Ocular Surface Cell and Tear Collection Composite Test Strip and Preparation Method

Abstract

The present invention provides an ocular surface cell and tear collection composite test strip and a preparation method thereof. The collection composite test strip of the present invention includes a water-absorbing filter paper layer and an adhesion filter membrane layer. The present invention innovatively uses a double-layer composite filter paper composed of a water-absorbing filter paper and an adhesion filter membrane as the collection test strip for ocular surface cells and tears, which increases the adhesion to the ocular surface tissue and regards the ocular surface diseases and tears as a unified whole as a whole. By using the test strip of the present invention in combination with the established standard for collecting ocular surface cells and tears, sufficient tears and partial ocular surface tissue cells can be obtained simultaneously, which is of great significance for the early diagnosis and treatment effect of diseases.

Description

Technical Field

[0001] The present invention relates to a composite test strip for collecting ocular surface cells and tear fluid and a preparation method thereof. Background Art

[0002] The ocular surface includes the cornea, conjunctiva and tear film, and ocular surface diseases include corneal, conjunctival, eyelid, lacrimal apparatus and lacrimal duct diseases, which are common diseases in ophthalmic clinical practice. Tear fluid, as the only external secretion in the eye, is distributed on the ocular surface and is closely related to both the physiological and pathological states of the ocular surface. Previous studies have shown that tear fluid can not only be used to examine and evaluate the function of the lacrimal gland, but also quantitatively study its specific components (such as proteins, inflammatory mediators, etc.) for the diagnosis of ocular surface diseases and the judgment of the severity of the diseases. Especially for ocular surface infectious diseases, the diagnostic value of tear fluid becomes even more important. By detecting the types and quantities of pathogenic microorganisms in tear fluid, not only can the pathogenic types be identified as early as possible to guide individualized clinical medication and reduce the decline in vision and visual function caused by misdiagnosis of diseases, but also the proliferation or replication levels of pathogenic microorganisms can be monitored to evaluate the treatment effect.

[0003] Currently, the commonly used tear fluid collection methods in clinical practice are divided into direct collection methods and indirect collection methods. The direct collection method refers to the collection method of directly obtaining tear fluid specimens through a hollow container. In this method, a capillary tube is placed in the tear fluid at the lower eyelid conjunctival sac or the semilunar fold. During the operation, the cornea and conjunctival tissues are not contacted, and the irritation to the conjunctiva is relatively small. It is mainly applicable to directly obtain and measure the physical and chemical properties of tear fluid or collect tear fluid without irritation. Currently, most of the direct collection methods still use glass capillary tubes, so the sampling process has relatively high requirements for both the operator and the patient's cooperation, and there is a risk of damaging the normal structure of the ocular surface. The indirect collection method is to place a material with a certain adsorption effect in the lower eyelid conjunctival sac. After the tear fluid soaks through the adsorption material, the adsorption material is taken out and detected. Filter paper is the most widely used adsorption material. Compared with the direct collection method, the indirect collection method has a lower sampling risk. In addition, for different detection components in tear fluid and ocular surface tissues, the selection of the adsorption material will significantly affect the detection results, posing higher requirements for the standardization and normalization of sampling.

[0004] Currently, existing research shows that for all sampling methods, the following factors may affect the sampling results, including but not limited to: (1) the use of eye medications by the subject; (2) a suitable sampling environment; (3) the proficiency of the sampling doctor; (4) sufficient patient education and the cooperation degree of the subject; (5) the sampling duration; (6) the specimen preservation method, temperature and time; (7) the specimen rewarming method and environment; (8) the sensitivity and specificity levels of the analysis and determination itself.

[0005] In summary, the existing sampling methods mainly have the following deficiencies:

[0006] First, the sampling process lacks an executable operation specification, and the test specimens are vulnerable to contamination introduced by humans during the operation process, increasing the uncertainty and reliability of the test results.

[0007] Secondly, there are certain risks in some sampling processes. For example, the capillary sampling method has high requirements for patient cooperation and there is a risk of damaging the normal structure of the ocular surface.

[0008] Most importantly, all existing sampling methods have always centered around tears, ignoring the close connection between tears and ocular surface tissues, and there is little discussion on the significance of obtaining ocular surface tissues during the sampling process.

