A detection reagent and kit for eosinophilic chronic sinusitis with nasal polyps

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a detection reagent and kit for eosinophilic chronic sinusitis with nasal polyps. The detection reagent includes: a substance for detecting Galectin-10 protein (Charcot-Leyden crystals, CLCs) in a crystalline state. The reagent for detecting CLCs includes a mixture of eosin, glacial acetic acid and water. The mass volume ratio of the eosin to glacial acetic acid is 1:1-50g / mL, and the volume ratio of the glacial acetic acid to water is 1:2-100. The present invention is the first to discover a reagent that can efficiently detect CLCs. CLCs is a biomarker for eosinophilic chronic sinusitis with nasal polyps, and therefore provides a detection reagent for eosinophilic chronic sinusitis with nasal polyps. By detecting CLCs, eosinophilic chronic sinusitis with nasal polyps can be predicted, which is simple and quick, and has good predictive performance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a detection reagent and a kit for eosinophilic chronic sinusitis with nasal polyps. Background Art

[0002] Chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous upper airway inflammatory disease encompassing two intrinsic subtypes: eosinophilic and non-eosinophilic. These two subtypes differ significantly in recurrence rates, clinical symptoms, and medical management. Brazilian researchers published a meta-analysis in the journal Rhinology to define eosinophilic CRSwNP. They screened 142 articles and classified 29 eosinophilic CRSwNP cutoffs based on indicators such as nasal polyp recurrence, allergic status, quality of life, immunohistochemistry, and cluster analysis. The top three were eosinophil counts >10% proposed by Chinese researchers, eosinophil counts >10 / HPF proposed by the EPOS guidelines, >70 / HPF proposed by Japanese researchers, and >5 / HPF proposed by American researchers (tied for third). The classification of eosinophilic CRSwNP lacks unified histopathological criteria and consistent cutoffs.

[0003] Disadvantages of Similar Techniques: Although 10% is the most commonly used cutoff for eosinophilic CRSwNP in Chinese patients, the following issues exist: 1. Due to the heterogeneous distribution of eosinophils within localized polyps, manual counting based on average or hotspots can introduce sampling errors. 2. While whole-slide counting can better reflect inflammatory cell infiltration, manual eosinophil counting in whole tissue sections is time-consuming and labor-intensive, making it difficult to implement in clinical practice. Recent studies have reported that galectin-10 protein and mRNA levels in nasal secretions or tissues, as measured by ELISA and PCR, can be effective predictors of nasal polyp recurrence and the diagnosis of eosinophilic CRSwNP. However, galectin-10 exists in three forms: extracellular vesicles, soluble protein, and crystalline protein. Crystalline galectin-10 is known as Charcot-Leyden crystals (CLCs). Only crystalline Galectin-10 proteins (CLCs) can drive type 2 immunity, allergic reactions, and neutrophilic inflammation. Although ELISA and PCR tests can show the protein and mRNA expression levels of Galectin-10, they cannot show crystalline CLCs. Existing eosin reagents can well display eosinophils, but cannot show the morphology of CLCs. Through preliminary experiments, we found that commercially available eosin stains cannot effectively detect CLCs. However, by adding different volumes of glacial acetic acid to the eosin solution prepared with eosin Y powder, the detection efficiency of CLCs was significantly improved. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a detection reagent and kit for eosinophilic chronic sinusitis with nasal polyps.

[0005] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0006] The detection reagent for eosinophilic chronic sinusitis with nasal polyps provided by the present invention can detect crystalline Galectin-10 protein (CLCs). CLCs is a biomarker diagnostic substance that saves time and labor and has good predictive performance.

[0007] The present invention provides a detection reagent for eosinophilic chronic sinusitis with nasal polyps, comprising a substance for detecting CLCs.

[0008] Furthermore, the substance for detecting CLCs includes eosin.

[0009] Preferably, the eosin is eosin Y.

[0010] Furthermore, the substance for detecting CLCs includes a mixture of eosin, glacial acetic acid and water.

[0011] Preferably, in the mixture of eosin, glacial acetic acid and water, the mass volume ratio of eosin to glacial acetic acid is 1:1-50 g / mL.

