WO2021189672A1 - 用于红斑狼疮分型的皮肤组织免疫细胞原位检测试剂盒及其应用 - Google Patents

用于红斑狼疮分型的皮肤组织免疫细胞原位检测试剂盒及其应用 Download PDF

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WO2021189672A1
WO2021189672A1 PCT/CN2020/095791 CN2020095791W WO2021189672A1 WO 2021189672 A1 WO2021189672 A1 WO 2021189672A1 CN 2020095791 W CN2020095791 W CN 2020095791W WO 2021189672 A1 WO2021189672 A1 WO 2021189672A1
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cd11b
dapi
antibody
staining
monoclonal antibody
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French (fr)
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陆前进
吴海竞
李倩文
陈凯丽
赵明
胡凯
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中南大学湘雅二医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the invention belongs to the technical field of immune detection, and specifically relates to a skin tissue immune cell in situ detection kit for lupus erythematosus typing and its application.
  • Lupus erythematosus (Lupus erythematosus, LE) is a chronic autoimmune disease that involves multiple factors and seriously affects the quality of life and endangers health. More than 80% of patients may have skin involvement at the beginning or in the progression of the disease. LE is a spectrum disease. One end is cutaneous lupus erythematosus (CLE), which is chronically benign and the lesions are mainly confined to the skin; the other end is systemic lupus erythematosus (SLE), which is more acute Onset, lesions can involve multiple systems, and severe cases can be life-threatening.
  • CLE cutaneous lupus erythematosus
  • SLE systemic lupus erythematosus
  • DLE is the most common and typical type of CLE. Clinically, 50%-85% of lupus erythematosus patients with skin involvement have persistent discoid lesions. SCLE is the most light sensitive CLE in the subtypes of lupus erythematosus, and it often manifests as ring-shaped plaques or papules and scaly lesions. ACLE is generally considered to be the manifestation of skin involvement in SLE patients. It is the most serious type of lupus erythematosus. Its typical skin lesions are erythema with butterfly-shaped distribution on the face. Most patients have multiple systems involved when they develop.
  • Immune cells mainly include T lymphocytes (CD4 + T cells and CD8 + T cells), B lymphocytes, granulocytes, NK cells, monocytes/macrophages, etc.
  • T lymphocytes CD4 + T cells and CD8 + T cells
  • B lymphocytes granulocytes
  • NK cells monocytes/macrophages, etc.
  • DLE skin lesions there are a large number of B cells in the inflammatory infiltrated area. Compared with normal skin, the inflammatory infiltration of SLE and SCLE skin lesions did not show a significant increase in B cells.
  • NK cell-related genes may be specific genes for skin inflammation in patients with DLE.
  • the prepared single-cell suspension is often difficult to perform flow analysis due to the small number of cells, and it is easy to contaminate and degrade, which makes it difficult to complete sequencing.
  • the results obtained by methods such as streaming lack the corresponding spatial distribution information, and the organization morphology data cannot be displayed intuitively.
  • the present invention designs a detection kit for the combination of four antibodies of CD4, CD8, CD19, and CD11b.
  • CD4 is a characteristic molecular marker of CD4 + T lymphocytes
  • CD8 is a characteristic molecular marker of CD8 + T cells
  • CD19 is a characteristic molecular marker of B lymphocytes
  • CD11b is a characteristic molecular marker of granulocytes, NK cells, and monocytes. / Molecular markers shared by macrophages.
  • Simultaneous detection of the four molecular markers CD4, CD8, CD19, and CD11b in a tissue section will obtain the expression and distribution of most immune cells involved in the occurrence of lupus erythematosus skin lesions.
  • the quadruple quantitative detection results of CD4-CD8-CD19-CD11b can be very good Distinguish discoid lupus erythematosus, subacute lupus erythematosus, systemic lupus erythematosus and healthy controls. Results At the same time, it meets the needs of clinical tissue morphology observation and quantitative detection of immune cell expression. It is simple and quick to operate, economical and applicable, and suitable for application and promotion in clinical and scientific research.
  • the primary objective of the present invention is to provide an immunostaining kit for in-situ labeling of skin tissue.
  • the present invention uses the simultaneous detection of the four molecular markers of CD4, CD8, CD19, and CD11b to obtain the expression and distribution of most immune cells involved in the occurrence of lupus erythematosus skin lesions, and is based on the quadruple of CD4-CD8-CD19-CD11b
  • the test results are different between lupus erythematosus and normal people, as well as between the various types of lupus erythematosus.
  • the designed supporting product fills the gap in the clinical diagnosis of lupus erythematosus kit.
  • the detection result of CD4-CD8-CD19-CD11b obtained by the kit of the present invention combined with Mantra multispectral imaging software, realizes the detection of the distribution and expression of immune cells with high accuracy, and can effectively distinguish the three subtypes of lupus erythematosus , High sensitivity and good specificity.
  • the kit of the present invention includes reagents required for detecting CD4, CD8, CD19, and CD11b positive immune cells by in-situ labeling immunostaining.
  • the reagents required for detecting CD4, CD8, CD19, and CD11b positive immune cells include: antigen retrieval solution, non-specific blocking agent, serum blocking agent, antibody diluent, monoclonal antibody, detergent, horseradish peroxide Enzyme-labeled secondary antibody, tyramine fluorescent stain and nuclear stain.
  • the antigen retrieval solution in the kit of the present invention includes an acidic antigen retrieval solution with pH 6.0 and/or an alkaline retrieval solution with pH 8.0.
  • the non-specific blocking agent is a 0.5% periodic acid solution alone or a combined 0.5% periodic acid solution and 3% H 2 O 2 solution, preferably a 0.5% periodic acid solution.
  • the key to the dyeing reaction of the kit is to use the horseradish peroxidase on the secondary antibody to catalyze the activation of the dye to develop color.
  • the presence of a large number of endogenous peroxidases in skin tissues (in the epidermis, blood vessels, and glands) will interfere with the specificity of the final color, which will affect the accuracy of signal analysis. Therefore, a suitable and effective non-specific blocking agent is essential to obtain specific detection results.
  • the most commonly used non-specific blocking method for immunostaining is 3% H 2 O 2 incubation for 15 minutes at room temperature.
  • the comparison is made with 3% H 2 O 2 blocking alone, 0.5% periodic acid solution alone, and combined use
  • the 3% H 2 O 2 and 0.5% periodic acid solutions were incubated for the same time (15 min at room temperature), which had the effect of reducing non-specific staining.
  • the results showed that the blocking effect of 3% H 2 O 2 alone was the worst, while There is no obvious difference between 0.5% periodic acid solution alone, 3% H 2 O 2 and 0.5% periodic acid solution in combination. Therefore, for the convenience of operation and saving reagent cost, the non-specific
  • the blocking agent is preferably a 0.5% periodic acid solution.
  • the serum blocking agent components include bovine serum albumin (Bovine Serum Album, BSA), phosphate buffer saline (PBS), Tween-20 (Tween-20), and polyethylene glycol octane. Benzyl Ether (Triton X-100).
  • BSA bovine serum albumin
  • PBS phosphate buffer saline
  • Tween-20 Tween-20
  • polyethylene glycol octane Triton X-100.
  • the effect of different serum blocking time on the staining positive rate was compared (15min, 30min, 60min, 90min), and it was found that there was no difference in staining positive rate between room temperature serum blocking for 15 minutes and room temperature serum blocking for 30 minutes, while room temperature serum blocking for 1 hour and 90 minutes In the two groups, the positive rate of staining decreased, indicating that too long serum blocking will block some specific binding sites, which will lead to false negative staining. Therefore, the serum blocking time here is 15-50min, preferably 15-30min, and more
  • the antibody diluent components include bovine serum albumin (Bovine Serum Album, BSA), phosphate buffer saline (phosphate buffer saline, PBS).
  • BSA bovine Serum Album
  • phosphate buffer saline phosphate buffer saline
  • the detergent components include phosphate buffer saline (PBS), Tween-20 (Tween-20), and polyethylene glycol octyl phenyl ether (Triton X-100).
  • PBS phosphate buffer saline
  • Tween-20 Tween-20
  • Triton X-100 polyethylene glycol octyl phenyl ether
  • horseradish peroxidase-labeled secondary antibody is a goat anti-rabbit/mouse horseradish peroxidase-labeled ready-to-use secondary antibody.
  • the tyramine fluorescent dye includes Opal520 dye solution, Opal570 dye solution, Opal650 dye solution, and Opal690 dye solution.
  • the tyramine fluorescent stain is matched with the specific antibodies in the monoclonal antibody group one by one to balance the signal intensity of each detection target in the sample to be detected after the fluorescent stain is bound to the fluorescent stain.
  • the monoclonal antibody in the kit of the present invention includes:
  • each monoclonal antibody staining sequence, antigen retrieval conditions, dilution ratio range, incubation time, corresponding secondary antibody incubation time, corresponding matching The high-degree tyramine fluorescent stain and the incubation time of the stain are as follows:
  • the first round of staining antibody CD11b monoclonal antibody; its antigen retrieval conditions: acidic antigen retrieval solution high pressure retrieval or acidic antigen retrieval solution microwave retrieval, preferably acidic antigen retrieval solution microwave retrieval; its dilution ratio range: 1:1000-1 :2000, followed by primary antibody and secondary antibody dressing, and then dressing with dye.
  • the CD11b monoclonal antibody dilution ratio is preferably 1:1400-1:1600, and more preferably 1:1500.
  • Primary antibody incubation time 1 hour at room temperature.
  • Secondary antibody incubation time 10min at room temperature.
  • Correspondingly matched tyramine fluorescent stain and incubation time of the stain Opal570 stain is preferred, incubation at room temperature for 10 minutes, and washing with detergent;
  • the second round of staining antibody CD8 monoclonal antibody; its antigen retrieval conditions: alkaline antigen retrieval solution high pressure repair or alkaline antigen retrieval solution microwave repair, preferably alkaline antigen retrieval solution high pressure repair; its dilution ratio range: 1: 2-1:5, incubate with primary antibody and secondary antibody in turn, and then use dye to incubate.
  • the CD8 monoclonal antibody dilution ratio is preferably 1:2-1:4, and more preferably 1:3.
  • Primary antibody incubation time 1 hour at room temperature.
  • Secondary antibody incubation time 10min at room temperature.
  • Correspondingly matched tyramine fluorescent stain and incubation time of the stain Opal 650 stain is preferred, incubation at room temperature for 10 minutes, and washing with detergent;
  • the third round of staining antibody CD4 monoclonal antibody; its antigen retrieval conditions: alkaline antigen retrieval solution high pressure repair or alkaline antigen retrieval solution microwave repair, preferably alkaline antigen retrieval solution high pressure repair; its dilution ratio range: 1: 2-1:5, incubate with primary antibody and secondary antibody in turn, and then use dye to incubate.
  • the CD4 monoclonal antibody dilution ratio is preferably 1:2-1:3, and more preferably 1:2.
  • the preferred incubation time for the primary antibody is 70-80min at room temperature, preferably 75min.
  • the secondary antibody incubation time is 15-25min at room temperature, preferably 20min at room temperature.
  • Opal520 stain is preferred, 25-35min at room temperature, preferably 30min, washing with detergent;
  • the fourth round of staining antibody CD19 monoclonal antibody; its antigen retrieval conditions: alkaline antigen retrieval solution high pressure repair or alkaline antigen retrieval solution microwave repair, preferably alkaline antigen retrieval solution high pressure repair; its dilution ratio range: 1: 200-1:500, followed by primary antibody and secondary antibody coating, and then using dye coating.
  • the CD19 monoclonal antibody dilution ratio is preferably 1:250-1:350, more preferably 1:300.
  • Primary antibody incubation time 70-80min at room temperature, preferably 75min.
  • the secondary antibody incubation time is 15-25min at room temperature, preferably 20min at room temperature. It corresponds to the matching degree of tyramine fluorescent stain and the incubation time of the stain: Opal690 stain is preferred, 15-25min at room temperature, preferably 20min, and detergent wash.
  • the room temperature range is 20-27°C.
  • nuclear counterstaining is used to detect all cells: an acidic antigen retrieval solution is used for microwave repair, and DAPI (4',6-diamidino-2-phenylindole) is used for staining. Specifically, drop nuclear stain on the slices, incubate for 10 minutes, mount the slices, and detect with a Mantra multispectral imager.
  • the monoclonal antibody skin tissue staining is prone to non-specific staining. Therefore, after the antigen retrieval, the monoclonal antibody needs to be blocked with a non-specific blocking agent before the primary antibody is applied. Wash with detergent, then block with serum blocking agent at room temperature, the blocking time of serum is 15-50min, preferably 15-30min, more preferably 15min.
  • the sample to be tested in the present invention is a paraffin section of the skin injury of the exposed part of the lupus erythematosus patient (head, face and neck and the front end of both upper limbs).
  • the skin tissue in-situ labeling immunostaining kit of the present invention performs multiple antibody staining
  • its preparation principle is that the added tyramine fluorescent stain (in an inactive state when added) is carried by the horseradish contained in the secondary antibody Peroxidase is catalyzed and activated, and then covalently coupled to the tyrosine residue of the epitope, thereby obtaining a stable binding epitope-fluorescent dye complex, and between the primary antibody and the epitope, the secondary antibody
  • the binding with the primary antibody is non-covalent and can be eluted by microwave or high pressure treatment.
  • the second objective of the present invention is to provide the application of the above-mentioned skin tissue immune cell in situ detection kit in preparing preparations for assisting the diagnosis and/or typing of lupus erythematosus.
  • the present invention is based on the first discovery that the total number of four positive immune cells in skin tissue, CD4 + cells, CD8 + cells, CD19 + cells and CD11b + cells, accounted for the total number of cells in the inflammatory infiltration area (DAPI stained cells) under a 200-fold visual field.
  • DAPI stained cells a inflammatory infiltration area
  • the percentage of the total number of CD4 + cells, CD8 + cells, CD19 + cells and CD11b + cells in the total number of cells is used for auxiliary erythema Lupus diagnosis and/or classification.
  • the present invention is specifically based on ROC curve analysis to obtain specific diagnosis and classification critical values for judgment.
  • samples to be tested are all detected in the dermal layer inflammation infiltration area under a single field of view under a 200-fold electron microscope to ensure that the dermal layer inflammation infiltration area covers the entire field of view.
  • the present invention uses the above-mentioned reagents to detect the quadruple detection result of CD4-CD8-CD19-CD11b ((CD4+CD8+CD19+CD11b)/DAPI), which is used to assist the diagnosis and/or analysis of lupus erythematosus
  • CD19 single positive test result (CD19/DAPI) and the CD8-CD19 dual test result ((CD8+CD19)/DAPI) can be used as auxiliary test conditions, and CD4-CD8-
  • the combined application of CD19-CD11b quadruple detection results can further improve the accuracy of the diagnosis of lupus erythematosus.
  • the critical value for the diagnosis and/or classification of lupus erythematosus after the above-mentioned reagent detection is as follows:
  • the present invention has been verified by samples and found that when the quadruple detection result of CD4-CD8-CD19-CD11b is between 77.7% and 86.5%, there is a certain probability that SCLE will be misjudged as DLE. In this case, the single positive detection of CD19 is combined Results (CD19/DAPI) can improve the diagnostic accuracy.
  • the patient can be judged
  • the source of the test sample is DLE; when the quadruple test result of CD4-CD8-CD19-CD11b is between 77% and 86.5%, and the single positive test result of CD19 is less than 12.1%, the source of the test sample can be judged
  • the patient is SCLE;
  • the patient from the source of the test sample is determined to be SCLE; when the quadruple of CD4-CD8-CD19-CD11b When the test result is between 55.4% and 67.7%, and the CD19 single positive test result is less than 2.5% and/or the CD8-CD19 dual test result is less than 25.2%, it is determined that the patient from which the sample to be tested comes from is SLE.