[0009] Therefore, it is urgently needed in the diagnosis of ocular surface diseases to develop a method that can simultaneously obtain tears and a certain amount of ocular surface tissues and make it as simple, non-invasive, standardized and procedural as possible. Summary of the Invention

[0010] The object of the present invention is to address the deficiencies of the prior art. Based on the above-mentioned filter paper adsorption method and combined with the principle of conjunctival impression cytology examination, a composite double-layer filter paper that can simultaneously obtain tears and ocular surface tissues is innovatively designed. The double-layer filter paper can adsorb tears, and at the same time, the filter membrane can adhere to a certain number of ocular surface cells (such as conjunctival epithelial cells, goblet cells, lymphocytes, etc.). At the same time, by establishing a standardized standard operation process for simultaneously collecting ocular surface cells and tears, in the research and verification of common ocular surface diseases, it is found that this detection method can effectively improve the collection quality of tear samples, improve the accuracy and reliability of the examination in the early diagnosis of diseases, and provide an auxiliary role for the diagnosis and treatment of ocular surface diseases.

[0011] The technical solution adopted by the present invention is as follows:

[0012] A composite test paper for collecting ocular surface cells and tears, comprising a laminated water-absorbing filter paper layer and an adhesion filter membrane layer, wherein the water-absorbing filter paper layer is used to absorb and collect tears, and the adhesion filter membrane layer is used to adhere and collect ocular surface cells.

[0013] Further, the water-absorbing filter paper layer and the adhesion filter membrane layer are laminated by edge binding.

[0014] Further, the water-absorbing filter paper layer is made of any material with adsorption function and biocompatibility, such as cotton fiber filter paper, etc.

[0015] Further, the adhesion filter membrane layer is made of any material with adhesion function and biocompatibility, such as nitrocellulose filter membrane, cellulose acetate filter membrane, hydrophilic polyethylene filter membrane, etc.

[0016] Further, the shape of the test paper is rectangular.

[0017] Further, the size of the test strip is 10 mm in length and 5 mm in width.

[0018] A method for preparing a composite test strip for collecting ocular surface cells and tear fluid, specifically: cutting the water-absorbing filter paper layer and the adhesion filter membrane layer into the same size, and binding the edges of the two layers of filter paper with the same size using a physical pressing method to make a composite test strip for collecting ocular surface cells and tear fluid.

[0019] The present invention is a simple, non-invasive, standardized, and programmed method for collecting ocular surface cells and tear specimens, which can obtain immune components such as cytokines and antibodies in tear fluid and ocular surface tissues, as well as potential pathogenic microorganisms, and is applied in the early diagnosis and treatment effect evaluation of ocular surface diseases. Compared with the traditional filter paper adsorption method, this technical invention regards tear fluid and other components of the ocular surface as a complete functional unity, adds a key step of "adhesion", significantly improves the accuracy and reliability of the test, and the standardization and programming reduce the risk of sample contamination, while the simplicity and non-invasiveness improve the comfort of patients.

[0020] The present invention is applicable to all patient populations in need of immunological diagnosis and etiological diagnosis of ocular surface diseases. The key technical points involved mainly include the following two parts: the adsorption of tear fluid and the adhesion of ocular surface tissues. The above two technical points are carried out simultaneously and promote each other during the collection process.

[0021] The present invention also provides a method for collecting the composite test strip of the present invention, specifically as follows:

[0022] (I) Collection stage

[0023] 1. Place the subject in a comfortable diagnosis and treatment environment, adjust the magnification of the slit lamp to 6X, adjust the slit lamp field of view under the low-power lens to be able to completely observe the palpebral fissure area and the lower eyelid of the subject, instruct the subject to look upward, and press a disposable medical sterilized cotton swab tightly against the skin of the lower eyelid and twist it downward to fully expose the inferior fornix as much as possible;

[0024] 2. Adhesion of ocular surface tissues: Use a non-toothed forceps to gently attach the adhesion filter membrane of the composite test strip to the surface of the bulbar conjunctiva near the inferior fornix of the examined eye. The head of the non-toothed forceps can gently press the composite test strip to obtain a certain amount of ocular surface cells (such as conjunctival epithelial cells, goblet cells, lymphocytes, etc.) by using the adhesion of the filter membrane;

[0025] Adsorption of tear fluid: Slowly release the medical cotton swab and relax the lower eyelid. The increased height of the tear river is conducive to the test strip continuing to adsorb the tear fluid in the conjunctival sac until it is observed that the composite test strip is completely infiltrated with tear fluid.