[0012] Further preferably, in the mixture of eosin, glacial acetic acid and water, the mass volume ratio of eosin to glacial acetic acid is 1:35 g / mL.

[0013] Preferably, in the mixture of eosin, glacial acetic acid and water, the volume ratio of the glacial acetic acid to water is 1:2-100.

[0014] Further preferably, in the mixture of eosin, glacial acetic acid and water, the volume ratio of the glacial acetic acid to water is 7:20.

[0015] More preferably, in the mixture of eosin, glacial acetic acid and water, the volume ratio of glacial acetic acid to water is 7:20, and the mass volume ratio of eosin to glacial acetic acid is 1:35 g / mL.

[0016] The present invention provides a kit for detecting eosinophil CRSwNP, which comprises the above-mentioned eosinophil CRSwNP detection reagent.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] The present invention first discovered that CLCs are a biomarker for diagnosing eosinophil CRSwNP, and therefore provides a detection reagent for eosinophil CRSwNP. By detecting CLCs, eosinophil CRSwNP can be identified, which takes less time than eosinophil detection, and thus has the characteristics of simplicity and speed, and good predictive performance. Compared with existing dyes, the mixture of eosin, glacial acetic acid and water used in the present invention can more efficiently detect CLCs while ensuring a sharp contrast with the cell nucleus. Compared with fluorescent staining methods, the method provided by the present invention is simpler, more convenient, easy to clinically promote, has low dye and reagent costs, and the stained sections are easy to preserve and stable for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1a This is a graph showing the results of detecting CLCs using Scheme A in an embodiment of the present invention;

[0020] Figure 1b This is a graph showing the results of detecting CLCs using Scheme B in an embodiment of the present invention;

[0021] Figure 1cThis is a graph showing the results of detecting CLCs using Scheme C in an embodiment of the present invention;

[0022] Figure 1d This is a graph showing the results of detecting CLCs using Scheme D in an embodiment of the present invention;

[0023] Figure 2 : is the ROC curve for predicting eosinophil CRSwNP by the number of CLCs in the embodiment of the present invention;

[0024] Figure 3a This is a graph showing the correlation between the number of CLCs in nasal polyps and the eosinophil count in nasal polyps according to an embodiment of the present invention;

[0025] Figure 3b This is a graph showing the correlation between the number of CLCs in nasal polyps and the percentage of eosinophils in peripheral blood in an embodiment of the present invention;

[0026] Figure 3c Graph showing the correlation between the number of CLCs in nasal polyps and the eosinophil count in peripheral blood in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] The following examples further illustrate the specific implementation of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes not specifically described below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0028] Charcot-Leyden crystals (CLCs), that is, crystalline Galectin-10 protein, are the product of hyperactivated eosinophils. They are often expressed in eosinophil-related diseases such as asthma, chronic sinusitis, and eosinophilic esophagitis. Typical CLCs are needle-shaped bipyramidal Galectin-10 crystals. Hyperactivated eosinophils release three different forms including crystalline Galectin-10 protein (CLCs), extracellular vesicles, and extracellular soluble Galectin-10 protein. However, only CLCs, not soluble Galectin-10 protein, can drive type 2 immune responses, allergies, and neutrophilic inflammation.

[0029] The present invention proposes for the first time that CLCs can be used to diagnose eosinophilic CRSwNP. CLCs is a biomarker with good predictive performance for diagnosing eosinophilic CRSwNP.

[0030] To address the clinical bottleneck of low CLC detection rates, the present invention discovered differences in CLC detection efficiency and color appearance when adding different amounts of glacial acetic acid to CLCs during HE staining. By selecting the optimal reagent ratio, bipyramidal hexagonal CLCs can be efficiently and accurately detected, ensuring a distinct color difference between them and the cell nucleus. CLC counting allows for a more efficient, simple, and rapid diagnosis of eosinophilic CRSwNP.

[0031] 1. Research Methods

[0032] Patient Enrollment and Recurrence Criteria: The study included 97 patients with bilateral nasal polyps who had undergone ESS surgery. Patients who had used oral / nasal corticosteroids, immunomodulators, or antibiotics within 1 month before enrollment were excluded. Patients with fungal sinusitis, cystic fibrosis, allergic fungal rhinitis, or primary ciliary dyskinesia were also excluded. CRSwNP was defined as eosinophilic CRSwNP when the percentage of eosinophils in the tissue was >10%; otherwise, it was defined as non-eosinophilic CRSwNP.