  • the third objective of the present invention is to provide the application of the above kit in the study of various types of lupus erythematosus and/or the characteristics of normal skin immune cells for non-diagnostic purposes.
  • the wax film covering the tissue will affect the subsequent epitope restoration and the combination of the primary antibody and the secondary antibody, which will result in the failure of staining. Therefore, during the use of the kit, when the indoor temperature is low, the turpentine used for dewaxing should be preheated to ensure that the wax film on the tissue can be completely eluted; in addition, the turpentine will be deposited on the tissue for too long. The paraffin wax in the turpentine will affect the dewaxing effect, so pay attention to timely replacement of the turpentine used for dewaxing (replacement frequency once 1-2 weeks);
  • the sections should be placed at room temperature to slowly return to room temperature, and the sections should not be placed in ice cubes to speed up cooling.
  • the process of cooling at room temperature is one to further complete the antigen retrieval process, and the other is to avoid sudden changes in temperature (such as putting the slice in ice water) to cause the tissue to fall off;
  • the components of the primary antibody diluent are monoclonal antibodies, serum, and buffer.
  • the serum is prone to bacterial contamination after being stored for a long time, which will affect the specificity of antibody staining. Therefore, do not use the primary antibody dilution that has been stored for more than two weeks (stored at 4°C);
  • the incubation time is shorter than 12 hours or longer than 16 hours;
  • the primary antibody incubation of the third round of staining can be incubated overnight at 4°C on the first night, or at room temperature for 1 hour in the morning of the next day. However, if the primary antibody is incubated overnight at 4°C, the incubation time is shorter than 12 hours, and the antibody cannot fully bind to the epitope, which may cause false negative results; if the incubation time is longer than 16 hours, the excess antibody will interact with the tissues. Non-specific binding site binding, resulting in false positive results;
  • the diagnosis of lupus erythematosus classification is mainly based on the patient’s medical history (presence of onset, predisposing factors, etc.), clinical manifestations (features of rash, light sensitivity, system involvement, etc.), laboratory examinations (blood and urine routine, serum autoantibody detection) ), skin pathological examination (HE stain immunofluorescence band examination of skin lesions).
  • lupus erythematosus is a progressive disease and is affected by multiple factors, clinically, medical history (some patients with DLE or SCLE can also show acute onset), clinical manifestations (some patients with DLE or SCLE can also show SLE butterfly erythema As well as mild systemic involvement), laboratory tests (some patients with DLE or SCLE may also show leukopenia, SLE-related pathogenic autoantibodies are positive), skin pathological examination (positive immunofluorescence band test can only diagnose lupus erythematosus, but not Distinguish between DLE, SCLE and SLE), these indications and laboratory tests are not effective in distinguishing subtypes of lupus erythematosus.
  • the present invention is the first skin tissue in-situ detection kit that can effectively distinguish subtypes of lupus erythematosus, and the detected characteristic molecular markers include: CD4, CD8, CD19, and CD11b.
  • CD4 is a characteristic molecular marker of CD4 + T lymphocytes
  • CD8 is a characteristic molecular marker of CD8 + T cells
  • CD19 is a characteristic molecular marker of B lymphocytes
  • CD11b is a characteristic molecular marker of neutrophils, NK cells, Molecular markers shared by monocytes/macrophages.
  • the CD4-CD8-CD19-CD11b quadruple detection results obtained by using the kit of the present invention can well reflect the distribution and expression differences of immune cells in the skin lesions of different subtypes of lupus erythematosus, and the in-situ immune cell detection results of the skin tissue of the sample can be Carry out accurate quantitative analysis to assist in the diagnosis of lupus erythematosus classification.
  • the skin tissue in situ multi-marker immunostaining kit prepared by the present invention can mark multiple characteristic marker molecules related to immune cells on a piece of skin tissue section, and the signals of different marker molecules do not interfere with each other, and the detection accuracy is low. High specificity.
  • the quadruple detection results of CD4-CD8-CD19-CD11b obtained by this kit, combined with specific photographic analysis software, can achieve high-precision detection of the distribution and expression of immune cells, and at the same time meet the needs of clinical tissue morphology and Quantitative detection of immune cell expression is required to effectively distinguish different subtypes of lupus erythematosus.
  • the kit is simple and quick to operate, economical and applicable, and suitable for application and promotion in clinical and scientific research work.
  • Figure 1 Comparison of monoclonal antibody staining signals under different antigen retrieval conditions and different antibody dilution ratios; a is the comparison of AS value and TS/BS value of the signal of monoclonal antibody CD11b under different antigen retrieval conditions and antibody dilution ratios ; B is the comparison of AS value and TS/BS value of the signal of monoclonal antibody CD8 under different antigen retrieval conditions and antibody dilution ratios; c is the signal of monoclonal antibody CD4 under different antigen retrieval conditions and antibody dilution ratios AS value is compared with TS/BS value; d is the comparison of AS value and TS/BS value of the signal of monoclonal antibody CD19 under different antigen retrieval conditions and antibody dilution ratios.
  • Figure 2 Comparison of monoclonal antibody staining results under different non-specific blocking agents and different serum blocking times; a is the staining result of blocking with 3% H 2 O 2 for 15 minutes; b is the 0.5% periodic acid solution alone Blocked staining results for 15 minutes; c is the combined use of 3% H 2 O 2 and 0.5% periodic acid solution to block the staining results for 15 minutes; d is the staining condition of serum blocking for 15 minutes; e is the staining condition of serum blocking for 30 minutes; f It is the staining condition of serum blocking for 60 minutes; g is the staining condition of serum blocking for 90 minutes.
  • Figure 3 Comparison of monoclonal antibody staining signals when matched with different tyramine fluorescent stains; a is the comparison of the AS value and TS/BS value of the signal when the monoclonal antibody CD11b is matched with different tyramine fluorescent stains; b is the match of the monoclonal antibody CD8 Comparison of the AS value and TS/BS value of the signal when different tyramine fluorescent stains; c is the comparison of the AS value and TS/BS value of the signal when the monoclonal antibody CD4 is matched with different tyramine fluorescent stains; d is the match of the monoclonal antibody CD19 Comparison of the AS value and TS/BS value of the signal with different tyramine fluorescent stains.
  • Figure 4 Comparison of the staining signals of the monoclonal antibody matching the corresponding tyramine fluorescent stain under different staining orders; a is the AS value and TS/ of the signal obtained when the monoclonal antibody CD11b matches the corresponding tyramine fluorescent stain when the staining order is different BS value comparison; b is the AS value and TS/BS value comparison of the signal obtained when the monoclonal antibody CD8 matches the corresponding tyramine fluorescent stain when the staining order is different; c is the monoclonal antibody CD4 matches the corresponding tyramine when the staining order is different Comparison of the AS value and TS/BS value of the signal obtained by the fluorescent stain; d is the comparison of the AS value and TS/BS value of the signal obtained by the monoclonal antibody CD19 matching the corresponding tyramine fluorescent stain when the staining sequence is different.
  • Figure 5 Comparison of monoclonal antibody staining signals when performing single staining and multiple staining; a is the comparison of AS value and TS/BS value of staining signal when each monoclonal antibody is single staining and multiple staining; b is adjustment Comparison of the AS value and TS/BS value of the staining signal during multiple staining for CD4 antibody or dye solution incubation time; c is the AS value and TS/BS value of the staining signal during multiple staining for adjusting the CD19 antibody or dye solution incubation time BS value comparison.
  • Figure 6 The final staining scheme is used to process the staining of multiple monoclonal antibodies without interference; the nucleus is blue, CD4 is red, CD8 is white, CD11b is yellow, and CD19 is green; a is multi-labeled immunofluorescence Staining results; b is the CD4 monochromatic image after the spectral resolution processing in Figure 6a; c is the CD8 monochromatic image after the spectral separation processing in Figure 6a; d is the CD11b monochrome image after the spectral separation processing in Figure 6a; e is Fig. 6a is a monochromatic image of CD19 after spectral separation processing; f is a two-color image of CD4 and CD19 after spectral separation processing of Fig. 6a.
  • Figure 7 Staining effects of multiple monoclonal antibodies in four skin tissue samples from different sources using the final protocol; the nucleus is blue, CD4 is red, CD8 is white, CD11b is yellow, and CD19 is green; a is DLE The results of multiple immunofluorescence staining using the final protocol for the skin lesions of patients; b is the multiple immunofluorescence staining results of the final protocol for the skin lesions of SCLE patients; c is the multiple immunofluorescence staining results of the final protocol for the skin lesions of SLE patients; d is the final use of normal skin tissue Scheme multiple immunofluorescence staining results.
  • Figure 8 ROC data analysis results; a is the quadruple detection ROC analysis of CD4-CD8-CD19-CD11b in normal and lupus erythematosus patients; b is the quadruple of CD4-CD8-CD19-CD11b in DLE and non-DLE patients Detection ROC analysis diagram; c is the quadruple detection ROC analysis diagram of CD4-CD8-CD19-CD11b in SCLE and SLE patients.
  • Figure 9 The application strategy of combined detection of multiple types of immune cells in the skin in the diagnosis of lupus erythematosus.
  • Acidic repair solution pH6.0: 3g trisodium citrate, 0.4g citric acid, 20ul Tween 20, 1000ml sterile deionized water;
  • Alkaline repair solution (pH8.0): 1.8g EDTA, 0.9g EDTA-2Na, 1000ml sterile deionized water, about 8ml 10% NaOH to adjust the pH to 8.0;
  • PBST-T solution phosphate Tween Triton buffer: 1xPBS powder, 5ul Tween 20, 1ul TritonX-100, 1000ml sterile deionized water;
  • Serum blocking solution 0.8g BSA powder, 10ml PBST-T solution, mix well, and can be stored at 4°C for 2 weeks after preparation;
  • Antibody diluent 0.1g BSA powder, 10ml 1xPBS solution, mix well, and can be stored at 4°C for 2 weeks after preparation;
  • Opal520 working solution the original solution of Opal520 staining solution and the matching amplification diluent 1:100 dilution;
  • Opal570 working solution the original solution of Opal570 staining solution and the supporting amplification diluent are diluted 1:100;
  • Opal650 working solution the original solution of Opal650 staining solution and the supporting amplification diluent are diluted 1:100;
  • Opal690 working solution the original solution of Opal690 staining solution and the supporting amplification diluent are diluted 1:100.
  • Example 1 Preparation method of in situ multi-label immunostaining kit for skin tissue
  • the result of multi-label immunostaining is captured by processing the filters of the four color channels (DAPI, FITC, Cy3, Cy5), and the emission spectra of different filters will overlap to a certain extent.
  • the multi-spectral analysis software judges the spectrum based on the peak of the spectrum when identifying and splitting the spectrum. When multiple monoclonal antibodies are co-stained on a tissue section, the spectra corresponding to the multiple antibodies will overlap to different degrees. When the color of a certain antibody is too strong, its color spectrum may cover the spectrum of neighboring antibodies, resulting in cross-color between two or more antibodies. In order to avoid this situation, the preparation of in-situ multi-labeled immunostaining kits for skin tissues requires optimization of the conditions for each key step.
  • Each monoclonal antibody has been compared with different antigen retrieval solutions (acid retrieval solution and alkaline retrieval solution), different antigen retrieval conditions (high pressure retrieval and microwave retrieval), and different dilution ratios in the following table (Table 1-4). Determine the best antigen retrieval conditions and the best dilution ratio range for each monoclonal antibody staining.
  • the average fluorescence signal intensity of cells (Average Signal of cell, referred to as AS in the subsequent graphs)
  • the ratio of the total cell fluorescence signal intensity to the total background signal intensity of the tissue area Total signal of cell/Background signal, subsequent graphs
  • TS/BS is used to refer to). While the AS is relatively highest, the condition with the highest TS/BS is the best condition that can ensure the strongest signal and the lowest background of the positive staining result (see Figure 1).
  • Figure 1a shows that the best repair conditions for monoclonal antibody CD11b are: acid microwave repair or acid high pressure repair, preferably acid microwave repair; the best antibody dilution ratio range is: 1:1000-1:2000, preferably 1:1400- 1600; More preferably 1:1500;
  • Figure 1b shows that the best repair conditions for monoclonal antibody CD8 are: alkaline microwave repair or alkaline high pressure repair, preferably alkaline high pressure repair; the best antibody dilution ratio range is: 1:2-1:5, preferably 1 :2-1:4, more preferably 1:3;
  • Figure 1c shows that the best repair conditions for monoclonal antibody CD4 are: alkaline microwave repair or alkaline high pressure repair, preferably alkaline high pressure repair; the optimal antibody dilution ratio range is: 1:2-1:5, preferably 1 :2-1:3, more preferably 1:2;
  • Figure 1d shows that the best repair conditions for monoclonal antibody CD19 are: alkaline microwave repair or alkaline high pressure repair, preferably alkaline high pressure repair; the optimal antibody dilution ratio range is: 1:200-1:500, preferably 1 :250-1:350, more preferably 1:300.
  • the non-specific blocking agent here is preferably a 0.5% periodic acid solution.
  • Figure 2d shows the staining condition of serum blocking for 15 minutes
  • Figure 2e shows the staining condition of serum blocking for 30 minutes.
  • Each monoclonal antibody is compared with different tyramine fluorescent stains in the following table (Table 5), so as to determine the best tyramide fluorescent stain with each monoclonal antibody.
  • Table 5 the average fluorescence signal intensity of cells (Average Signal of cell, referred to as AS in the subsequent graphs), the ratio of the total fluorescence signal intensity of the cells to the total background signal intensity of the tissue area (Total signal of cell/Background signal, subsequent graphs)
  • AS Average Signal of cell
  • Total signal of cell/Background signal, subsequent graphs Total signal of cell/Background signal, subsequent graphs
  • the condition with the highest AS and the highest TS/BS is the tyramine fluorescent stain that best matches the corresponding monoclonal antibody (see Figure 3).
  • Figure 3a When CD11b matches Opal570 stain and Opa650 stain, its AS value and TS/BS value are relatively high; Figure 3b When CD8 matches Opal570 stain and Opa650 stain, its AS value and TS/BS value are relatively higher High; Figure 3c When CD4 matches Opal520 stain, Opal570 stain and Opa650 stain, its AS value and TS/BS value are relatively high; Figure 3d When CD19 matches Opal570 stain, Opa650 stain and Opa690 stain, Its AS value and TS/BS value are relatively high.
  • tyramine fluorescent stains that can be matched with monoclonal antibody CD11b are: Opal570 stain and Opal650 stain;
  • tyramine fluorescent stains that can be matched with the monoclonal antibody CD8 are: Opal570 stain and Opal650 stain;
  • the tyramine fluorescent stains that can be matched with monoclonal antibody CD4 are: Opal520 stain, Opal570 stain and Opal650 stain;
  • the tyramine fluorescent stains that can be matched with the monoclonal antibody CD19 are: Opal570 stain, Opa650 stain and Opa690 stain.
  • the tyramine fluorescent stain corresponding to each monoclonal antibody is sequentially covalently coupled to the corresponding epitope of the monoclonal antibody, and multiple rounds of microwave or high pressure treatment are required to remove the previous round of non-covalent binding Primary antibody and secondary antibody, therefore, it is necessary to determine the staining order of each monoclonal antibody to ensure that the integrity of the epitope corresponding to the monoclonal antibody is not affected after multiple rounds of microwave or high-pressure treatment. In addition, it is necessary to ensure the earliest The fluorescent stain of tyramine coupled to the epitope can withstand the subsequent heating process.
  • the present invention uses the average cell fluorescence signal intensity (Average Signal of cell, referred to as AS in the subsequent graphs), and the ratio of the total cell fluorescence signal intensity to the total background signal intensity of the tissue area (Total signal of cell).