[0026] (II) Preservation stage

[0027] After adhesion and adsorption are completed, gently remove the composite test paper soaked with tears using the above-mentioned non-toothed forceps. At this time, the outer edge of the composite test paper should be clamped with the non-toothed forceps to completely remove the filter paper, which can effectively prevent the non-toothed forceps from contacting and damaging the ocular surface tissue. Place it in a sterilized 1.5 ml centrifuge tube, tightly close the tube mouth to prevent aerosol contamination, accurately mark the sampling time, eye side, location, and subject information, etc. It can be temporarily stored in an ice box and transferred to a -80°C refrigerator for storage within 2 hours. The low-temperature freezing time should not exceed 30 days.

[0028] The present invention innovatively uses a double-layer composite test paper composed of a water-absorbing filter paper and an adhesion filter membrane as a sampling test paper for analyzing samples of ocular surface infectious diseases, increasing the adhesion to the ocular surface tissue. Considering the ocular surface diseases and tears as a unified whole, using the composite test paper of the present invention and the established ocular surface cell and tear sampling standards, it is possible to simultaneously obtain sufficient tears and part of the ocular surface tissue, which is of great significance for the early diagnosis of diseases and the evaluation of treatment effects, and can be applied to:

[0029] 1. Nucleic acid detection of ocular surface pathogens, such as bacteria, fungi, viruses, amoeba, chlamydia, mycoplasma, rickettsia, spirochetes, etc.;

[0030] 2. Ocular surface protein analysis, such as cytokines, inflammatory factors, abnormal ocular surface proteins, etc.;

[0031] 3. Detection of cell morphology and nucleic acid analysis of abnormal ocular surface cells, etc.

[0032] Thus, it plays a role in the early rapid diagnosis of ocular surface infectious diseases and the evaluation of disease treatment effects; the evaluation of dry eye types and disease treatment effects; the diagnosis, condition assessment, and allergen identification of ocular surface allergic diseases; the condition and disease treatment effect assessment of thyroid-related ophthalmopathy; the detection of abnormal cell morphology and nucleic acid analysis of ocular surface tumors, etc.

[0033] Compared with the traditional clinical tear detection methods, the advantages of this technology are as follows:

[0034] 1. Simplicity: It is simple in material acquisition - using easily available water-absorbing filter paper and adhesion filter membrane in clinical tests and improving them, and using the most commonly used sterile instruments, cotton swabs, etc. in clinics. It is simple and non-invasive in operation - obtaining ocular surface cells and tears simultaneously in one sampling, minimizing the discomfort of the subjects to the greatest extent;

[0035] 2. High sensitivity: Adopting the key technology of "adhesion", increasing the acquisition of ocular surface tissue and improving the test sensitivity of the sample;

[0036] 3. Standardization: Standardizing and streamlining the above sampling methods, and minimizing the human errors and sample contamination introduced by non-standard operation steps as much as possible. Description of the Drawings

[0037] Figure 1 It is the structural diagram of the collection test strip of the present invention;

[0038] Figure 2 It is the flow chart of collecting ocular surface cells and tear fluid using the collection test strip of the present invention. Detailed implementation manners

[0039] The present invention provides a composite test strip for collecting ocular surface cells and tear fluid, which comprises a water-absorbing filter paper layer and an adhesion filter membrane layer that are adhered to each other. The water-absorbing filter paper in the present invention mainly adsorbs tear fluid by adsorption means to achieve tear fluid collection. Generally, any material that does not harm the eyes and has an adsorption function can be used as the filter paper of the present invention, such as various filter papers containing cotton fibers; the adhesion filter membrane in the present invention non-invasively collects ocular surface tissues by adhesion means. Similarly, generally, any material that does not harm the eyes and has an adhesion function can be used as the filter membrane of the present invention, such as nitrocellulose filter membrane, cellulose acetate filter membrane, hydrophilic polyethylene filter membrane, etc.; the test strip of the present invention is made into a composite double-layer test strip by physical adhesion to simultaneously collect ocular surface cells and tear fluid; the physical adhesion of the test strip only needs to ensure that the water-absorbing filter paper and the adhesion filter membrane are an integral structure, and all four edges can be adhered, or only one edge can be adhered, as Figure 1 shown.