[0033] 2. Specimen collection and processing:

[0034] (1) Sampling and fixation: During ESS surgery, polyp tissue specimens are collected and small pieces of nasal polyps are clamped with sterile ethmoid sinus forceps. In order to maintain the integrity of tissue cells, the tissue is immersed in 10% neutral formalin for fixation after sampling.

[0035] (2) Dehydration, transparency and wax immersion: First, the water in the tissue is replaced by gradient alcohol, then transparentized by xylene and immersed in liquid paraffin.

[0036] (3) Embedding: Use hard wax (preferably 56-58°C or 60-62°C), pour it into a metal frame, bury the tissue embedding box in the hot wax, and place it on an ice table to cool for 4-6 hours. Remove the wax block after it is completely solidified.

[0037] (4) Sectioning: Fix the wax block on the microtome. Hold the brush in your left hand and turn the microtome with your right hand. First, trim the tissue block and make the section flat. Then set the section thickness to 3.5 μm and section.

[0038] (5) Spreading, scooping, and baking the slices: Lay the slices face up in warm water. The slices will naturally unfold under the action of surface tension. Insert a glass slide vertically into the water, close to the slices, and gently pull them out. Attach the slices to the slide, mark the information, and bake them for later use.

[0039] 3. Experimental methods: CLCs and eosinophils were stained with hematoxylin-eosin (H&E) and then quantitatively counted.

[0040] The property of being easily stained by basic or acidic dyes is called basophilia and acidophilia; while the phenomenon of having a relatively weak affinity for both basic and acidic dyes is called neutrophilia.

[0041] There are many different types of amino acids that make up proteins within tissues, each with a different isoelectric point. Acidic substances within cells, such as chromatin in the nucleus, the rough endoplasmic reticulum within glandular cells and nerve cells, and the hyaline cartilage matrix, are stained by basic dyes; these substances are called basophils. Other proteins within the cytoplasm, such as hemoglobin in red blood cells, eosinophil granules, and collagen fibers, are stained by acidic dyes; these substances are called eosinophils. If the pH of the staining solution is altered, substances stained with acidic dyes become basophils when the pH increases, while substances stained with basic dyes become eosinophils when the pH decreases. Therefore, the pH of the staining solution can influence the staining reaction.

[0042] The phosphate groups on both strands of deoxyribonucleic acid (DNA) face outward, carry a negative charge, and are acidic. They easily bind to the positively charged alkaline dye hematoxylin through ionic bonds and become stained. Hematoxylin appears blue in alkaline solutions, so the cell nucleus is stained blue. Eosin Y is a chemically synthesized acidic dye that dissociates into negatively charged anions in water. It binds to the positively charged cations of protein amino groups to stain the cytoplasm. The cytoplasm, red blood cells, connective tissue, eosinophilic granules, and crystalline Galectin-10 proteins (CLCs) are stained in varying degrees of red or pink, forming a sharp contrast with the blue cell nucleus. Eosin is an excellent dye for the cytoplasm.

[0043] Different tissue or cell components have varying affinities for hematoxylin and staining properties. After hematoxylin staining, cell nuclei and calcium salt mucus appear blue. Hydrochloric acid and alcohol differentiation and weak alkaline solutions can be used to develop the blue. Proper treatment can result in a clear dark blue stain for the cell nucleus and decolorize other components, such as the cytoplasm. The cytoplasmic dye eosin is then used to stain the cytoplasm, causing the various cytoplasmic components to appear in varying shades of pink. This reveals the general morphological and structural characteristics of various tissue or cell components and lesions. Quantification is then performed by blindly reading five hotspots, each with different target cells and structures.