  • AS Average Cell fluorescence signal intensity
  • TS/BS Total background signal intensity of the tissue area
  • Figure 4a shows that when CD11b matches Opal570 stain or Opal650 stain and is in the position of the first round of staining, its AS value and TS/BS value are relatively high. Among them, CD11b-Opal570 stain-the first round of staining The AS value and TS/BS value obtained by collocation are the highest;
  • Figure 4b shows that when CD8 matches Opal570 stain or Opal650 stain and is in the second round of staining, its AS value and TS/BS value are relatively high. Among them, CD8-Opal650 stain-the second round of staining The AS value and TS/BS value obtained by collocation are the highest;
  • Figure 4c shows that when CD4 matches Opal520 stain or Opal570 stain and is in the third or fourth round of staining, its AS value and TS/BS value are relatively high, of which CD4-Opal520 stain-third round Or the highest AS value and TS/BS value obtained by the fourth round of dyeing combination;
  • Figure 4d shows that when CD19 matches Opa690 stain and is in the third or fourth round of staining, its AS value and TS/BS value are relatively high. Among them, the combination of CD19-Opal690 stain-fourth round of staining The obtained AS value and TS/BS value are the highest.
  • the first round of staining is the monoclonal antibody CD11b, and the corresponding tyramine fluorescent stain is Opal570;
  • the second round of staining is the monoclonal antibody CD8, and the corresponding tyramine fluorescent stain is Opal650;
  • the first round of staining is the monoclonal antibody CD4, and the corresponding tyramine fluorescent stain is Opal520;
  • the first round of staining is the monoclonal antibody CD19, and the corresponding tyramine fluorescent stain is Opal690.
  • the application time of each monoclonal antibody in the multi-label immunostaining scheme is further determined, which corresponds to the application time of the secondary antibody and the stain.
  • the difference in antibody staining signal was compared for each monoclonal antibody after a single antigen retrieval or multiple antigen retrieval. While performing superimposed staining of multiple monoclonal antibodies, the binding of the previous monoclonal antibody corresponding to the tyramine fluorescent stain and the epitope may affect the subsequent monoclonal antibody corresponding to the tyramine fluorescent stain and the antigen epitope.
  • Figure 5a shows that the CD11b signal is not significantly different between single staining and multiple staining, so the antibody incubation time and the secondary antibody staining time do not need to be adjusted;
  • Figure 5a shows that the CD8 signal is not significantly different between single staining and multiple staining, so the antibody incubation time and the secondary antibody staining time do not need to be adjusted;
  • Figure 5b shows that the signal of CD4 signal in multiple staining is significantly weaker than the signal in single staining, and when the CD4 primary antibody incubation time is extended to 75min, the secondary antibody incubation time is extended to 20min, and the Opal520 staining agent incubation time is extended At 30 minutes, the AS value and TS/BS value are relatively high, and the adjusted CD4 signal is close to the signal when performing single staining;
  • Figure 5c shows that the signal of CD19 signal in multiple staining is significantly weaker than that in single staining.
  • Figure 6a shows the results of multi-label immunofluorescence staining; the nucleus is blue, CD4 is red, CD8 is white, CD11b is yellow, and CD19 is green. It can be seen that the four monoclonal antibody signals are not significantly missing in multiple staining.
  • Figure 6b shows that there is no obvious loss of CD4 signal in a single stain;
  • Figure 6c shows that there is no obvious loss of CD8 signal in a single stain;
  • Figure 6d shows that there is no obvious loss of CD11b signal in a single stain;
  • Figure 6e shows that CD4 signal is not obvious in a single stain Deletion;
  • Figure 6f shows that the CD4 and CD19 signals are not significantly missing in the double staining.
  • the nucleus is blue, CD4 is red, CD8 is white, CD11b is yellow, and CD19 is green; it can be seen that the final solution can achieve stable and interference-free staining in the four source samples.
  • Example 2 Using method of in situ multi-label immunostaining kit for skin tissue.
  • step 6 configure CD11b antibody working solution (1:1500 dilution): 1ul CD11b antibody stock solution (Abcam#ab52478), 25ul goat serum, 1474ul antibody dilution solution (can be stored at 4°C for 2 weeks);
  • step 5 configure CD8 antibody working solution (1:3 dilution): 500ul CD8 antibody stock solution (Fuzhou Maixin, MAB-0021), 25ul goat serum, 975ul antibody dilution solution (can be stored at 4°C Two weeks);
  • step 6 configure CD4 antibody working solution (1:2 dilution): 750ul CD4 antibody stock solution (Fuzhou Maixin, RMA-0620), 25ul goat serum, 725ul antibody dilution (can be stored at 4°C Two weeks);
  • step 6 configure CD19 antibody working solution: 5ul CD19 antibody stock solution (Abcam, #ab227688), 25ul goat serum, 1470ul antibody diluent (can be stored at 4°C for 2 weeks);
  • the next step is to use the function of manually dividing the organization area to divide the organizational vision that you need to analyze. Because this kit is used for the analysis of immune cells in the inflammatory infiltration area of lupus erythematosus skin lesions, the epidermis, glands, etc. should be divided in this step and not included in the analysis;
  • the cell positioning and segmentation function to select the type of organelle to be analyzed, such as nucleus, cytoplasm or cell membrane (the molecular markers used in this kit are all expressed and located on the cell membrane, so check the cell membrane), and select the tissue area to be analyzed (tick Select the immune cell infiltration area determined in the previous step), and select the staining spectrum of the specific organelle type (check Fitc, Cy3, Cy5 and DAPI). Click the cell segmentation button at the bottom left to get the organelle type analysis results;
  • the type of organelle to be analyzed such as nucleus, cytoplasm or cell membrane (the molecular markers used in this kit are all expressed and located on the cell membrane, so check the cell membrane), and select the tissue area to be analyzed (tick Select the immune cell infiltration area determined in the previous step), and select the staining spectrum of the specific organelle type (check Fitc, Cy3, Cy5 and DAPI).
  • the next step is to use the cell typing function to self-mark 10 positive results for each monoclonal antibody.
  • this kit manually label CD4 + cells, CD19 + cells, CD8 + cells, CD11b + cells, 10 other cells (CD4 - CD8 - CD19 - CD11b - cells), and then click the cell typing button in the lower left corner, software is started in accordance with positive cells manually marked self-learning, and treat all tissue regions analyzed for cell type, and finally to obtain CD4 + cells, CD19 + cells, CD8 + cells, CD11b + cells, Other cells (CD4 - CD8 - CD19 - CD11b - cells) the cell count results of these five types of cells;
  • Example 3 Determination of the detection index of the skin tissue in situ multi-label immunostaining kit that can assist the diagnosis and typing of lupus erythematosus
  • CD4-CD8 dual test results (CD4+CD8)/DAPI
  • CD4-CD19 dual test results (CD4+CD19)/DAPI
  • CD4-CD11b dual test results (CD4+CD11b)/DAPI)
  • CD8-CD19 dual test results (CD8+CD19)/DAPI
  • CD8-CD11b dual test results (CD8+CD11b)/DAPI)
  • CD4-CD19-CD11b triple test results ((CD4+CD19+CD11b)/DAPI)
  • CD4-CD8-CD19 triple test results ((CD4+CD8+CD19)/DAPI)
  • CD4-CD19-CD11b triple test results ((CD4+CD19+CD11b)/DAPI)
  • CD4-CD8-CD19 triple test results ((CD4+CD8+CD19)/DA
  • CD4/DAPI The single positive detection result of CD4 (CD4/DAPI), the single positive detection result of CD8 (CD8/DAPI), the single positive detection result of CD19 (CD19/DAPI), the single positive detection result of CD11b (CD11b/DAPI) , CD4-CD8 dual test results ((CD4+CD8)/DAPI), CD4-CD19 dual test results ((CD4+CD19)/DAPI), CD4-CD11b dual test results ((CD4+CD11b) )/DAPI), CD8-CD19 dual test results ((CD8+CD19)/DAPI), CD8-CD11b dual test results ((CD8+CD11b)/DAPI), CD19-CD11b dual test results ( (CD19+CD11b)/DAPI), CD4-CD8-CD19 triple test results ((CD4+CD8+CD19)/DAPI), CD4-CD19-CD11b triple test results ((CD4+CD19+CD11b)/DAPI) , CD4
  • CD4/DAPI single positive detection result of CD4
  • CD19/DAPI single positive detection result of CD19/DAPI
  • CD11b/DAPI dual detection result of CD4-CD19
  • CD8-CD19 dual test results (CD8+CD19)/DAPI)
  • CD19-CD11b dual test results (CD19+CD11b)/DAPI)
  • CD4-CD8-CD19 triple test Results ((CD4+CD8+CD19)/DAPI)
  • CD4-CD19-CD11b triple test results ((CD4+CD19+CD11b)/DAPI)
  • CD8-CD19-CD11b triple test results ((CD8+CD19+CD11b)/DAPI)
  • CD4-CD8-CD19-CD11b quadruple test results are statistically significant between the DLE and non-DLE groups.
  • the 8 groups of data with higher predictive diagnostic value selected in step 5 are in There
  • CD4 single positive test results CD4/DAPI
  • CD8 single positive test results CD8/DAPI
  • CD19 single positive detection result CD19/DAPI
  • CD11b single positive detection result CD11b/DAPI
  • CD4-CD8 dual detection result (CD4+CD8)/DAPI)
  • CD4-CD19 dual detection result Test result (CD4+CD19)/DAPI
  • CD4-CD11b dual test result (CD4+CD11b)/DAPI)
  • CD8-CD19 dual test result ((CD8+CD19)/DAPI)
  • CD8-CD19 dual test result CD8+CD19/DAPI
  • CD8- The double test result of CD11b ((CD8+CD11b)/DAPI), the double test result of CD19-CD11b ((CD19+CD11b)/DAPI), the triple test result of CD4-CD8-CD19 ((CD4+CD8+CD19) )/DAPI)
  • CD4-CD19 dual detection result (CD4+CD8+
  • test index that distinguishes any group (LEvsNC, DLEvs non-DLE, SCLEvsSLE) in AUC ⁇ 0.7, then the test index is deemed to be unable to effectively diagnose and distinguish different subtypes of lupus erythematosus, and it is marked as ⁇ in the feasibility column , That is not feasible;
  • test index that can distinguish between any two groups and above (LEvsNC, DLEvs non-DLE, SCLEvsSLE), 0.7 ⁇ AUC ⁇ 0.8, then the test index is deemed to be unable to effectively diagnose and distinguish different subtypes of lupus erythematosus.
  • the column is marked with ⁇ , which means it is not feasible;
  • test index that distinguishes all groups (LEvsNC, DLEvs non-DLE, SCLEvsSLE) in AUC> 0.7, and any two or more AUC> 0.8, then the test index is considered to be effective in diagnosing and distinguishing erythema For different subtypes of lupus, mark it as ⁇ in the feasibility column.
  • CD19 single positive detection CD19/DAPI
  • CD8-CD19 The double detection ((CD8+CD19)/DAPI)
  • the triple detection of CD4-CD8-CD19 (CD4+CD8+CD19)/DAPI)
  • the quadruple detection of CD4-CD8-CD19-CD11b (CD4+CD8) +CD19+CD11b)/DAPI)
  • these four detection indicators are only CD4-CD8-CD19-CD11b quadruple detection AUC>0.8
  • the non-parametric test P value ⁇ 0.001 that is When comparing the results of the quadruple detection of CD4-CD8-CD19-CD11b between the two sets of data of SCLE and SLE, there are significant differences between the two sets of data.
  • CD4-CD8 is used compared to the other three detection indicators. -The AUC value obtained by the quadruple detection index of CD19-CD11b is the largest. It is suggested that the quadruple detection index of CD4-CD8-CD19-CD11b is the most preferred detection index for the diagnosis of lupus erythematosus;
  • the critical value for distinguishing SCLE and SLE corresponds to a specificity of 88.89%, which means that when distinguishing SCLE and SLE, CD4 -The quadruple detection of CD8-CD19-CD11b can be combined with the single positive detection of CD19 and/or the double detection of CD8-CD19, so as to reduce the possibility of SLE being misjudged as SCLE as much as possible.
  • the present invention will obtain the quadruple detection result of CD4-CD8-CD19-CD11b ((CD4+CD8+CD19+CD11b)/DAPI) after the detection of the above reagents, and the detection result is used to assist An essential test for the diagnosis and/or classification of lupus erythematosus.
  • the CD19 single positive test result (CD19/DAPI) and the CD8-CD19 dual test result ((CD8+CD19)/DAPI) can be used as auxiliary test conditions .
  • the obtained quadruple test results of CD4-CD8-CD19-CD11b can be used for the diagnosis and/or classification of lupus erythematosus: when the quadruple test result of CD4-CD8-CD19-CD11b is ⁇ 77.7%, it is judged to be tested
  • the patient from the sample source is highly likely to have DLE; 55.4% ⁇ CD4-CD8-CD19-CD11b and the quadruple test result ⁇ 77.7%, the patient from the source of the sample to be tested is more likely to be SCLE; 41.2% ⁇ CD4-CD8 -When the quadruple test result of CD19-CD11b is less than or equal to 55.4%, it is highly likely that the patient from the source of the sample to be tested is determined to be SLE; when the quadruple test result of CD4-CD8-CD19-CD11b is less than or equal to 41.2%, the sample to be tested is judged The source is highly likely to be a normal person;
  • the samples to be tested are all detected in the dermal inflammatory infiltration area under a single field of view under a 200-fold electron microscope.
  • sample number 1 is DLE skin lesion No. 2
  • sample number 2 is SLE patient skin lesion No.
  • CD19 single positive test results CD19/DAPI
  • CD8-CD19 dual test results CD8+CD19/DAPI
  • CD4-CD8-CD19 triple test results CD4+CD8+CD19/DAPI
  • CD4-CD8-CD19-CD11b quadruple test results (CD4+CD8+CD19+CD11b)/DAPI)
  • the dual detection result of CD8-CD19 ((CD8+CD19)/DAPI) can also be used, so CD4-CD8-CD19 -The quadruple detection result of CD11b combined with the single positive detection result of CD19 and the detection result of CD8-CD19 are set as combined detection 2.

