Positive control wax strip and preparation method thereof
By mixing Mycobacterium abscess and Candida albicans, inactivate and embed them in paraffin, a positive control wax strip that can satisfy both acid-resistant staining, hexamine silver staining and PAS staining was prepared, and the solution was solved The problem of poor stability of positive control wax blocks in the prior art improves the detection consistency and reliability of pathological diagnosis.
Patent Information
- Application Number
- CN202411491590.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2025-02-07
AI Technical Summary
In pathological diagnosis, especially in the detection of tuberculosis and fungal infections, the prior art is difficult to provide stable positive control wax blocks, resulting in poor detection consistency and difficulty in obtaining and preserving positive control wax blocks.
By mixing Mycobacterium abscess and Candida albicans, inactivate and embed in paraffin, a positive control wax bar was prepared that satisfies both acid-fast staining, hexamine silver staining and PAS staining.
This method can effectively provide positive control wax blocks that meet three special dyeing needs, reduce the possibility of dyeing failure, improve the stability and reliability of detection, and simplify the preparation and preservation of positive control wax blocks.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pathology and relates to a positive control wax strip and a preparation method thereof. Background Art
[0002] In clinical pathological diagnosis, to confirm whether the lesion is caused by tuberculosis / fungal infection, it is necessary to find tuberculosis / fungus in the patient's body fluids and related tissue sections, which is the "gold standard" for diagnosis. Since it is not easy to observe tuberculosis / fungus in tissue cells on HE sections, special pathological staining is an important means of examination for the pathological diagnosis of tuberculosis and fungal infections. Ziehl-Neelsen staining can show tuberculosis and leprosy, and PAS and hexamine silver staining can clearly show fungi, which are widely used in pathological examination. Since most laboratory special staining is manual, it is easily affected by many factors such as reagent configuration, staining operation, and reagent storage, and the detection consistency is poor. When the tissue slice tested is negative, if there is no clear control, it is difficult to judge whether the tissue is originally negative or the staining experiment has failed. This problem is particularly prominent in special staining of tuberculosis and fungi. When caseous necrosis and / or granulomatous diseases are found under the microscope, they are usually considered to be related to tuberculosis or fungal infection. Diagnostic physicians often apply for three special stains for the same specimen: acid-fast staining, PAS staining, and hexamine silver staining. Since each staining requires different positive control photos, when there are a large number of special stains, the positive control photos are often taken incorrectly, resulting in staining failure. When the staining results are inconsistent with clinical considerations, physicians often doubt whether the special staining results are reliable. Therefore, clear positive controls are mounted next to the test tissue for staining to control the staining results throughout the process, which requires a large number of positive control tissues. In daily work, special staining positive control wax blocks are generally known positive tissue wax blocks, but tuberculosis patients are generally not diagnosed by biopsy, and the positive rate of acid-fast staining is only 20% to 40%. It is very difficult to find acid-fast staining positive control tissues with stable bacterial counts and can be repeatedly sectioned; some laboratories use the method of culturing tuberculosis to obtain a stable source of positive controls, but the tuberculosis culture cycle is long, the culture risk is high, and the laboratory requirements are high. Fungal infection specimens are relatively common in clinical practice, but fungal infections are either treated and special stains are negative; or the specimens themselves are small and the bacterial count cannot be guaranteed after repeated re-cutting. In addition, in order to ensure the integrity of the archived wax block tissue, the number of wax blocks that can be used as positive controls is obviously insufficient. Summary of the invention
[0003] The purpose of the invention is to provide a positive control wax strip and a preparation method thereof.