[0040] The test strip can be of any shape. Considering the size of the eye collection area, it is generally appropriate to have a length of 10 mm and a width of 5 mm.

[0041] Its preparation method is as follows: Cut the water-absorbing filter paper layer and the adhesion filter membrane layer into the same size, and use physical pressing method to bind the edges of the two layers of filter paper with the same size to make a double-layer composite test strip, as Figure 1 shown. Sterilize the above double-layer composite test strip with low-temperature ethylene oxide and store it in a sterilized 1.5 ml microcentrifuge tube to obtain the collection test strip of the present invention.

[0042] Research results show that tear fluid contains various components secreted by ocular surface tissues. Among them, the immune components include cytokines, inflammatory factors, abnormal ocular surface proteins, etc. Its main component is protein, and the commonly used detection techniques are solid-phase immunosorption (including enzyme-linked immunosorbent assay, immunoblotting, etc.). When pathogenic microorganisms (such as bacteria, fungi, viruses, amoeba, chlamydia, mycoplasma, rickettsia, spirochetes, etc.) invade the ocular surface tissues and cause infectious keratitis and conjunctivitis, in addition to detecting immune components in tear fluid such as specific IgA and IgM, polymerase chain reaction technology can also be used to detect nucleic acid fragments of pathogenic microorganisms in tear fluid. Detection of abnormal ocular surface cell morphology and its nucleic acid analysis can be performed by impression cytology.

[0043] Specifically, it can be divided into the following major detection directions according to disease categories:

[0044] Infectious diseases of the ocular surface: The polymerase chain reaction technology can be used to detect the DNA of bacteria, fungi, viruses, amoeba, chlamydia, mycoplasma, rickettsia, spirochetes, etc. in tears, and the specific Ig can be detected by immunoelectrophoresis, which is helpful for the diagnosis of infectious ocular surface and intraocular diseases;

[0045] Dry eye: The levels of inflammatory factors such as interleukin IL-1, IL-2, IL-6 and tumor necrosis factor TNF- in tears can be detected, which is helpful for the diagnosis of dry eye. Proteomics technologies such as iTRAQ can be used to detect the protein levels in various tears, which is beneficial for the classification of dry eye;

[0046] Allergic diseases of the ocular surface: The levels of IgE, histamine, inflammatory factors, N-glycans, etc. in tears can be detected, which is helpful for the diagnosis of diseases such as vernal keratoconjunctivitis and atopic conjunctivitis;

[0047] Thyroid-associated ophthalmopathy: The levels of inflammatory factors in tears can be detected, which is helpful for the evaluation of the condition of thyroid-associated ophthalmopathy and the evaluation of the therapeutic effect of hormone shock therapy;

[0048] Corneal ectatic diseases: The total protein, lactoferrin, and secretory IgA in tears can be detected, which is beneficial for the diagnosis of diseases. The level of interleukin IL-6 in tears indicates the progress of the disease;

[0049] Detection of abnormal cell morphology and nucleic acid analysis of ocular surface cells in ocular surface tumors.

[0050] The collection test strip of the present invention can collect tears and ocular surface tissues at the same time, which can greatly increase the concentration of components in the collected sample, thereby effectively improving the diagnostic accuracy of the above diseases.

[0051] The following further illustrates the effect of the present invention by taking the affected eyes clinically diagnosed as "viral infectious ophthalmopathy" as the research object:

[0052] (I) Collection preparation stage

[0053] The composite test strip has been previously placed in a 1.5 ml microcentrifuge tube and sterilized by low-temperature ethylene oxide. Prepare the disposable medical sterilized cotton swabs required during the collection process, and the non-toothed forceps sterilized by high-temperature and high-pressure sterilization.