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Abstract

用于红斑狼疮分型的皮肤组织免疫细胞原位检测试剂盒及其应用。试剂盒运用原位多标记免疫染色及多光谱成像技术可检测CD4、CD8、CD19、CD11b这四种分子标记。CD4-CD8-CD19-CD11b的四联检测结果能很好的区分盘状红斑狼疮(DLE)、亚急性红斑狼疮(SCLE)、系统性红斑狼疮(SLE)与健康对照，配合CD19的单阳检测结果和/或CD8-CD19的二联检测结果，准确率更高。操作简单、经济实用，结果对于区分不同类型红斑狼疮的皮损有较高的特异性，便于其在红斑狼疮机制研究和临床诊断工作中的应用和推广。

Description

用于红斑狼疮分型的皮肤组织免疫细胞原位检测试剂盒及其应用 技术领域
本发明属于免疫检测技术领域，具体涉及用于红斑狼疮分型的皮肤组织免疫细胞原位检测试剂盒及其应用。
背景技术
红斑狼疮(Lupus erythematosus,LE)是一种多因素参与，严重影响生活质量和危害健康的慢性自身免疫性疾病。80％以上的患者可在发病初始或者进展中出现皮肤受累。LE为一病谱性疾病，一端为皮肤型红斑狼疮(cutaneous lupus erythematosus,CLE),呈慢性良性经过，病变主要局限于皮肤；另一端为系统性红斑狼疮(systemic lupus erythematosus,SLE)，多急性发病，病变可累及多系统，严重者可危及生命。临床上，医生根据红斑狼疮皮损表现和相应组织病理改变，将LE分为急性皮肤型红斑狼疮(acute cutaneous lupus erythematosus,ACLE)，亚急性皮肤型红斑狼疮(subacute cutaneous lupus erythematosus,SCLE)和盘状红斑狼疮(discoid lupus erythematosus,DLE)三大类。
DLE是最常见也最典型的一类CLE，临床上50％-85％的有皮肤受累的红斑狼疮患者均表现为持久的盘状皮损。SCLE是红斑狼疮亚型中光敏感性最强的一类CLE，常表现为环状斑块或丘疹鳞屑样皮损。ACLE通常被认为是SLE患者皮肤受累的表现，是红斑狼疮各类型中最为严重的一型，其典型皮损为面部蝶形分布的红斑，绝大多数患者发病时即有多系统受累。临床上，对那些皮损典型的红斑狼疮患者，医生可以进行清晰的诊断分型以及早期精准的干预治疗。但红斑狼疮是一个进行性疾病且受多因素影响，临床上仍有较多患者皮损不典型，难以进行明确的诊断分型，从而导致治疗上的过度用药或病情的延误。因此，红斑狼疮不同亚型的诊断及其鉴别诊断对于临床早期干预和精准治疗至关重要。目前缺少简便快捷、特异性和灵敏度高的红斑狼疮辅助诊断试剂盒。因此建立有效的临床适用的试剂盒就显得尤为重要。
免疫细胞主要包括T淋巴细胞(CD4 +T细胞和CD8 +T细胞)、B淋巴细胞、粒细胞、NK细胞、单核/巨噬细胞等。大量研究证实，免疫细胞的异常活化 和致病性自身抗体的大量产生在LE的发生发展中其重要作用。有研究发现在DLE皮损中，其炎症浸润区域有大量的B细胞的存在。而SLE和SCLE皮损的炎症浸润相较于正常皮肤则没有表现出明显的B细胞增多。此外，一项不同亚型狼疮皮损与外周血的差异基因表达研究发现，与NK细胞相关的差异基因仅在DLE皮损中有显著表达，而在SLE皮损和外周血以及DLE外周血中的表达均不明显，提示NK细胞相关基因可能是DLE患者皮肤炎症的特异性基因。已有研究证实细胞毒性CD8 +T细胞参与了狼疮皮损中表皮基底及血管内皮细胞的凋亡坏死过程，从而导致真表皮交界处及血管周的炎症浸润。
目前关于红斑狼疮皮肤原位免疫细胞的研究有很多，但大多都是利用免疫组化单染或者免疫荧光双染技术。对于皮肤组织原位的免疫细胞检测，传统的免疫组化单染或双染技术存在其局限性，无法实现组织原位标志物的多重标记，不能对多种免疫细胞进行同时观察和定量检测。而流式细胞技术和二代测序等方法虽可以识别皮肤组织中的细胞种类，但必须先将组织解离成单细胞悬液方能进行。而临床上人的皮肤组织样本较难获得，且取材体积一般较小，制备成的单细胞悬液常因细胞数目过少难以进行流式分析，以及容易污染和降解而难以完成测序。并且通过流式等方法获得的结果缺少相应的空间分布信息，不能直观的显示其组织形态学数据。此外，虽然有一些研究表明某些免疫细胞在红斑狼疮亚型间存在一定差异，但因受传统染色的技术局限，无法做到多重标记和定量检测，目前尚未有利用免疫细胞的表达差异来辅助红斑狼疮诊断分型的相关应用。
本发明设计了CD4、CD8、CD19、CD11b四种抗体组合的检测试剂盒。CD4为CD4 +T淋巴细胞的特征性分子标记物、CD8为CD8 +T细胞的特征性分子标记物、CD19为B淋巴细胞的特征性分子标记物、CD11b则为粒细胞、NK细胞、单核/巨噬细胞共有的分子标记。在一张组织切片中同时检测CD4、CD8、CD19、CD11b这四种分子标记，将获得参与红斑狼疮皮损发生的绝大多数免疫细胞的表达和分布情况。相较于传统免疫组化或荧光染色进行单个指标检测获得的单个分子标记的表达情况(如CD19的定性、半定量结果)，CD4-CD8-CD19-CD11b的四联定量检测结果能很好的区分盘状红斑狼疮、亚急性红斑狼疮、系统性红斑狼疮与健康对照。结果同时满足临床上组织形态学观察和免疫细胞表达情况的定量检测需求，操作简单快捷，经济适用，适合在临床和 科研工作中应用和推广。
发明内容
本发明的首要目的是提供一种皮肤组织原位标记免疫染色试剂盒。本发明是利用同时检测CD4、CD8、CD19、CD11b这四种分子标记，获得参与红斑狼疮皮损发生的绝大多数免疫细胞的表达和分布情况，并依据CD4-CD8-CD19-CD11b的四联检测结果在红斑狼疮与正常人之间，以及红斑狼疮各种分型之间存在差异设计的配套产品，填补了目前临床上红斑狼疮诊断分型试剂盒的空白。利用本发明试剂盒获得的CD4-CD8-CD19-CD11b的检测结果，搭配Mantra多光谱成像软件，实现了精准度较高的免疫细胞分布及表达情况检测，可以有效地区分红斑狼疮三个亚型，灵敏度高、特异性好。
本发明所述的试剂盒包括：利用原位标记免疫染色检测CD4、CD8、CD19、CD11b阳性免疫细胞所需的试剂。
所述的检测CD4、CD8、CD19、CD11b阳性免疫细胞所需的试剂包括：抗原修复液、非特异性阻断剂、血清封闭剂、抗体稀释剂、单克隆抗体、洗涤剂、辣根过氧化物酶标记的二抗、酪胺荧光染色剂和核染色剂。
本发明所述的试剂盒中所述抗原修复液包括pH6.0的酸性抗原修复液和/或pH8.0的碱性修复液。所述非特异性阻断剂为单独的0.5％高碘酸溶液或者联合使用的0.5％高碘酸溶液和3％H 2O 2溶液，优选0.5％高碘酸溶液。
进一步的，不同的单克隆抗体其对应最佳的组织切片抗原修复的条件不同，通常来讲，酸性抗原修复液的修复效果弱于碱性修复液的修复效果，而反之来说使用酸性抗原修复液修复后的组织行免疫染色后，其非特异性染色的程度亦弱于碱性抗原修复液，故本发明每种单克隆抗体都需进行抗原修复液的最优化比对。
进一步的，本试剂盒染色反应的关键是借助二抗上带有的辣根过氧化物酶来催化染色剂活化进而显色。而皮肤组织中大量内源性过氧化物酶的存在(表皮，血管，腺体中)会对最后显色的特异性产生干扰，进而影响信号分析的精准度。故合适有效的非特异性阻断剂对获取特异性检测结果至关重要。免疫染色最常用的非特异性阻断方式为3％H 2O 2室温敷育15min。本试剂盒制备中，为了更好的进行非特异性阻断，获得更精准更特异的染色结果，比对了单用3％H 2O 2 阻断、单用0.5％高碘酸溶液、联合使用3％H 2O 2和0.5％高碘酸溶液敷育同样时间(室温15min)，其在减少非特异性染色方面的效果，结果表明单用3％H 2O 2阻断的效果最差，而单用0.5％高碘酸溶液、联合使用3％H 2O 2和0.5％高碘酸溶液阻断两者之间没有明显差别，故为了操作的简便性和节约试剂成本，此处的非特异性阻断剂优选为0.5％的高碘酸溶液。
进一步的，所述的血清封闭剂成分包括牛血清白蛋白(Bovine Serum Albumin，BSA)、磷酸缓冲盐溶液(phosphate buffer saline，PBS)、吐温-20(Tween-20)、聚乙二醇辛基苯基醚(Triton X-100)。比对了不同血清封闭时间对染色阳性率的影响(15min，30min，60min，90min)，结果发现室温血清封闭15min与室温血清封闭30min在染色阳性率上没有区别，而室温血清封闭1小时和90min的两组，染色阳性率下降，说明过长的血清封闭会封闭一些特异性结合位点，进而导致染色的假阴性，故此处血清封闭时间为15-50min，优选15-30min，进一步优选15min。
进一步的，所述的抗体稀释剂成分包括牛血清白蛋白(Bovine Serum Albumin，BSA)、磷酸缓冲盐溶液(phosphate buffer saline，PBS)。
进一步的，所述洗涤剂成分包括磷酸缓冲盐溶液(phosphate buffer saline，PBS)、吐温-20(Tween-20)、聚乙二醇辛基苯基醚(Triton X-100)。
进一步的，所述的辣根过氧化物酶标记的二抗为羊抗兔/鼠辣根过氧化物酶标记的即用型二抗。
进一步的，所述酪胺荧光染色剂包括Opal520染液、Opal570染液、Opal650染液、Opal690染液。
进一步的，所述酪胺荧光染色剂与单克隆抗体组中的特定抗体一一匹配，以此来平衡待检测样品中各检测靶点结合荧光染色剂后的信号强度。
本发明所述的试剂盒中所述单克隆抗体包括：
兔源CD4单克隆抗体、鼠源CD8单克隆抗体、兔源CD19单克隆抗体、兔源CD11b单克隆抗体；CD11b单克隆抗体匹配Opal570染液；CD8单克隆抗体匹配Opal650染液；CD4单克隆抗体匹配Opal520染液；CD19单克隆抗体匹配Opal690染液。
本发明所述的试剂盒中检测CD4、CD8、CD19、CD11b四种细胞时，每种单克隆抗体染色顺序、抗原修复条件、稀释比范围、敷育时间、对应二抗敷育 时间，对应匹配度的酪胺荧光染色剂及染色剂敷育时间如下：
a)第一轮染色抗体：CD11b单克隆抗体；其抗原修复条件：酸性抗原修复液高压修复或酸性抗原修复液微波修复，优选酸性抗原修复液微波修复；其稀释比范围：1:1000-1:2000，依次经过一抗和二抗敷育，然后采用染色剂敷育。优选CD11b单克隆抗体稀释比1:1400-1:1600，进一步优选1:1500。一抗敷育时间：室温1小时。二抗敷育时间：室温10min。其对应匹配度的酪胺荧光染色剂及染色剂敷育时间：优选Opal570染色剂，室温敷育10min，洗涤剂洗涤；
b)第二轮染色抗体：CD8单克隆抗体；其抗原修复条件：碱性抗原修复液高压修复或碱性抗原修复液微波修复，优选碱性抗原修复液高压修复；其稀释比范围：1:2-1:5，依次经过一抗和二抗敷育，然后采用染色剂敷育。优选CD8单克隆抗体稀释比1:2-1:4，进一步优选1:3。一抗敷育时间：室温1小时。二抗敷育时间：室温10min。其对应匹配度的酪胺荧光染色剂及染色剂敷育时间：优选Opal650染色剂，室温敷育10min，洗涤剂洗涤；
c)第三轮染色抗体：CD4单克隆抗体；其抗原修复条件：碱性抗原修复液高压修复或碱性抗原修复液微波修复，优选碱性抗原修复液高压修复；其稀释比范围：1:2-1:5，依次经过一抗和二抗敷育，然后采用染色剂敷育。优选CD4单克隆抗体稀释比1:2-1:3，进一步优选1:2。一抗敷育时间优选：室温70-80min，优选75min。二抗敷育时间室温15-25min，优选室温20min。其对应匹配度的酪胺荧光染色剂及染色剂敷育时间：优选Opal520染色剂，室温敷育25-35min，优选30min，洗涤剂洗涤；
d)第四轮染色抗体：CD19单克隆抗体；其抗原修复条件：碱性抗原修复液高压修复或碱性抗原修复液微波修复，优选碱性抗原修复液高压修复；其稀释比范围：1:200-1:500，依次经过一抗和二抗敷育，然后采用染色剂敷育。优选CD19单克隆抗体稀释比1:250-1:350，进一步优选1:300。一抗敷育时间：室温70-80min，优选75min。二抗敷育时间室温15-25min，优选室温20min。其对应匹配度的酪胺荧光染色剂及染色剂敷育时间：优选Opal690染色剂，室温敷育15-25min，优选20min，洗涤剂洗涤。
上述试剂盒使用时，所述的室温范围：20-27℃。
本发明采用核复染来对全体细胞进行检测：取酸性抗原修复液微波修复， 采用DAPI(4',6-二脒基-2-苯基吲哚)进行染色。具体是将切片上滴加核染色剂，敷育10min，封片，用Mantra多光谱成像仪检测。
本发明所述的试剂盒，单克隆抗体皮肤组织染色容易出现非特异性着色，因此，单克隆抗体在抗原修复后，一抗敷育之前，需行非特异性阻断剂阻断，完成阻断后洗涤剂洗涤，再血清封闭剂室温封闭，血清封闭时间为15-50min，优选15-30min，进一步优选15min。
本发明待测样本为红斑狼疮患者曝光部位(头面颈部及双上肢前端)皮肤损伤的石蜡切片。
本发明所述的皮肤组织原位标记免疫染色试剂盒在进行多种抗体染色时，其制备原理在于加入的酪胺荧光染色剂(加入时为非活化态)被二抗所带有的辣根过氧化物酶催化活化，进而跟抗原表位的酪氨酸残基共价偶联，从而获得稳定结合的抗原表位-荧光染色剂复合物，而一抗与抗原表位之间、二抗与一抗之间的结合为非共价结合，可通过微波或高压处理来洗脱。故在一种单克隆抗体完成一抗-二抗-酪胺荧光染色剂的结合后，进行微波或高压处理，与该抗原表位非共价结合的一抗-二抗复合物被洗掉，而酪胺荧光染色剂仍共价结合于抗原表位并留存在样品上。此时再根据需要挑选另外一种单克隆抗体来第二轮孵育，周而复始。从而实现了一张组织切片原位多标记免疫染色。
本发明的第二个目的是提供上述皮肤组织免疫细胞原位检测试剂盒在制备辅助红斑狼疮诊断和/或分型制剂中的应用。
本发明是基于首次发现皮肤组织中CD4 +细胞、CD8 +细胞、CD19 +细胞和CD11b +细胞四种阳性免疫细胞数目总和在200倍视野下炎症浸润区全体细胞(DAPI染色细胞)中所占的百分比在不同亚型红斑狼疮皮损间有显著差异，并据此开发出新的辅助红斑狼疮诊断和/或分型的制剂。
具体是将CD4 +细胞、CD8 +细胞、CD19 +细胞和CD11b +细胞四种细胞数目总和在全体细胞中所占的百分比，即(CD4+CD8+CD19+CD11b)/DAPI的结果用于辅助红斑狼疮诊断和/或分型。
进一步的，在依据(CD4+CD8+CD19+CD11b)/DAPI结果进行判断的基础上，依据CD19 +细胞数目在全体细胞中所占的百分比；即CD19/DAPI的结果对SLE、SCLE和DLE进一步分型；或者依据CD19/DAPI和/或(CD8+CD19)/DAPI 结果对SCLE和SLE进一步分型。三个指标判别趋势均为：正常人＜SLE＜SCLE＜DLE。
本发明具体是根据ROC曲线分析获得具体的诊断和分型临界值进行判断。
进一步的，待测样品均是在200倍电镜单个视野下，真皮层炎症浸润区进行的检测，保证真皮层炎症浸润区布满整个视野。
本发明利用上述的试剂检测后将获得CD4-CD8-CD19-CD11b的四联检测结果((CD4+CD8+CD19+CD11b)/DAPI)，该检测结果是用于辅助红斑狼疮诊断和/或分型诊断的必备检测，此外，CD19的单阳检测结果(CD19/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)，可作为辅助检测条件，与CD4-CD8-CD19-CD11b的四联检测结果联合应用，来进一步提高红斑狼疮分型诊断的准确率。
进一步的，本发明经过上述的试剂检测后红斑狼疮诊断和/或分型的判定临界值：