[0004] There is no method or technology for mixing two or more bacteria to make wax blocks in the prior art. The two bacteria selected by the present invention are easy to obtain in the laboratory, culture quickly, and do not require special culture. The morphology, size, and staining characteristics do not interfere with each other. Mycobacterium abscessus is positive by acid-fast staining, and is negative by hexamine silver staining and PAS staining, while Candida albicans is positive by hexamine silver staining and PAS staining, and is negative by acid-fast staining. The control wax block made by the present invention can at least meet the control of acid-fast staining, hexamine silver staining, and PAS staining at the same time, making up for the current situation where these three special staining controls are insufficient in daily use. A positive control wax sheet can meet the three special staining positive controls, which can effectively avoid the situation of carelessly taking the wrong control wax block and reduce the number of times the special staining is redone.
[0005] The present invention provides a method for preparing a positive control wax strip, comprising the following steps: 1) taking Mycobacterium abscessus and Candida albicans and inoculating and culturing them separately; 2) Mix the cultured Mycobacterium abscessus and Candida albicans in 10% neutral formalin to inactivate them; 3) centrifuging the inactivated system in step 2) to remove formalin, and then dehydrating with anhydrous ethanol; 4) centrifuging the system after dehydration in step 3) to remove anhydrous ethanol, adding xylene and shaking to mix; 5) The system treated in step 4) is heated, and embedding paraffin is injected and mixed, and the paraffin is sucked up with a pipette, and then cooled. After the paraffin solidifies, the pipette is cut to obtain a positive control wax strip.
[0006] In the above method, in step 1), the culture conditions are as follows: in a 5% carbon dioxide incubator, the culture temperature is 37° C., and the culture time is 48 h to 72 h.
[0007] In the above method, in step 2), the colony count ratio of the abscess mycobacterium and the candida albicans can be 5:2; since the colonies of candida albicans are significantly larger and easily soluble in 10% neutral formalin, in order to ensure that the bacterial amount is sufficient without overlapping, the present invention preferably uses a mixture of 4 colonies of candida albicans and 10 colonies of mycobacterium abscessus; The soaking time may be 6 to 10 hours.
[0008] In the above method, in step 3), the centrifugal speed may be 3500-4000 rpm, specifically 3800 rpm; The process of dehydration with anhydrous ethanol is as follows: centrifugation, discarding the supernatant, adding anhydrous ethanol, and oscillating to mix; The dehydration process with anhydrous ethanol may be performed 2 to 6 times.
[0009] In the above method, in step 5), the heating temperature may be 58-62°C, specifically 60°C.
[0010] The present invention also provides a positive control wax strip prepared by the above method.
[0011] The present invention further provides a method for using the positive control wax strip, comprising the following steps: heating a glass slide, contacting the positive control wax strip with the heated glass slide, melting the paraffin on the glass slide, and obtaining a comparison photograph; 2) Each of the photographs was stained using HE staining, phenol-basic fuchsin method, hexamine silver method, and periodic acid-leucofuchsin method to evaluate the bacterial count.
[0012] The present invention has the following beneficial effects: 1. The present invention uses common conditional pathogens - Mycobacterium abscessus and Candida albicans as raw materials for rapid culture, and utilizes the characteristics of each bacterium to produce a control wax block that can meet the needs of three special stainings (phenol alkaline fuchsin method, hexamine silver method, and periodic acid-colorless fuchsin method).
[0013] 2. This method is simple to prepare, does not require embedding and slicing, is suitable for large-scale production, is easy to preserve, and is highly practical. It can solve the problem of severe shortage of special staining control wax blocks in grassroots hospitals.
[0014] 3. The present invention does not require slicing and scooping, which can avoid partial tissue loss caused by slicing. Scooping may cause positional bias. The control bacteria are wrapped in a wax strip, and when used, it is only necessary to gently touch the positive control wax strip with the heated slide. Once the positive control wax strip is made, the positive control photo does not need to be completed by professional pathology technicians, and the operation is simple.
[0015] 4. The positive control wax block of the present invention, which integrates three special dyes, can also reduce the situation of carelessly taking the wrong control wax piece and improve the stability of special dyeing. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 HE staining shows that Mycobacterium abscessus and Candida albicans are both red (magnification 400 times).
[0017] Figure 2 Acid-fast staining shows Mycobacterium abscessus in red and Candida albicans in blue (magnification 1000x).
