left and right sides are perpendicular to the cutting edge. All angles in the preparation and formal sectioning are consistent and must not be changed at will.
Prepare the slice: Apply the rotary microtome (with the second-stage sampler) to install the old disposable blade, and remove the excess paraffin and uneven tissue on the surface of the wax block until the tissue is exposed to the maximum surface. Brush and refreeze for formal slicing. In the case of calcified or hardened tissue, the surface of the wax block may be softened with 5N hydrochloric acid.
Formal sectioning: Clean the microtome with a brush and replace it with a new one. The thickness of the slice is usually specified to be 4 μm, and the integrity of the slice requires uniform tissue, uniform thickness, smooth and wrinkle-free, and no knife marks. The smooth, non-contaminated slices are placed on the surface of the water, and the slides are wiped clean with gauze. When the slices are taken, the slices are parallel to the slide along the long axis. The warm water of the drifter must be changed daily and kept clean, and the temperature range is between 45 °C and 49 °C. Write the correct number on the slide and quickly put it into the oven. The temperature of the oven is set to about 80 °C ~ 90 °C. It is usually baked for 15 minutes to 30 minutes.
3, the choice of slicing tools, tweezers and brushes
Tweezers recommends using the dental 20CM long bend used in the dentistry to facilitate the operation of the sliced ​​piece. The ophthalmology curve is too small and the operation flexibility is lower than the former. It is recommended to use oil brush and water chalk for the brush. The ordinary brush is loose and easy to puncture the tissue section, which is not conducive to operation.
4, the operation of the hand wheel during the roughing of the slice
It is recommended to recommend full rotation of the handwheel instead of the handwheel. When the handwheel turns up and down halfway, the wax block is not easy to "sickle"
5, the safety operation when slicing
The microtome is generally equipped with a handwheel lock that locks the handwheel in any position to prevent accidental access to the handwheel during idle periods, resulting in accidental injury or colliding of the tool. The more user-friendly slicer is often equipped with a second handwheel lock, and the handwheel handle can only be locked at the highest point, making it easy to change samples or tools at any time during the slicing process.
Regardless of the steel tool holder or the disposable blade holder, it should be equipped with a strong knife guard. When changing samples or when the machine is idle, the technical operator must develop good habits, covering the knife guard over the blade, for personnel and Tools are a kind of protection. A good knife guard must safely cover the entire length of the blade.
6, the storage of disposable sharp blades such as sliced ​​on-site
Safety is based on good organization, and in a clean and tidy laboratory, there are few opportunities for accidents. After the specimen is processed, the work surface and the equipment should be disinfected in time. After the disposable blade is used up, once the glass product is damaged, it should be discarded in the container marked with the word "sharp".
7, fishing tablets and lifting
The action of the slice is the feeling of holding up the tissue slice with a slide, and it is easy to leave a lot of water on the slide. The action of the lifting piece is to lift the slide glass forward and upward, and pick up the tissue section. This method has little residual water on the glass slide.
8, baking sheet and baking sheet
The tissue slice is placed on the hot table for heating, which is called the baked sheet; the space with the heat source is heated, which is called baking sheet. Baked slices require the tissue to be sliced ​​and then inverted to drain the water. After the tissue is slightly whitened, place the hot plate for 5-10 minutes. This method requires that the tissue slice can not carry water, and the tissue slice is prevented from spreading due to the moisture of the tissue. When the sheet is picked up, the tissue section can be erected and inserted into the dyeing rack and placed in the box to be heated. The effect of moisture is not too great.
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