(CD4+CD8+CD19+CD11b)/DAPI≥77.7％时，判断该待测样品来源于DLE；55.4％<(CD4+CD8+CD19+CD11b)/DAPI<77.7％时，判断该待测样品来源于SCLE；41.2％<(CD4+CD8+CD19+CD11b)/DAPI≤55.4％时，判断该待测样品来源于SLE；(CD4+CD8+CD19+CD11b)/DAPI≤41.2％，判断该待测样品来源于正常人。
当77.7％≤(CD4+CD8+CD19+CD11b)/DAPI≤86.5％，CD19/DAPI检测结果≥12.1％时，判断该待测样品来源于DLE；CD19/DAPI检测结果<12.1％时，判断该待测样品来源于SCLE；
当55.4％≤(CD4+CD8+CD19+CD11b)/DAPI≤67.7％时，CD19/DAPI检测结果≥2.5％和/或(CD8+CD19)/DAPI检测结果≥25.2％时，判断该待测样品来源于SCLE；CD19/DAPI检测结果<2.5％和/或(CD8+CD19)/DAPI检测结果<25.2％时，判断该待测样品来源于SLE。
本发明经样本验证发现，CD4-CD8-CD19-CD11b的四联检测结果在77.7％与86.5％之间时，有一定几率把SCLE误判为DLE的可能，此时联合应用CD19的单阳检测结果(CD19/DAPI)可提高诊断准确率，当CD4-CD8-CD19-CD11b 的四联检测结果在77.7％与86.5％之间，同时CD19的单阳检测结果≥12.1％时，可判断该待测样品来源的患者为DLE；当CD4-CD8-CD19-CD11b的四联检测结果在77％与86.5％之间，同时CD19的单阳检测结果<12.1％时，可判断该待测样品来源的患者为SCLE；
经样本验证发现，CD4-CD8-CD19-CD11b的四联检测结果在55.4％与67.7％之间时，有一定几率把SLE误判为SCLE的可能，此时联合应用CD19的单阳检测结果(CD19/DAPI)和/或CD8-CD19的二联检测结果((CD8+CD19)/DAPI)可提高诊断准确率，当CD4-CD8-CD19-CD11b的四联检测结果在55.4％与67.7％之间，同时CD19的单阳检测结果≥2.5％和/或CD8-CD19的二联检测结果≥25.2％时，判断该待测样品来源的患者为SCLE；当CD4-CD8-CD19-CD11b的四联检测结果在55.4％与67.7％之间，同时CD19的单阳检测结果<2.5％和/或CD8-CD19的二联检测结果<25.2％时，判断该待测样品来源的患者为SLE。
本发明的第三个目的是提供使用上述的试剂盒在非诊断目的的研究各种红斑狼疮分型和/或正常皮肤免疫细胞特征中的应用。
本发明试剂盒的使用中发现还有以下这些因素也会影响最后染色结果的特异性：
1)石蜡切片在脱蜡过程中，组织上覆盖的蜡膜未完全洗脱干净；
当组织脱蜡这一步骤脱蜡不彻底时，覆盖在组织上的蜡膜会影响后续的抗原表位修复以及一抗二抗的结合，进而造成着色失败的情况。故在试剂盒使用过程中，当室内温度较低时，应先对脱蜡用的松节油进行预热，从而确保组织上覆盖的蜡膜可完全洗脱干净；此外松节油使用时间过久，沉积于松节油中的石蜡会影响脱蜡效果，故应注意及时更换脱蜡用的松节油(更换频率1-2周一次)；
2)抗原修复结束后放置入冰块中加速冷却；
在抗原修复结束后，应放置室温使切片缓慢恢复至室温，不可为加快冷却而将切片放置入冰块中。室温冷却这个过程，一是为了进一步完成抗原修复过程，二是避免温度的骤变(如将切片放入冰水中)导致组织的脱片情况；
3)在一轮完整的染色结束前，将切片停置在某个环节过久；
在一轮完整的染色结束前(即进行新一轮抗原修复之前)，所有环节都应严格按照使用说明进行。血清封闭环节停置太久(超过1小时)，容易导致假阴性结果； 一抗环节敷育太久(超过90min)，容易导致假阳性结果；二抗及染液环节本身有信号放大作用，敷育太久(超过30min)，容易出现非特异性染色；一抗与二抗之间、二抗与染色剂之间的洗涤环节，若放置太久，会出现染色信号弱或假阴性的结果；
4)使用放置超过两周以上的一抗稀释液；
因一抗稀释液的成分是单克隆抗体、血清、缓冲液。其中的血清在长时间放置后，容易发生细菌污染，进而影响抗体染色的特异性，故不可使用放置超过两周以上(4℃存放)的一抗稀释液；
5)使用4℃过夜敷育一抗时，敷育时间短于12小时或长于16小时；
本试剂盒的四轮染色完成需要1-2天，第三轮染色的一抗敷育环节可以选择在第一天晚上行4℃敷育过夜，也可以选择在第二天上午室温1小时。但若使用4℃过夜敷育一抗时，敷育时间短于12小时，抗体不能充分与抗原表位结合，易造成假阴性结果；敷育时间长于16小时，多余的抗体会与组织中的非特异性结合位点结合，进而造成假阳性结果；
6)在操作的任何环节，组织发生干片；
在整个操作环节中，全程应避免组织发生干片，一旦组织干片，会导致抗体无法有效的与抗原表位结合，进而造成染色过浅或不染色的情况出现。
目前，红斑狼疮分型的诊断主要是由患者病史(发病缓急，有无诱发因素等)、临床表现(皮疹特点，光敏感，系统受累情况等)、实验室检查(血尿常规，血清自身抗体检测)、皮肤病理检查(皮损的HE染色剂免疫荧光带检查)综合确定的。由于红斑狼疮是一个进行性疾病且受多因素影响，临床上，病史(部分DLE或者SCLE患者也可表现为急性起病)、临床表现(部分DLE或者SCLE患者也可表现为SLE的蝶形红斑以及轻度系统受累)、实验室检查(部分DLE或者SCLE患者也可表现为白细胞减少，SLE相关致病自身抗体阳性)、皮肤病理检查(免疫荧光带检查阳性只能确诊是红斑狼疮，并不能区分DLE，SCLE和SLE)，这些指征和实验室检查均不能有效的区分红斑狼疮亚型。又由于不同亚型的红斑狼疮在治疗、随访及预后方面均不同，若不能明确的进行诊断分型，有可能会导致治疗上的过度用药或病情的延误。因此，红斑狼疮不同亚型的诊断及其鉴别诊断对于临床早期干预和精准治疗至关重要。目前缺少简便快捷、特异性 和灵敏度高的红斑狼疮辅助诊断试剂盒。因此建立有效的临床适用的试剂盒就显得尤为重要。
本发明是首个可有效区分红斑狼疮亚型的皮肤组织原位检测试剂盒，所检测的特征性分子标记包括：CD4、CD8、CD19、CD11b。CD4为CD4 +T淋巴细胞的特征性分子标记物、CD8为CD8 +T细胞的特征性分子标记物、CD19为B淋巴细胞的特征性分子标记物、CD11b则为中性粒细胞、NK细胞、单核/巨噬细胞共有的分子标记。利用本发明试剂盒获得的CD4-CD8-CD19-CD11b四联检测结果可很好的体现不同亚型红斑狼疮皮损中免疫细胞的分布与表达差异，样本的皮肤组织原位免疫细胞检测结果可进行精准的定量分析，从而进行红斑狼疮分型的辅助诊断。
本发明制备的皮肤组织原位多标记免疫染色试剂盒能够在一张皮肤组织切片上标记多个免疫细胞相关的特征性标记分子，并且不同标记分子信号之间不相互干扰，检测的准确度和特异度高。利用本试剂盒获得的CD4-CD8-CD19-CD11b的四联检测结果，搭配特定的拍照分析软件，可以做到精准度较高的免疫细胞分布及表达情况检测，同时满足临床上组织形态学和免疫细胞表达情况的定量检测需求，从而可有效区分红斑狼疮不同亚型。本试剂盒操作简单快捷，经济适用，适合在临床和科研工作中应用和推广。
附图说明
图1：在不同抗原修复条件和不同抗体稀释比条件下单克隆抗体染色信号比较；a为单克隆抗体CD11b在不同抗原修复条件和抗体稀释比的情况下信号的AS值和TS/BS值比较；b为单克隆抗体CD8在不同抗原修复条件和抗体稀释比的情况下信号的AS值和TS/BS值比较；c为单克隆抗体CD4在不同抗原修复条件和抗体稀释比的情况下信号的AS值和TS/BS值比较；d为单克隆抗体CD19在不同抗原修复条件和抗体稀释比的情况下信号的AS值和TS/BS值比较。
图2：在不同非特异性阻断剂和不同血清封闭时间下单克隆抗体染色结果比较；a为单用3％H 2O 2阻断15min的染色结果；b为单用0.5％高碘酸溶液阻断15min的染色结果；c为联合使用3％H 2O 2和0.5％高碘酸溶液阻断15min的染色结果；d为血清封闭15min的染色情况；e为血清封闭30min的染色情况；f为血清封闭60min的染色情况；g为血清封闭90min的染色情况。
图3：匹配不同酪胺荧光染色剂时单克隆抗体染色信号比较；a为单克隆抗体CD11b匹配不同酪胺荧光染色剂时信号的AS值和TS/BS值比较；b为单克隆抗体CD8匹配不同酪胺荧光染色剂时信号的AS值和TS/BS值比较；c为单克隆抗体CD4匹配不同酪胺荧光染色剂时信号的AS值和TS/BS值比较；d为单克隆抗体CD19匹配不同酪胺荧光染色剂时信号的AS值和TS/BS值比较。
图4:在不同染色顺序下单克隆抗体匹配相应酪胺荧光染色剂的染色信号比较；a为在染色顺序不同时单克隆抗体CD11b匹配相应酪胺荧光染色剂获得的信号的AS值和TS/BS值比较；b为在染色顺序不同时单克隆抗体CD8匹配相应酪胺荧光染色剂获得的信号的AS值和TS/BS值比较；c为在染色顺序不同时单克隆抗体CD4匹配相应酪胺荧光染色剂获得的信号的AS值和TS/BS值比较；d为在染色顺序不同时单克隆抗体CD19匹配相应酪胺荧光染色剂获得的信号的AS值和TS/BS值比较。
图5：在进行单个染色和进行多重染色时单克隆抗体染色信号比较；a为当每种单克隆抗体进行单个染色和进行多重染色时染色信号的AS值和TS/BS值比较；b为调整CD4抗体或染液敷育时间在进行多重染色时染色信号的AS值和TS/BS值比较；c为调整CD19的抗体或染液敷育时间在进行多重染色时染色信号的AS值和TS/BS值比较。
图6使用最终方案染色处理多种单克隆抗体的染色之间相互无干扰效果图；其中细胞核为蓝色，CD4为红色，CD8为白色，CD11b为黄色，CD19为绿色；a为多标记免疫荧光染色结果；b为图6a光谱拆分处理后的CD4单色图；c为图6a光谱拆分处理后的CD8单色图；d为图6a光谱拆分处理后的CD11b单色图；e为图6a光谱拆分处理后的CD19单色图；f为图6a光谱拆分处理后的CD4和CD19双色图。
图7：使用最终方案染色处理四种不同来源皮肤组织样本多种单克隆抗体的染色效果图；其中细胞核为蓝色，CD4为红色，CD8为白色，CD11b为黄色，CD19为绿色；a为DLE患者皮损使用最终方案多重免疫荧光染色结果；b为SCLE患者皮损使用最终方案多重免疫荧光染色结果；c为SLE患者皮损使用最终方案多重免疫荧光染色结果；d为正常人皮肤组织使用最终方案多重免疫荧光染色结果。
图8：ROC数据分析结果；a为正常人与红斑狼疮患者的CD4-CD8-CD19-CD11b的四联检测ROC分析图；b为DLE与非DLE患者的CD4-CD8-CD19-CD11b的四联检测ROC分析图；c为SCLE与SLE患者的CD4-CD8-CD19-CD11b的四联检测ROC分析图。
图9：皮肤原位多类型免疫细胞联合检测在诊断分型红斑狼疮中的应用策略。
具体实施方式
以下实施例将有助于本领域的普通技术人员进一步理解本发明。基于本发明中的实施方式，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有实施案例，都属于本发明保护的范围。
试剂的准备：
1)抗原修复液：
酸性修复液(pH6.0)：3g柠檬酸三钠，0.4g柠檬酸，20ul吐温20，1000ml灭菌去离子水；
碱性修复液(pH8.0)：1.8g EDTA，0.9g EDTA-2Na，1000ml灭菌去离子水，约8ml10％NaOH调pH值至8.0；
2)PBST-T溶液(磷酸盐吐温曲拉通缓冲液)：1xPBS粉末，5ul吐温20，1ul TritonX-100，1000ml灭菌去离子水；
3)血清封闭液：0.8g BSA粉末，10ml PBST-T溶液，充分混匀，配置好后可4度存放2周；
4)抗体稀释液：0.1g BSA粉末，10ml 1xPBS溶液，充分混匀，配置好后可4度存放2周；
5)TSA染液(购自PerkinElmer)，现配现用：
Opal520工作液：Opal520染色液原液与配套放大稀释液1:100稀释；
Opal570工作液：Opal570染色液原液与配套放大稀释液1:100稀释；
Opal650工作液：Opal650染色液原液与配套放大稀释液1:100稀释；
Opal690工作液：Opal690染色液原液与配套放大稀释液1:100稀释。
实施例1：皮肤组织原位多标记免疫染色试剂盒的制备方法
多标记免疫染色的结果拍摄是通过四个颜色通道的滤光片的处理来实现的 (DAPI，FITC,Cy3,Cy5)，不同滤光片的发射光谱之间会发生一定重叠。而多光谱分析软件在进行光谱的识别和拆分时是依据光谱的峰值来判断的，在多种单克隆抗体共染于一张组织切片时，多抗体对应的光谱会发生不同程度的重叠。当某种抗体显色过强，则其显色光谱可能会覆盖临近抗体的光谱，从而导致两种或多种抗体间相互串色的结果发生。为了避免这种情况的出现，皮肤组织原位多标记免疫染色试剂盒的制备，各个关键步骤的条件优化至关重要。
1.1确定单克隆抗体染色最佳抗原修复条件及最佳稀释比范围。
每种单克隆抗体都如下表(表1-4)经过了不同抗原修复液(酸性修复液和碱性修复液)，不同抗原修复条件(高压修复和微波修复)，不同稀释比的比较，从而确定每种单克隆抗体染色最佳抗原修复条件及最佳稀释比范围。本发明选用了细胞平均荧光信号强度(Average Signal of cell，后续图表中以AS来指代)、细胞总荧光信号强度与组织区域总背景信号强度的比值(Total signal of cell/Background signal，后续图表中以TS/BS来指代)来确定，AS相对最高的同时，TS/BS相对最高的条件即为可确保染色阳性结果信号最强而背景最低的最佳条件(见附图1)。
表1.单克隆抗体CD11b不同抗原修复条件和抗体稀释比的染色结果
Figure PCTCN2020095791-appb-000001
表2 单克隆抗体CD8不同抗原修复条件和抗体稀释比的染色结果
Figure PCTCN2020095791-appb-000002
表3 单克隆抗体CD4不同抗原修复条件和抗体稀释比的染色结果
Figure PCTCN2020095791-appb-000003
表4 单克隆抗体CD19不同抗原修复条件和抗体稀释比的染色结果
Figure PCTCN2020095791-appb-000004
通过比较，每种单克隆抗体染色最佳抗原修复条件及最佳稀释比范围如下：
a)图1a显示单克隆抗体CD11b的最佳修复条件为：酸性微波修复或酸性高压修复，优选酸性微波修复；最佳抗体稀释比范围为：1:1000-1:2000，优选1:1400-1600；进一步优选1:1500；
b)图1b显示单克隆抗体CD8的最佳修复条件为：碱性微波修复或碱性高压修复，优选碱性高压修复；最佳抗体稀释比范围为：1:2-1:5，优选1:2-1:4，进一步优选1:3；
c)图1c显示单克隆抗体CD4的最佳修复条件为：碱性微波修复或碱性高压修复，优选碱性高压修复；最佳抗体稀释比范围为：1:2-1:5，优选1:2-1:3，进一步优选1:2；
d)图1d显示单克隆抗体CD19的最佳修复条件为：碱性微波修复或碱性高压修复，优选碱性高压修复；最佳抗体稀释比范围为：1:200-1:500，优选1:250-1:350， 进一步优选1:300。
1.2确定单克隆抗体染色非特异性背景最低的非特异性阻断条件及血清封闭液封闭时间。
为确定单克隆抗体染色非特异性背景最低的非特异性阻断条件及血清封闭液封闭时间，我们选择了四种单克隆抗体中，最容易有非特异性染色的单克隆抗体CD11b来进行非特异性阻断剂血清封闭的条件优化。在抗原修复条件及抗体稀释比相同的条件下(酸性微波修复，稀释比1:1500)，我们对比了单用3％H 2O 2阻断、单用0.5％高碘酸溶液、联合使用3％H 2O 2和0.5％高碘酸溶液阻断在减少非特异性背景上的效果。图2a为单用3％H 2O 2阻断15min的染色结果，可见皮肤的腺体区域有较亮的非特异性着色，且整个组织背景较高；图2b为单用0.5％高碘酸溶液阻断15min的染色结果，图2c为联合使用3％H 2O 2和0.5％高碘酸溶液阻断15min的染色结果，两种阻断方式对减少非特异性染色的效果均较理想，可见皮肤腺体区域的非特异性着色显著减少，且整个组织背景干净，阳性染色清晰，对比度高。