[0018] Figure 3 The hexadecene silver stain shows Candida albicans as black (magnification 400 times).
[0019] Figure 4 PAS staining shows Candida albicans as purple-red (magnification 400 times). DETAILED DESCRIPTION
[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0022] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings, in which preferred embodiments of the present invention are shown.
[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.
[0024] The special staining described in the present invention refers to the need to select a corresponding staining method for staining in order to clarify the special substances, lesions and pathogenic microorganisms that appear in the process of pathological diagnosis. The commonly used special staining method for tuberculosis bacilli is the phenol alkaline fuchsin method (modified Wade-Fite method), and the commonly used special staining method for fungi is the hexamine silver method (GMS method) and the periodic acid-leucofuchsin method (PAS method).
[0025] Phenol-alkaline fuchsin method (modified Wade-Fite method): The cell wall of Mycobacterium tuberculosis / Mycobacterium leprae contains varying amounts of lipids, which form a complex with phenol-alkaline fuchsin, which can resist decolorization by acid reagents and remains red after decolorization with sulfuric acid.
[0026] Hexamine silver method (GMS method): Chromic acid is used to oxidize the polysaccharides in the fungal wall to expose aldehyde groups, which then reduce the silver ions in the hexamine silver to black metallic silver and develop color.
[0027] Periodic acid-colorless fuchsin method (PAS method): Use periodic acid to oxidize the polysaccharides in the fungal cell wall to release aldehyde groups, which then combine with colorless fuchsin to form a new magenta complex and develop color.
[0028] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available (weak acid-fast staining kit (Zhuhai Beso Company, catalog number: BA4213), PAS staining kit (Fuzhou Maixin Company, catalog number: MST-8038), hexamine silver staining kit (Zhuhai Beso Company, catalog number BA4094)); unless otherwise specified, the experimental methods are conventional experimental methods in the art.
[0029] Example 1. Obtain fungi: Use Mycobacterium abscessus and Candida albicans cultured and identified by the Laboratory Department of Shenzhen People's Hospital. Both are cultured by transfer, without other bacteria, and the culture conditions are: 5% carbon dioxide incubator, 37℃, and culture for 72h.
[0030] 2. Bacterial fixation and inactivation: In a biosafety cabinet, gently scrape up 10 colonies of Mycobacterium abscessus and 4 colonies of Candida albicans and place them in 1.5 ml centrifuge tubes, add 1 ml of 10% neutral formalin, oscillate to mix, and fix and inactivate at room temperature for 6 hours.
[0031] 3. Centrifuge at 3800 rpm for 2 min and discard the supernatant; add 1 ml of anhydrous ethanol and shake to mix for 1 min.
[0032] 4. Centrifuge at 3800 rpm for 2 min and discard the supernatant; add 1 ml of anhydrous ethanol and shake to mix for 1 min.
[0033] 5. Centrifuge at 3800 rpm for 2 min and discard the supernatant; add 0.2 ml of xylene (to fully dissolve with the paraffin used in subsequent steps) and oscillate to mix for 1 min.
[0034] 6. Make control wax strips: Place the centrifuge tube in 60℃ water to heat in advance, inject 1ml of melted embedding paraffin, blow and mix with a disposable pipette, be careful not to produce bubbles, suck the paraffin completely into the pipette, maintain the pressure of the rubber head, place the pipette in ice water, and quickly solidify the paraffin. After 2 minutes, the paraffin is completely solidified, and the pipette is gently cut with a sharp blade. The wax strip is ready. For easy storage, divide the wax strip into about 3-5cm.
[0035] 7. Storage conditions: Randomly select 4 wax strips made in step 6 and divide them into two groups. Group A is stored in a sealed bag in a 4℃ refrigerator; Group B is stored in a wax block cabinet at room temperature (25℃). After 6 months of storage, the special staining of the two groups of wax blocks after sectioning is stable, the positioning is clear, and no bacterial denaturation or coloring ability decrease is observed.