[0044] After various attempts, the present invention finally adopted four different eosin dye ratio schemes (as the substance for detecting CLCs): (1) Scheme A: a mixture of 0.1g eosin Y + 10ml distilled water + 100μl glacial acetic acid; (2) Scheme B: a mixture of 0.1g eosin Y + 10ml distilled water + 2.5ml glacial acetic acid; (3) Scheme C: a mixture of 0.1g eosin Y + 10ml distilled water + 3.5ml glacial acetic acid; (4) Scheme D: a mixture of 0.1g eosin Y + 10ml distilled water + 5ml glacial acetic acid. By comparing the four different ratios, the present invention found that the above scheme C can detect CLCs, which appear red and have a clear contrast with the color of the cell nucleus, which appears dark blue. The results are as follows Figure 1a 、 Figure 1b 、 Figure 1c and Figure 1d As shown; According to the final result ( Figure 1a 、 Figure 1b 、 Figure 1c and Figure 1d ), the present invention uses a mixture of 0.1 g eosin Y + 10 ml distilled water + 3.5 ml glacial acetic acid (the aforementioned scheme C) to stain and count CLCs.

[0045] 2. Results of the CLCs test in nasal polyp tissue to diagnose eosinophilic CRSwNP

[0046] The study subjects were 97 patients with bilateral nasal polyps who underwent ESS surgery, of whom 43 patients (44.33%) were diagnosed with eosinophilic CRSwNP; the remaining 54 patients (55.67%) were diagnosed with non-eosinophilic CRSwNP.

[0047] Patients in the two groups presented with different demographic and clinical characteristics (see Table 1). Patients with eosinophilic CRSwNP had significantly higher rates of allergic rhinitis (P < 0.001), a history of allergies (P < 0.001), and clinical symptom scores (olfactory impairment, P = 0.025) than those in the non-eosinophilic CRSwNP group. Patients with eosinophilic CRSwNP had significantly higher peripheral blood eosinophil counts (P = 0.001), percentages (P = 0.007), and tissue CLC counts (P < 0.001) than those in the non-eosinophilic CRSwNP group. Age, sex, smoking history, and the percentage and count of peripheral blood neutrophils did not differ significantly between the two groups (Table 1).

[0048] Table 1. Patient demographics and clinical characteristics.

[0049]

[0050] EOS, eosinophil; eosinophilic CRSwNP, ECRSwNP; non-eosinophilic CRSwNP, nECRSwNP; NEU, neutrophil; CLCs, Charcot-Leyden Crystals; NPs, nasal polyps; PB, peripheral blood; #, count number, absolute count.

[0051] 3. The predictive factors of eosinophilic CRSwNP were evaluated by ROC curve. The number of CLCs in nasal polyps (AUC = 0.833, 95% CI = 0.738-0.929) had a high predictive value for eosinophilic CRSwNP, while the peripheral blood eosinophil count (AUC = 0.705, 95% CI = 0.587-0.823) and percentage (AUC = 0.67295% CI = 0.552-0.793; as shown in Figure 3). Figure 2 The predictive value of the polyp is relatively low. Data analysis shows that once CLCs are present in polyp tissue, the sensitivity and specificity for predicting eosinophilic CRSwNP in patients are the highest. Compared to the existing technology of diagnosing acidic CRSwNP by counting eosinophils, the detection substance provided by the embodiments of the present invention is more simple and easy to diagnose acidic CRSwNP by detecting the presence of CLCs.

[0052] 4. CLCs are products of highly activated eosinophils. Through Spearman correlation analysis, the present invention conducted a correlation analysis between CLCs and eosinophils. The results showed that the number of CLCs in nasal polyps was positively correlated with the eosinophil count (R = 0.755, P < 0.001, Figure 3a In addition, the CLCs count was significantly correlated with the percentage of eosinophils in peripheral blood (R=0.214, P=0.035, Figure 3b ) and count (R=0.388, P=0.001, Figure 3c ) are positively correlated.

[0053] In summary, the present invention discovered for the first time that CLCs is a biomarker of eosinophil CRSwNP, and thus provides a detection reagent for eosinophil CRSwNP. By detecting CLCs, eosinophil CRSwNP can be predicted, which is simple, fast and has good predictive performance.

[0054] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. Use of a composition in the preparation of a detection reagent for eosinophilic chronic sinusitis with nasal polyps, for detecting crystalline Galectin-10 protein, characterized in that: The composition is a mixture of eosin Y, glacial acetic acid and water; the volume ratio of the glacial acetic acid to water is 7:20, and the mass volume ratio of the eosin Y to glacial acetic acid is 1:35 g / mL.

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