可见，单用3％H 2O 2阻断的效果最差，而单用0.5％高碘酸溶液、联合使用3％H 2O 2和0.5％高碘酸溶液阻断两者之间没有明显差别，故为了操作的简便性和节约试剂成本，此处的非特异性阻断剂优选为0.5％的高碘酸溶液。
此外我们还比对了不同血清封闭时间对染色阳性率的影响(15min，30min，60min，90min)，图2d为血清封闭15min的染色情况，图2e为血清封闭30min的染色情况，两种封闭时间对染色阳性率的影响差别不大，组织整体染色背景干净，阳性染色清晰；图2f为血清封闭60min的染色情况，图2g为血清封闭90min的染色情况，相较于封闭15-30min的染色结果，这两种血清封闭时间下的阳性染色明显减少，且信号略弱，染色阳性率下降，说明过长的血清封闭会封闭一些特异性结合位点，进而导致染色的假阴性，故为了节约操作中的时间成本，此处的血清封闭时间优选为室温封闭15min。
1.3确定与每个单克隆抗体匹配度最佳的酪胺荧光染色剂。
每种单克隆抗体如下表(表5)经过了与不同酪胺荧光染色剂匹配的比较，从而确定与每个单克隆抗体匹配度最佳的酪胺荧光染色剂。本发明选用了细胞平均荧光信号强度(Average Signal of cell，后续图表中以AS来指代)、细胞总荧 光信号强度与组织区域总背景信号强度的比值(Total signal of cell/Background signal，后续图表中以TS/BS来指代)来确定，AS相对最高的同时，TS/BS相对最高的条件即为与对应单克隆抗体匹配度最佳的酪胺荧光染色剂(见附图3)。
图3a当CD11b匹配Opal570染色剂和Opa650染色剂时，其AS值及TS/BS值相对较高；图3b当CD8匹配Opal570染色剂和Opa650染色剂时，其AS值及TS/BS值相对较高；图3c当CD4匹配Opal520染色剂、Opal570染色剂和Opa650染色剂时，其AS值及TS/BS值相对较高；图3d当CD19匹配Opal570染色剂、Opa650染色剂和Opa690染色剂时，其AS值及TS/BS值相对较高。
表5 每种单克隆抗体匹配不同酪胺荧光染色剂的染色结果比较
Figure PCTCN2020095791-appb-000005
通过比较，与每个单克隆抗体匹配度最佳的酪胺荧光染色剂分别为：
a)单克隆抗体CD11b可匹配的酪胺荧光染色剂为：Opal570染色剂和Opal650染色剂；
b)单克隆抗体CD8可匹配的酪胺荧光染色剂为：Opal570染色剂和Opal650染色剂；
c)单克隆抗体CD4可匹配的酪胺荧光染色剂为：Opal520染色剂、Opal570染色剂和Opal650染色剂；
d)单克隆抗体CD19可匹配的酪胺荧光染色剂为：Opal570染色剂、Opa650染色剂和Opa690染色剂。
1.4确定每个单克隆抗体的染色顺序。
由于每种单克隆抗体对应的酪胺荧光染色剂是依次共价偶联在单克隆抗体对应的抗原表位上的，且需要通过多轮微波或高压处理来去除上一轮非共价结合的一抗和二抗，因此需要确定每个单克隆抗体的染色顺序，以保证在多轮微波 或高压处理后，单克隆抗体对应的抗原表位的完整性不受影响，此外，还需确保最早偶联于抗原表位上的酪胺荧光染色剂能耐受后续加热过程。关于如何量化的去比较，本发明选用了细胞平均荧光信号强度(Average Signal of cell，后续图表中以AS来指代)、细胞总荧光信号强度与组织区域总背景信号强度的比值(Total signal of cell/Background signal，后续图表中以TS/BS来指代)来确定，AS相对最高的同时，TS/BS相对最高的组即为单克隆抗体对应最佳的染色顺序。
表6 单克隆抗体匹配相应酪胺荧光染色剂，在不同染色顺序下的染色结果比较
Figure PCTCN2020095791-appb-000006
基于以上要点，根据表格6进行了每一种单克隆抗体-匹配相应酪胺荧光染色剂，在不同染色顺序下的信号值比较(见附图4)。有如下发现：
1)图4a显示当CD11b匹配Opal570染色剂或Opal650染色剂，处于第一轮染色的位置时，其AS值及TS/BS值相对较高，其中，CD11b-Opal570染色剂-第一轮染色的搭配获得的AS值及TS/BS值最高；
2)图4b显示当CD8匹配Opal570染色剂或Opal650染色剂，处于第二轮染色的位置时，其AS值及TS/BS值相对较高，其中，CD8-Opal650染色剂-第二轮染色的搭配获得的AS值及TS/BS值最高；
3)图4c显示当CD4匹配Opal520染色剂或Opal570染色剂，处于第三或第四轮染色的位置时，其AS值及TS/BS值相对较高，其中CD4-Opal520染色剂-第三轮或第四轮染色的搭配获得的AS值及TS/BS值最高；
4)图4d显示当CD19匹配Opa690染色剂，处于第三轮或第四轮染色的位置时，其AS值及TS/BS值相对较高，其中CD19-Opal690染色剂-第四轮染色的搭配获得的AS值及TS/BS值最高。
从而确定了每个单克隆抗体的最佳染色顺序及匹配度最佳酪胺荧光染色剂如下：
a)染色第一轮为单克隆抗体CD11b，对应酪胺荧光染色剂为Opal570；
b)染色第二轮为单克隆抗体CD8，对应酪胺荧光染色剂为Opal650；
c)染色第一轮为单克隆抗体CD4，对应酪胺荧光染色剂为Opal520；
d)染色第一轮为单克隆抗体CD19，对应酪胺荧光染色剂为Opal690。
1.5通过每个单克隆抗体的单个染色和多重染色中信号的比较，进一步确定多标记免疫染色的方案中各单克隆抗体的敷育时间，对应二抗及染色剂敷育时间。
上一步骤中，比较了每种单克隆抗体进行单次抗原修复或进行多次抗原修复后，抗体染色信号的差异。而在进行多种单克隆抗体的叠加染色种，前续单克隆抗体对应酪胺荧光染色剂与抗原表位的结合，有可能会影响到后续单克隆抗体对应酪胺荧光染色剂与抗原表位的结合，通过对每种单克隆抗体进行单个染色(即不叠加其它的抗体，只是染色和多轮抗原修复)和进行多重染色(即按照步骤1.4确定的配色方案完成整个多标记染色过程)时，其染色信号的AS值和TS/BS值的比较，我们进一步确定多标记免疫染色的方案中各单克隆抗体的敷育时间，对应二抗及染色剂敷育时间，如下所示(见附图5)：
a)图5a显示CD11b信号在单个染色和多重染色比较中无明显差异，故抗体敷育时间及二抗染色剂敷育时间无需调整；
b)图5a显示CD8信号在单个染色和多重染色比较中无明显差异，故抗体敷育时间及二抗染色剂敷育时间无需调整；
c)图5b显示CD4信号在多重染色中的信号明显弱于单个染色中的信号，而当CD4一抗敷育时间延长至75min、二抗敷育时间延长至20min、Opal520染色剂 敷育时间延长至30min，其AS值及TS/BS值相对较高，调整后CD4信号与进行单个染色时的信号接近；
d)图5c显示CD19信号在多重染色中的信号明显弱于单个染色中的信号，当CD19一抗敷育时间延长至75min、二抗敷育时间延长至20min、Opal520染色剂敷育时间延长至20min，其AS值及TS/BS值相对较高，调整后CD4信号与进行单个染色时的信号接近。
1.6确定最终方案所获结果中，多种单克隆抗体的染色之间相互无干扰，无串色(见附图6-7)。
图6a为多标记免疫荧光染色结果；其中细胞核为蓝色，CD4为红色，CD8为白色，CD11b为黄色，CD19为绿色，可见四种单克隆抗体信号在多重染色中均无明显缺失。图6b显示CD4信号在单个染色中无明显缺失；图6c显示CD8信号在单个染色中无明显缺失；图6d显示CD11b信号在单个染色中无明显缺失；图6e显示CD4信号在单个染色中无明显缺失；图6f显示CD4和CD19信号在双重染色中无明显缺失。
图7中细胞核为蓝色，CD4为红色，CD8为白色，CD11b为黄色，CD19为绿色；可见最终方案在四种来源样本中染色均能达到稳定无干扰效果。
实施例2：皮肤组织原位多标记免疫染色试剂盒的使用方法。
2.1石蜡切片预处理
选取皮肤组织石蜡切片，60℃烤片2小时；2小时后取出，放入预热的松节油中浸泡15分钟，然后依次在100％乙醇、95％乙醇、70％乙醇、灭菌水中各浸泡10分钟。
2.2染色第一轮：CD11b-Opal570
1)取酸性修复液(pH6.0)200ml，放入盛有切片的抗原修复盒中，将抗原修复盒放入微波炉中，调至高火档位，加热2-3min(具体以操作环境和使用电器品牌不同而定，时间的确定根据观察的修复盒液面出现沸腾为准)，待修复盒里修复液开始沸腾，迅速调至低火档位，继续修复20min；
2)修复结束后，关闭微波炉，使切片自然冷却至室温；
3)取0.5％高碘酸溶液200ml，浸泡处理切片15min；
4)灭菌去离子水浸泡洗涤切片2min；
5)PBST-T溶液浸泡洗涤切片5min；
6)擦干切片组织周围液体，用免疫组化笔圈出玻片上的样本区域，每个样本区域滴加配置好的血清封闭液150-200ul，湿盒内室温敷育15min；
7)在步骤6)进行过程中，配置CD11b抗体工作液(1:1500稀释)：1ul CD11b抗体原液(Abcam#ab52478)，25ul羊血清，1474ul抗体稀释液(可在4℃存放2周)；
8)血清封闭液敷育结束后，弃去切片上的封闭液，滴加配置好的CD11b抗体工作液，使其充分覆盖组织，室温敷育1小时；
9)PBST-T溶液浸泡洗涤切片3次，每次5min；
10)滴加二抗溶液(羊抗兔/鼠即用型二抗，PerkinElmer)，室温孵育10分钟；
11)PBST-T溶液浸泡洗涤切片3次，每次5min；
12)滴加配置好的Opal570染液，室温保湿孵育10分钟；
13)PBST-T溶液浸泡洗涤切片3次，每次5min。
2.3染色第二轮：CD8-Opal650
1)取碱性修复液(pH8.0)200ml，放入抗原修复盒中，然后放入已完成CD11b染色的切片，确保修复液已完全浸没切片的组织区域；
2)将抗原修复盒放入高压锅中(高压锅预先盛有500ml自来水)，调节电磁炉功率在1000W，待高压锅上汽后，继续修复7min，；
3)修复结束后，关闭高压锅，使切片自然冷却至室温；
4)取0.5％高碘酸溶液200ml，浸泡处理切片15min；
4)灭菌去离子水浸泡洗涤切片2min；
5)擦干切片组织周围液体，用免疫组化笔圈出玻片上的样本区域，每个样本区域滴加配置好的血清封闭液150-200ul，湿盒内室温敷育15min；
6)在步骤5)进行过程中，配置CD8抗体工作液(1:3稀释)：500ul CD8抗体原液(福州迈新，MAB-0021)，25ul羊血清，975ul抗体稀释液(可在4℃存放2周)；
7)血清封闭液敷育结束后，弃去切片上的封闭液，滴加配置好的CD8抗体工作液，使其充分覆盖组织，室温敷育1小时；
8)PBST-T溶液浸泡洗涤切片3次，每次5min；
9)滴加二抗溶液(羊抗兔/鼠即用型二抗，PerkinElmer)，室温孵育10分钟；
10)PBST-T溶液浸泡洗涤切片3次，每次5min；
11)滴加配置好的Opal650染液，室温保湿孵育10分钟；
12)PBST-T溶液浸泡洗涤切片3次，每次5min。
2.4染色第三轮：CD4-Opal520
1)取碱性修复液(pH8.0)200ml，放入抗原修复盒中，然后放入已完成CD8染色的切片，确保修复液已完全浸没切片的组织区域；
2)将抗原修复盒放入高压锅中(高压锅预先盛有500ml自来水)，调节高压锅功率在1000W，待高压锅上汽后，继续修复7min，；
3)修复结束后，关闭高压锅，使切片自然冷却至室温；
4)取0.5％高碘酸溶液200ml，浸泡处理切片15min；
5)灭菌去离子水浸泡洗涤切片2min；
6)擦干切片组织周围液体，用免疫组化笔圈出玻片上的样本区域，每个样本区域滴加配置好的血清封闭液150-200ul，湿盒内室温敷育15min；
7)在步骤6)进行过程中，配置CD4抗体工作液(1:2稀释)：750ul CD4抗体原液(福州迈新，RMA-0620)，25ul羊血清，725ul抗体稀释液(可在4℃存放2周)；
8)血清封闭液敷育结束后，弃去切片上的封闭液，滴加配置好的CD4抗体工作液，使其充分覆盖组织，室温敷育75min；
9)PBST-T溶液浸泡洗涤切片3次，每次5min；
10)滴加二抗溶液(羊抗兔/鼠即用型二抗，PerkinElmer)，室温孵育20分钟；
11)PBST-T溶液浸泡洗涤切片3次，每次5min；
12)滴加配置好的Opal520染液，室温保湿孵育30分钟；
13)PBST-T溶液浸泡洗涤切片3次，每次5min。
2.5染色第四轮：CD19-Opal690
1)取碱性修复液(pH8.0)200ml，放入抗原修复盒中，然后放入已完成CD4染色的切片，确保修复液已完全浸没切片的组织区域；
2)将抗原修复盒放入高压锅中(高压锅预先盛有500ml自来水)，调节高压锅功率在1000W，待高压锅上汽后，继续修复7min，；
3)修复结束后，关闭高压锅，使切片自然冷却至室温；
4)取0.5％高碘酸溶液200ml，浸泡处理切片15min；
5)灭菌去离子水浸泡洗涤切片2min；
6)擦干切片组织周围液体，用免疫组化笔圈出玻片上的样本区域，每个样本区域滴加配置好的血清封闭液150-200ul，湿盒内室温敷育15min；
7)在步骤6)进行过程中，配置CD19抗体工作液：5ul CD19抗体原液(Abcam,#ab227688)，25ul羊血清，1470ul抗体稀释液(可在4℃存放2周)；
8)血清封闭液敷育结束后，弃去切片上的封闭液，滴加配置好的CD19抗体工作液，使其充分覆盖组织，室温敷育75min；
9)PBST-T溶液浸泡洗涤切片3次，每次5min；
10)滴加二抗溶液(羊抗兔/鼠即用型二抗，PerkinElmer)，室温孵育20分钟；
11)PBST-T溶液浸泡洗涤切片3次，每次5min；
12)滴加配置好的Opal690染液，室温保湿孵育20分钟；
13)PBST-T溶液浸泡洗涤切片3次，每次5min。
2.6核复染及切片的存放
1)取酸性修复液(pH6.0)200ml，放入盛有切片的抗原修复盒中，将抗原修复盒放入微波炉中，调至高火档位，加热2-3min(具体以操作环境和使用电器品牌不同而定，时间的确定根据观察的修复盒液面出现沸腾为准)，待修复盒里修复液开始沸腾，迅速调至低火档位，继续修复20min；
2)修复结束后，关闭电热炉，使切片自然冷却至室温；
3)灭菌去离子水浸泡洗涤切片2min；
4)滴加DAPI(Vector Laboratories),湿盒内室温敷育10min，盖片并用无色指甲油封住切片边缘。存放于-20℃冰箱。需要读取结果时，随时从冰箱中取出，置于显微镜下观察。
2.7染色结果拍照和保存
1)启动电脑，打开电子显微镜电源及荧光机箱电源，荧光机箱启动后不可马上使用，应待机箱前部指示灯稳定后(即不再滚动闪烁)，再开启电脑主界面的Mantra操作软件(购自Perkinelmer)；
2)将完成染色的切片放置于显微镜卡座中，将切片平稳固定住后，将切片的组织 区域移至显微镜的正下方；
3)鼠标左键单击Mantra操作界面右上角的设置按钮，点击“新的操作”选项新建一个操作；
4)选择荧光模式，点击“添加通道”按钮来添加所需要的荧光通道，本试剂盒需要的是Fitc(Opal520染色剂经此通道)，Cy3(Opal570染色剂经此通道)，Cy5(Opal650和Opal690染色剂经此通道)，DAPI(核复染剂DAPI经此通道，为本操作命名为“CD4-CD8-CD19-CD11b”；