[0036] The method for using the control wax strip prepared above comprises the following steps: heating the glass slide to about 60°C, gently contacting the wax strip with the glass slide, and the paraffin on the surface melts on the glass slide, and the subsequent steps are: baking the slide at 80°C for 20 minutes, dewaxing with xylene, eluting xylene with gradient alcohol, hydrating, and staining. application: Evaluation of bacterial count: Randomly select 2 wax strips, cut them 1 cm apart, and make 5 pairs of photos for each wax strip to evaluate bacterial count. According to the HE staining method, phenol alkaline fuchsin method (modified Wade-Fite method), hexamine silver method (GMS method), and periodic acid-colorless fuchsin method (PAS method), the specific results are as follows.
[0037] HE staining: The staining results are as follows Figure 1 As shown by Figure 1 It can be seen that the bacterial counts in all sections are similar and evenly distributed. In the low-power field of view, the round or oval ones are Candida albicans, which are of different sizes, red or purple, and the number is >50. The abscess mycobacterium is smaller in size, and red rods can be seen in the high-power field of view, and the number is >100.
[0038] Phenol basic fuchsin method (modified Wade-Fite method) staining: The staining results are as follows Figure 2 As shown by Figure 2 It can be seen that the abscess mycobacterium is red, the fungi are blue (counterstained with aniline blue), and the number of abscess mycobacteria in the oil microscope field of view is greater than 50.
[0039] Hexamine silver method (GMS method) staining: The staining results are as follows Figure 3 As shown by Figure 3 It can be seen that the fungi are black and of varying sizes, the background is light green (restained bright green), and the number of fungi in the high-power field of view is >30.
[0040] Periodic acid-leucofuchsin method (PAS method) staining: The staining results are as follows Figure 4 As shown by Figure 4 It can be seen that Candida albicans is purple-red, the background is light pink, and the number of fungi in the high-power field of view is >30.
Claims
1. A method for preparing a positive control wax strip, characterized in that: The method comprises the following steps: 1) taking Mycobacterium abscessus and Candida albicans for inoculation and culture separately; 2) Mix the cultured Mycobacterium abscessus and Candida albicans in 10% neutral formalin to inactivate them; 3) centrifuging the inactivated system in step 2) to remove formalin, and then dehydrating with anhydrous ethanol; 4) centrifuging the system after dehydration in step 3) to remove anhydrous ethanol, adding xylene and shaking to mix; 5) The system treated in step 4) is heated, and embedding paraffin is injected and mixed, and the paraffin is sucked up with a pipette, and then cooled. After the paraffin solidifies, the pipette is cut to obtain a positive control wax strip.
2. The method according to claim 1, characterized in that In step 1), the culture conditions are as follows: in a 5% carbon dioxide incubator, the culture temperature is 37° C., and the culture time is 48 h to 72 h.
3. The method according to claim 1 or 2, characterized in that: In step 2), the colony count ratio of the Mycobacterium abscessus to the Candida albicans is 5:2; The soaking time is 6 to 10 hours.
4. The method according to any one of claims 1 to 3, characterized in that In step 3), the centrifugal speed is 3500-4000 rpm; The process of dehydration with anhydrous ethanol is as follows: centrifugation, discarding the supernatant, adding anhydrous ethanol, and oscillating to mix; The dehydration is performed with anhydrous ethanol 2 to 6 times.
5. The method according to any one of claims 1 to 4, characterized in that In step 5), the heating is performed to a temperature of 58-62°C.
6. The positive control wax strip prepared by the method according to any one of claims 1 to 5.
7. A method for using the positive control wax strip according to claim 6, characterized in that: The method comprises the following steps: heating a glass slide, contacting the heated glass slide with the positive control wax strip, melting the paraffin on the glass slide, and obtaining a comparison photograph; 2) Each of the photographs was stained using HE staining, acid-fast staining, hexamine silver method, and periodic acid-colorless fuchsin method to evaluate the bacterial count.