[0054] (II) Collection stage, as Figure 2 shown, including the following steps:

[0055] 1. Adjust the magnification of the slit lamp to 6X. Under the low-power lens, adjust the slit lamp field of view to be able to completely observe the palpebral fissure area and lower eyelid of the subject. It can be observed that the subject's conjunctiva has moderate mixed congestion. Instruct the subject to look upward, and press the disposable medical sterilized cotton swab tightly against the skin of the lower eyelid and twist it downward to expose the lower fornix as fully as possible;

[0056] 2. Use the above-mentioned forceps without teeth to attach the adhesive filter membrane side of the above-mentioned test strip to the bulbar conjunctiva in the inferior fornix of the eye to be examined, and it can be observed that the tear fluid gradually wets the test strip;

[0057] 3. Slowly release the medical cotton swab and relax the lower eyelid;

[0058] 4. After 1 minute, gently twist the skin of the lower eyelid to expose the fornix, and it can be observed that the tear fluid has completely soaked the test strip. Use the above-mentioned forceps without teeth to take out the test strip.

[0059] (III) Preservation stage

[0060] Use the above-mentioned forceps without teeth to take out the test strip soaked with tear fluid and place it in the above-mentioned sterilized 1.5 ml centrifuge tube. Close the tube mouth tightly, accurately mark the sampling time, eye, location, and information of the subject, etc., and temporarily store it in an ice box, and then immediately transfer it to a -80°C refrigerator for preservation.

[0061] (IV) Detection stage

[0062] Collect 451 ocular surface cell and tear samples from clinically suspected viral infectious eye diseases, and use polymerase chain reaction to detect 7 common herpes viruses (human herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), human herpesvirus 8 (HHV-8)). The results are shown in Table 1. In the test specimens of the present invention, the positive rate of inflammatory viral keratitis was 37.8% (85 / 225), which was significantly higher than the positive rate of 26.8% (118 / 407) reported in the literature (ALIABADI N, JAMALIDOUST M, ASAEI S, et al. Diagnosing of herpes simplex virus infections in suspected patients using real-time PCR[J]. Jundishapur J Microbiol, 2015, 8(2):e16727.) for tear specimens; the positive rate of necrotic viral keratitis was 94.7% (18 / 19), which was significantly higher than the positive rate of 0.77% (4 / 13) reported in the literature (MAJ-X, WANG L-N, ZHOU R-X, et al. Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis[J]. Int J Ophthalmol, 2016, 9(5):682-6.) for tear specimens. Therefore, the collection method of the ocular surface cell and tear collection composite test strip of the present invention has high sensitivity for the detection of ocular viral infectious eye diseases.

[0063] Table 1: Positive results of human herpes virus detection in ocular surface cells and tear specimens of ocular viral infectious diseases

[0064]

[0065] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An eye surface cell and tear collection composite test strip, characterized in that, It includes a bonded absorbent filter paper layer and an adhesive filter membrane layer. Among them, the absorbent filter paper layer is used to absorb and collect tears, and the adhesive filter membrane layer is used to adhere and collect ocular surface cells; the absorbent filter paper layer and the adhesive filter membrane layer are bonded through edge binding.

2. The test strip according to claim 1, wherein The absorbent filter paper layer is a cotton fiber filter paper.

3. The test strip according to claim 1, characterized in that, The adhesive filter membrane layer is a nitrocellulose filter membrane, an acetate filter membrane or a hydrophilic polyethylene filter membrane.

4. The test strip according to claim 1, wherein, The shape of the test strip is rectangular.

5. The test strip according to claim 4, wherein, The size of the test strip is 10 mm in length and 5 mm in width.

6. A method for preparing a composite test strip for collecting ocular surface cells and tear fluid, characterized in that, Specifically: cut the absorbent filter paper layer and the adhesive filter membrane layer into the same size, and use the physical pressing method to bind the edges of the two layers of the absorbent filter paper layer and the adhesive filter membrane layer with the same size to make a composite test strip for collecting ocular surface cells and tears. Among them, the absorbent filter paper layer is used to absorb and collect tears, and the adhesive filter membrane layer is used to adhere and collect ocular surface cells.

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