5)向前推显微镜下方的挡光板，确认明场光源处于遮挡状态，调节荧光机箱光强度至25％的位置；
6)选择合适的物镜，本试剂盒使用中选择的是20x物镜，一边观察电脑中显示的视野一遍移动载物台，直至选定合适视野，调整好焦距和曝光时间，点击拍照；
7)拍照完成的结果用Inform软件打开，单击左上方配置按钮，新建一个程序，勾选手动划分组织区域，细胞定位分割，细胞分型，结果导出这4个选项组成一个名为“CD4-CD8-CD19-CD11b”的程序；
8)选择所需的光谱，本试剂盒需要的是Fitc(Opal520染色剂经此通道)，Cy3(Opal570染色剂经此通道)，Cy5(Opal650和Opal690染色剂经此通道)，DAPI(核复染剂DAPI经此通道)，然后点击左下角的“准备全部”按钮，对所有结果图片进行去背景处理；
9)下一步利用手动划分组织区域功能，划分出自己需要分析的组织视野。因本试剂盒是用于红斑狼疮皮损的炎症浸润区免疫细胞的分析，故此步应将表皮、腺体等划分出去，不纳入分析；
10)下一步利用细胞定位分割功能，选择待分析细胞器类型如细胞核、细胞质或细胞膜(本试剂盒使用的分子标记均表达定位在细胞膜上，故勾选细胞膜)，选择待分析的组织区域(勾选上一步确定的免疫细胞浸润区域)，选择特定细胞器类型的染色光谱(勾选Fitc，Cy3，Cy5和DAPI)。单击左下方的细胞分割按钮获得细胞器类型分析结果；
11)下一步利用细胞分型功能，自行标记出10个每种单克隆抗体对应的阳性结果。本试剂盒中，手动标记CD4 +细胞，CD19 +细胞，CD8 +细胞，CD11b +细胞，Other细胞(CD4 -CD8 -CD19 -CD11b -细胞)各10个，然后点击左下角的细胞分型按钮， 分析软件开始根据手动标记出的阳性细胞进行自学习，并对待分析组织区域的所有细胞进行细胞分型，最后获得CD4 +细胞，CD19 +细胞，CD8 +细胞，CD11b +细胞，Other细胞(CD4 -CD8 -CD19 -CD11b -细胞)这五种细胞的细胞计数结果；
12)点击下一步，导出细胞计数的结果至文件夹，拷出数据，进一步根据细胞计数结果，用excel表格计算出各类细胞占整体细胞的阳性百分比。
实施例3：可辅助红斑狼疮诊断分型的皮肤组织原位多标记免疫染色试剂盒检测指标的确定
3.1区分正常人和狼疮
1)选择来自不同患者DLE、SLE、SCLE皮损样本各18例，并选择45例正常人皮肤作为对照，利用皮肤组织原位多标记免疫染色试剂盒进行样本检测，获得相应数据；
2)使用IBM SPSS Statistics 23构建ROC分析模型，将54例狼疮患者数据设为病例组LE，45例正常人数据设为正常对照NC；
3)以CD4的单阳检测结果(CD4/DAPI)、CD8的单阳检测结果(CD8/DAPI)、CD19的单阳检测结果(CD19/DAPI)、CD11b的单阳检测结果(CD11b/DAPI)、CD4-CD8的二联检测结果((CD4+CD8)/DAPI)、CD4-CD19的二联检测结果((CD4+CD19)/DAPI)、CD4-CD11b的二联检测结果((CD4+CD11b)/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD8-CD11b的二联检测结果((CD8+CD11b)/DAPI)、CD19-CD11b的二联检测结果((CD19+CD11b)/DAPI)、CD4-CD8-CD19的三联检测结果((CD4+CD8+CD19)/DAPI)、CD4-CD19-CD11b的三联检测结果((CD4+CD19+CD11b)/DAPI)、CD4-CD8-CD11b的三联检测结果((CD4+CD8+CD11b)/DAPI)、CD8-CD19-CD11b的三联检测结果((CD8+CD19+CD11b)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果，各为一个数据组，整理成表导入软件；
4)构建每个数据组的ROC模型，导出ROC曲线及所有分界值数据(最小分界值为最小实测检验值减1，最大分界值为最大实测检验值加1，所有其他分界值均为两个连续的有序实测检验值的平均值)；
5)计算出每个分界值的灵敏度、特异度、约登指数、阴性似然比及阳性似然比，以约登指数最大的分界值作为临界值(cut off值)；
6)根据ROC曲线下面积(AUC)判断模型诊断价值，筛选出诊断预测价值较高的几组数据(AUC>0.90，诊断预测价值较高；AUC＝[0.7-0.9],诊断预测价值中等；AUC＝[0.5-0.7],诊断预测价值较低；AUC＝0.5，模型没有诊断预测价值)，包括：CD8的单阳检测结果(CD8/DAPI)、CD19的单阳检测结果(CD19/DAPI)、CD4-CD8的二联检测结果((CD4+CD8)/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD8-CD11b的二联检测结果((CD8+CD11b)/DAPI)、CD4-CD8-CD19的三联检测结果((CD4+CD8+CD19)/DAPI)、CD4-CD8-CD11b的三联检测结果((CD4+CD8+CD11b)/DAPI)、CD8-CD19-CD11b的三联检测结果((CD8+CD19+CD11b)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果，这9组数据的AUC值均>0.9，具有较高的诊断区分效能(结果见表7)；
7)使用GraphPad Prism 8.0对54例LE及45例NC数据做非参数检验，设α为0.05，以p值<0.05认为两组间差异有统计学意义。结果发现CD4的单阳检测结果(CD4/DAPI)、CD8的单阳检测结果(CD8/DAPI)、CD19的单阳检测结果(CD19/DAPI)、CD11b的单阳检测结果(CD11b/DAPI)、CD4-CD8的二联检测结果((CD4+CD8)/DAPI)、CD4-CD19的二联检测结果((CD4+CD19)/DAPI)、CD4-CD11b的二联检测结果((CD4+CD11b)/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD8-CD11b的二联检测结果((CD8+CD11b)/DAPI)、CD19-CD11b的二联检测结果((CD19+CD11b)/DAPI)、CD4-CD8-CD19的三联检测结果((CD4+CD8+CD19)/DAPI)、CD4-CD19-CD11b的三联检测结果((CD4+CD19+CD11b)/DAPI)、CD4-CD8-CD11b的三联检测结果((CD4+CD8+CD11b)/DAPI)、CD8-CD19-CD11b的三联检测结果((CD8+CD19+CD11b)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果在正常人与红斑狼疮患者两组间差异均有统计学意义，其中步骤6)中筛选出的预测诊断价值较高的9组数据在正常人与红斑狼疮患者两组间统计学差异显著(p值<0.0001)。
表7 正常人与狼疮患者ROC分析结果
Figure PCTCN2020095791-appb-000007
Figure PCTCN2020095791-appb-000008
3.2区分DLE与非DLE
1)将18例DLE患者数据设为DLE组，将SLE和SCLE患者数据共36例设为非DLE组；
2)以CD4的单阳检测结果(CD4/DAPI)、CD8的单阳检测结果(CD8/DAPI)、CD19的单阳检测结果(CD19/DAPI)、CD11b的单阳检测结果(CD11b/DAPI)、CD4-CD8的二联检测结果((CD4+CD8)/DAPI)、CD4-CD19的二联检测结果((CD4+CD19)/DAPI)、CD4-CD11b的二联检测结果((CD4+CD11b)/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD8-CD11b的二联检测结果((CD8+CD11b)/DAPI)、CD19-CD11b的二联检测结果((CD19+CD11b)/DAPI)、CD4-CD8-CD19的三联检测结果((CD4+CD8+CD19)/DAPI)、CD4-CD19-CD11b的三联检测结果((CD4+CD19+CD11b)/DAPI)、CD4-CD8-CD11b的三联检测结果((CD4+CD8+CD11b)/DAPI)、CD8-CD19-CD11b的三联检测结果((CD8+CD19+CD11b)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果，各为 一个数据组，整理成表导入软件；
3)使用IBM SPSS Statistics 23构建每个数据组的ROC模型，导出ROC曲线及所有分界值数据(最小分界值为最小实测检验值减1，最大分界值为最大实测检验值加1，所有其他分界值均为两个连续的有序实测检验值的平均值)；
4)计算出每个分界值的灵敏度、特异度、约登指数、阴性似然比及阳性似然比，以约登指数最大的分界值作为临界值(cut off值)；
5)根据ROC曲线下面积(AUC)判断模型诊断价值，筛选出诊断预测价值较高的几组数据(AUC>0.90，诊断预测价值较高；AUC＝[0.7-0.9],诊断预测价值中等；AUC＝[0.5-0.7],诊断预测价值较低；AUC＝0.5，模型没有诊断预测价值)，包括：CD19的单阳检测结果(CD19/DAPI)、CD11b的单阳检测结果(CD11b/DAPI)、CD4-CD19的二联检测结果((CD4+CD19)/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD9-CD11b的二联检测结果((CD9+CD11b)/DAPI)、CD4-CD8-CD19的三联检测结果((CD4+CD8+CD19)/DAPI)、CD4-CD19-CD11b的三联检测结果((CD4+CD19+CD11b)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果，这8组数据的AUC值均>0.8，具有较高的诊断区分效能(结果见表8)；
6)使用GraphPad Prism 8.0对18例DLE及36例非DLE数据做非参数检验，设α为0.05，以p值<0.05认为两组间差异有统计学意义。结果发现CD4的单阳检测结果(CD4/DAPI)、CD19的单阳检测结果(CD19/DAPI)、CD11b的单阳检测结果(CD11b/DAPI)、CD4-CD19的二联检测结果((CD4+CD19)/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD19-CD11b的二联检测结果((CD19+CD11b)/DAPI)、CD4-CD8-CD19的三联检测结果((CD4+CD8+CD19)/DAPI)、CD4-CD19-CD11b的三联检测结果((CD4+CD19+CD11b)/DAPI)、CD8-CD19-CD11b的三联检测结果((CD8+CD19+CD11b)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果在DLE与非DLE两组间差异均有统计学意义，其中以步骤5)筛选出的预测诊断价值较高的8组数据在DLE与非DLE两组间统计学差异显著(p值<0.001)。
表8.DLE与非DLE患者ROC分析结果
Figure PCTCN2020095791-appb-000009
3.3区分SLE与SCLE
1)将18例SCLE患者数据设为病例组SCLE，18例SLE患者数据设为对照组SLE；2)以CD4的单阳检测结果(CD4/DAPI)、CD8的单阳检测结果(CD8/DAPI)、CD19的单阳检测结果(CD19/DAPI)、CD11b的单阳检测结果(CD11b/DAPI)、CD4-CD8的二联检测结果((CD4+CD8)/DAPI)、CD4-CD19的二联检测结果((CD4+CD19)/DAPI)、CD4-CD11b的二联检测结果((CD4+CD11b)/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD8-CD11b的二联检测结果((CD8+CD11b)/DAPI)、CD19-CD11b的二联检测结果((CD19+CD11b)/DAPI)、CD4-CD8-CD19的三联检测结果((CD4+CD8+CD19)/DAPI)、CD4-CD19-CD11b的三联检测结果((CD4+CD19+CD11b)/DAPI)、CD4-CD8-CD11b的三联检测结 果((CD4+CD8+CD11b)/DAPI)、CD8-CD19-CD11b的三联检测结果((CD8+CD19+CD11b)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果，各为一个数据组，整理成表导入软件；
3)使用IBM SPSS Statistics 23构建每个数据组的ROC模型，，导出ROC曲线及所有分界值数据(最小分界值为最小实测检验值减1，最大分界值为最大实测检验值加1，所有其他分界值均为两个连续的有序实测检验值的平均值)；
4)计算出每个分界值的灵敏度、特异度、约登指数、阴性似然比及阳性似然比，以约登指数最大的分界值作为临界值(cut off值)；
5)根据ROC曲线下面积(AUC)判断模型诊断价值，筛选出诊断预测价值较高的几组数据(AUC>0.90，诊断预测价值较高；AUC＝[0.7-0.9],诊断预测价值中等；AUC＝[0.5-0.7],诊断预测价值较低；AUC＝0.5，模型没有诊断预测价值)，包括：CD4的单阳检测结果(CD4/DAPI)、CD19的单阳检测结果(CD19/DAPI)、CD4-CD8的二联检测结果((CD4+CD8)/DAPI)、CD4-CD19的二联检测结果((CD4+CD19)/DAPI)、CD4-CD11b的二联检测结果((CD4+CD11b)/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD8-CD11b的二联检测结果((CD8+CD11b)/DAPI)、CD4-CD8-CD19的三联检测结果((CD4+CD8+CD19)/DAPI)、CD4-CD19-CD11b的三联检测结果((CD4+CD19+CD11b)/DAPI)、CD4-CD8-CD11b的三联检测结果((CD4+CD8+CD11b)/DAPI)、CD8-CD19-CD11b的三联检测结果((CD8+CD19+CD11b)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果，这12组数据的AUC值均>0.7，具有较高的诊断区分效能，其中只有CD4-CD8-CD19-CD11b的四联检测结果的AUC>0.8(结果见表9)；
6)使用GraphPad Prism 8.0对18例SCLE及18例SLE数据做非参数检验，设α为0.05，以p值<0.05认为两组间差异有统计学意义。结果发现步骤5)中获得的预测诊断价值较高的12组数据在SCLE与SLE两组间的差异均有统计学意义(p值<0.05)，其中，只有CD4-CD8-CD19-CD11b的四联检测结果在SCLE与SLE两组间的差异显著(p值<0.001)。
表9.SCLE与SLE患者ROC分析结果
Figure PCTCN2020095791-appb-000010
3.4综合LEvsNC，DLEvs非DLE，SCLEvsSLE的结果，确定可辅助红斑狼疮诊断分型的皮肤组织原位多标记免疫染色试剂盒检测指标
1)将LEvsNC，DLEvs非DLE，SCLEvsSLE的ROC分析结果(AUC)以及非参数检验的分析结果(P值：*p<0.05，**p<0.01，***p<0.001，****p<0.0001，ns表示p>0.05无统计学差异)汇总，见表10。通过汇总表格来筛选对LEvsNC，DLEvs非DLE，SCLEvsSLE均有较好诊断区分效能的检测指标。鉴于AUC>0.90，诊断预测价值较高，AUC＝[0.7-0.9],诊断预测价值中等，AUC＝[0.5-0.7]，诊断预测价值较低，故评估可行性方法如下：
a)若有检测指标在区分任意一组(LEvsNC，DLEvs非DLE，SCLEvsSLE)中AUC<0.7，则该检测指标被视为不能有效诊断区分红斑狼疮不同亚型，在可行性 一栏标记为×，即不可行；
b)若有检测指标在区分任意两组及以上(LEvsNC，DLEvs非DLE，SCLEvsSLE)中0.7<AUC<0.8，则该检测指标被视为不能有效诊断区分红斑狼疮不同亚型，在可行性一栏标记为×，即不可行；
c)若有检测指标在区分所有组别(LEvsNC，DLEvs非DLE，SCLEvsSLE)中AUC均>0.7，且有任意两组及以上的AUC>0.8，则该检测指标被视为可有效诊断区分红斑狼疮不同亚型，在可行性一栏标记为√，即可行。
表10.LEvsNC，DLEvs非DLE，SCLEvsSLE的ROC分析及非参数检验结果汇总
Figure PCTCN2020095791-appb-000011
2)经过表10的汇总评估，获得四种可用于红斑狼疮诊断分型的皮肤组织原位多标记免疫染色试剂盒检测指标，分别为：CD19的单阳检测(CD19/DAPI)、CD8-CD19的二联检测((CD8+CD19)/DAPI)、CD4-CD8-CD19的三联检测((CD4+CD8+CD19)/DAPI)与CD4-CD8-CD19-CD11b的四联检测((CD4+CD8+CD19+CD11b)/DAPI)，其中在区分SCLE和SLE时，这4种检测指标只有CD4-CD8-CD19-CD11b的四联检测的AUC>0.8，且非参数检验的P值<0.001，即SCLE和SLE两组数据在进行CD4-CD8-CD19-CD11b的四联检测结果的比较时，两组数据间存在显著差异，且ROC分析时，相较于其它三种检测指标，使用CD4-CD8-CD19-CD11b的四联检测指标获得的AUC值最大。提示 CD4-CD8-CD19-CD11b的四联检测指标为用于红斑狼疮诊断分型的最优选检测指标；
3)在确定了CD4-CD8-CD19-CD11b的四联检测指标为用于红斑狼疮诊断分型的最优选检测指标后，仍需确定在红斑狼疮分型的应用中，单独使用CD4-CD8-CD19-CD11b的四联检测指标或联合使用其它检测指标的优劣。表11中我们汇总了步骤2)获得的4种检测指标的临界值及相应的灵敏度和特异度。由表11可知，单独使用CD4-CD8-CD19-CD11b的四联检测时，其区分DLE和非DLE的临界值对应特异度为88.89％，此处特异度越高，非DLE数据被误判为DLE的可能就越小，而四种检测指标中，CD19的单阳检测区分DLE和非DLE的临界值对应特异度为97.22％，这意味着在区分DLE和非DLE时，CD4-CD8-CD19-CD11b的四联检测可联合使用CD19的单阳检测，从而尽可能的减少非DLE被误判为DLE的可能。此外，由表12中的DLEvs非DLE的ROC曲线坐标信息可知，当CD4-CD8-CD19-CD11b的四联检测结果>86.5％时，其特异度为97.22％，即若一待测样本的四联检测结果在此范围内(>86.5％)，非DLE被误判为DLE的可能性极低，可判定此待测样本来源自DLE患者。而当CD4-CD8-CD19-CD11b的四联检测结果介于77.7％和86.5％之间时，只能认为该样本为DLE的可能性大，但仍有将非DLE样本误判为DLE样本的可能，此时可联合CD19单阳检测指标来检测，从而进一步提高检测的正确率；
由表11可知，单独使用CD4-CD8-CD19-CD11b的四联检测时，其区分SCLE和SLE的临界值对应特异度为66.67％，此处特异度越高，SLE数据被误判为SCLE的可能就越小，而四种检测指标种，CD19的单阳检测和CD8-CD19二联检测区分SCLE和SLE的临界值对应特异度均为88.89％，这意味着在区分SCLE和SLE时，CD4-CD8-CD19-CD11b的四联检测可联合CD19的单阳检测和/或CD8-CD19二联检测，从而尽可能的减少SLE被误判为SCLE的可能。此外，由表12中的SCLEvsSLE的ROC曲线坐标信息可知，当CD4-CD8-CD19-CD11b的四联检测结果>67.7％时，其特异度为94.44％，即若一待测样本的四联检测结果在此范围内(67.7％-77.7％)，SLE被误判为SCLE的可能性极低，可判定此待测样本来源自SCLE患者。而当CD4-CD8-CD19-CD11b的四联检测结果介于55.4％和67.7％之间时，只能认为该样本为SCLE的可能性大，但仍有将SLE样 本误判为SCLE样本的可能，此时可联合联合CD19的单阳检测和/或CD8-CD19二联检测，从而进一步提高检测的正确率。
表11.四种检测指标的临界值及相应的灵敏度和特异度
Figure PCTCN2020095791-appb-000012
表12.DLEvs非DLE，SCLEvsSLE的ROC曲线坐标信息
Figure PCTCN2020095791-appb-000013
4)综上所述，本发明利用上述的试剂检测后将获得CD4-CD8-CD19-CD11b的四联检测结果((CD4+CD8+CD19+CD11b)/DAPI)，该检测结果是用于辅助红斑狼疮诊断和/或分型诊断的必备检测，此外，CD19的单阳检测结果(CD19/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)可作为辅助检测条件，与CD4-CD8-CD19-CD11b的四联检测结果联合应用，来进一步提高红斑狼疮分型诊断的准确率，具体如下：
a)获得的CD4-CD8-CD19-CD11b的四联检测结果可用于红斑狼疮诊断和/或分型的判定：CD4-CD8-CD19-CD11b的四联检测结果≥77.7％时，判断该待测样品来源的患者为DLE可能性大；55.4％<CD4-CD8-CD19-CD11b的四联检测结果<77.7％时，判断该待测样品来源的患者为SCLE可能性大；41.2％<CD4-CD8-CD19-CD11b的四联检测结果≤55.4％时，判断该待测样品来源的患者为SLE可能性大；CD4-CD8-CD19-CD11b的四联检测结果≤41.2％时，判断该待测样品来源与正常人可能性大；
b)当CD4-CD8-CD19-CD11b的四联检测结果在77.7％与86.5％之间时，有一定几率把SCLE误判为DLE的可能，此时联合应用CD19的单阳检测结果(CD19/DAPI)可提高诊断准确率，当77.7％≤(CD4+CD8+CD19+CD11b)/DAPI≤86.5％，，同时CD19的单阳检测结果≥12.1％时，判断该待测样品来源的患者为DLE；当77.7％≤(CD4+CD8+CD19+CD11b)/DAPI≤86.5％，，同时CD19的单阳检测结果<12.1％时，判断判断该待测样品来源的患者为SCLE；
c)当CD4-CD8-CD19-CD11b的四联检测结果在55.4％与67.7％之间时，有一定几率把SLE误判为SCLE的可能，此时联合应用CD19的单阳检测结果(CD19/DAPI)和/或CD8-CD19的二联检测结果((CD8+CD19)/DAPI)可提高诊断准确率，当55.4％≤(CD4+CD8+CD19+CD11b)/DAPI≤67.7％时，同时CD19的单阳检测结果≥2.5％和/或CD8-CD19的二联检测结果≥25.2％时，判断判断该待测样品来源的患者为SCLE；当55.4％≤(CD4+CD8+CD19+CD11b)/DAPI≤67.7％时，同时CD19的单阳检测结果<2.5％和/或CD8-CD19的二联检测结果<25.2％时，判断判断该待测样品来源的患者为SLE；
待测样品均是在200倍电镜单个视野下，真皮层炎症浸润区进行的检测。
3.5试剂盒检测指标的样本验证
1)为验证和比较试剂盒检测指标检测样本的特异性，另选取了临床及病理学组织确诊并长期随访获得肯定诊断的DLE、SLE、SCLE皮损样本各10例，由技术员甲来对这30例样本进行随机编号(例：编号1样本为2号DLE皮损，编号2样本为5号SLE患者皮损)，并进行试剂盒检测，获得可用于红斑狼疮分型的四组检测指标的结果：CD19的单阳检测结果(CD19/DAPI)、CD8-CD19的二联检测结果((CD8+CD19)/DAPI)、CD4-CD8-CD19的三联检测结果 ((CD4+CD8+CD19)/DAPI)与CD4-CD8-CD19-CD11b的四联检测结果((CD4+CD8+CD19+CD11b)/DAPI)，将获得的检测结果整合在一张Excel表中，并删除这些检测结果对应的诊断，只余对应的随机编号；
2)将Excel表交给技术员乙，由其利用四组检测指标临界值以及3.4中最终确定的联合检测诊断方法来对excel表中这30例样本的数据进行诊断，诊断结果再交由技术员甲来进行评估，最终获得各种检测指标的样本检测正确率见表13。因联合检测中，当77.7％≤(CD4+CD8+CD19+CD11b)/DAPI≤86.5％，或55.4％≤(CD4+CD8+CD19+CD11b)/DAPI≤67.7％时，需联合应用CD19的单阳检测结果(CD19/DAPI)，因此，将CD4-CD8-CD19-CD11b的四联检测结果联合CD19的单阳检测结果设置为联合检测1。当55.4％≤(CD4+CD8+CD19+CD11b)/DAPI≤67.7％时，还可以联用CD8-CD19的二联检测结果((CD8+CD19)/DAPI)，因此，将CD4-CD8-CD19-CD11b的四联检测结果联合CD19的单阳检测结果和CD8-CD19检测结果设置为联合检测2。
由表13的数据验证结果可知，目前确定的联合检测诊断方法，一定程度上减少了单个检测指标在诊断上的误判，大大提高了红斑狼疮分型诊断的正确率。
表13 使用不同检测指标诊断红斑狼疮分型的正确率比较
Figure PCTCN2020095791-appb-000014
Figure PCTCN2020095791-appb-000015

Claims (12)

  1. 一种用于红斑狼疮分型的皮肤组织免疫细胞原位检测试剂盒，其特征在于，包括利用原位标记免疫染色检测CD4、CD8、CD19、CD11b阳性免疫细胞所需的试剂，所需的试剂包括：抗原修复液、非特异性阻断剂、血清封闭剂、抗体稀释剂、单克隆抗体、洗涤剂、辣根过氧化物酶标记的二抗、酪胺荧光染色剂、核染色剂。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述抗原修复液包括pH6.0的酸性抗原修复液和/或pH8.0的碱性修复液；所述非特异性阻断剂为单独的0.5％高碘酸溶液或者联合使用的0.5％高碘酸溶液和3％H 2O 2溶液。
  3. 根据权利要求1所述的试剂盒，其特征在于，所述单克隆抗体包括：兔源CD4单克隆抗体、鼠源CD8单克隆抗体、兔源CD19单克隆抗体、兔源CD11b单克隆抗体；CD11b单克隆抗体匹配Opal570染液；CD8单克隆抗体匹配Opal650染液；CD4单克隆抗体匹配Opal520染液；CD19单克隆抗体匹配Opal690染液。
  4. 根据权利要求3所述的试剂盒，其特征在于，检测CD4、CD8、CD19、CD11b四种细胞时，每种单克隆抗体染色步骤如下：
    a)第一轮染色抗体：CD11b单克隆抗体；其抗原修复条件：酸性抗原修复液高压修复或酸性抗原修复液微波修复；其稀释比范围：1:1000-1:2000，依次经过一抗和二抗敷育，然后采用染色剂敷育；
    b)第二轮染色抗体：CD8单克隆抗体；其抗原修复条件：碱性抗原修复液高压修复或碱性抗原修复液微波修复；其稀释比范围：1:2-1:5，依次经过一抗和二抗敷育，然后采用染色剂敷育；
    c)第三轮染色抗体：CD4单克隆抗体；其抗原修复条件：碱性抗原修复液高压修复或碱性抗原修复液微波修复；其稀释比范围：1:2-1:5，依次经过一抗和二抗敷育，然后采用染色剂敷育；
    d)第四轮染色抗体：CD19单克隆抗体；其抗原修复条件：碱性抗原修复液高压修复或碱性抗原修复液微波修复；其稀释比范围：1:200-1:500，依次经过一抗和二抗敷育，然后采用染色剂敷育；
    室温范围：20-27℃；
    核复染是对全体细胞的检测：取酸性抗原修复液微波修复，采用DAPI进行染色。
  5. 根据权利要求1所述的试剂盒，其特征在于，单克隆抗体在抗原修复后，一抗敷育之前，进行非特异性阻断剂阻断，完成阻断后洗涤剂洗涤，再采用血清封闭剂室温封闭15-50min。
  6. 权利要求1-5任一项所述的试剂盒在制备辅助红斑狼疮诊断和/或分型制剂中的应用，所述的免疫细胞包括：CD4 +、CD8 +、CD19 +、CD11b +细胞。
  7. 根据权利要求6所述的应用，其特征在于，将CD4 +细胞、CD8 +细胞、CD19 +细胞和CD11b +细胞四种细胞数目总和在全体细胞中所占的百分比，即(CD4+CD8+CD19+CD11b)/DAPI的结果用于辅助红斑狼疮诊断和/或分型。
  8. 根据权利要求7所述的应用，其特征在于，在依据(CD4+CD8+CD19+CD11b)/DAPI结果进行判断的基础上，依据CD19 +细胞数目在全体细胞中所占的百分比；即CD19/DAPI的结果对SLE、SCLE和DLE进一步分型；或者依据CD19/DAPI和/或(CD8+CD19)/DAPI结果对SCLE和SLE进一步分型。
  9. 根据权利要求6所述的应用，其特征在于，根据ROC曲线分析获得具体的诊断和分型临界值进行判断。
  10. 根据权利要求9所述的应用，其特征在于，红斑狼疮诊断和/或分型的判定临界值：(CD4+CD8+CD19+CD11b)/DAPI≥77.7％时，判断该待测样品来源于DLE；55.4％<(CD4+CD8+CD19+CD11b)/DAPI<77.7％时，判断该待测样品来源于SCLE；41.2％<(CD4+CD8+CD19+CD11b)/DAPI≤55.4％时，判断该待测样品来源于SLE；(CD4+CD8+CD19+CD11b)/DAPI≤41.2％，判断该待测样品来源于正常人。
  11. 根据权利要求10所述的应用，其特征在于，
    当77.7％≤(CD4+CD8+CD19+CD11b)/DAPI≤86.5％，CD19/DAPI检测结果≥12.1％时，判断该待测样品来源于DLE；CD19/DAPI检测结果<12.1％时，判断该待测样品来源于SCLE；
    当55.4％≤(CD4+CD8+CD19+CD11b)/DAPI≤67.7％时，CD19/DAPI检测结果≥2.5％和/或(CD8+CD19)/DAPI检测结果≥25.2％时，判断该待测样品来源于SCLE；CD19/DAPI检测结果<2.5％和/或(CD8+CD19)/DAPI检测结果<25.2％时，判断该待测样品来源于SLE。
  12. 使用权利要求1-5任一项所述的试剂盒在非诊断目的的研究各种红斑狼疮分型和/或正常皮肤组织免疫细胞特征中的应用。
PCT/CN2020/095791 2020-03-25 2020-06-12 用于红斑狼疮分型的皮肤组织免疫细胞原位检测试剂盒及其应用 WO2021189672A1 (zh)

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