DEVICE FOR THE SAMPLING OF THE EYE SURFACE BY IMPRINTING

Abstract

The present invention relates to a device for the sampling of the eye surface, to the uses thereof in research and basic diagnostics in the veterinary, agro-feeding and medical field, in particular Ophthalmology. The object of the present invention is to provide a device and a method for the sampling and analysis of the outer layers of the ocular surface, by impression cytology, solving the disadvantages of the devices of known art.

Claims

1. A device for the ocular sampling, in particular for the sampling and analysis of the outer layers of the ocular surface by impression cytology, comprising: a first element with substantially elongated shape having at one end a hollow portion configured to house a membrane suitable for the ocular sampling; and a second element comprising guiding means of said first element configured to allow a relative sliding of said first element relative to said second element, and elastic means positioned in such a way as to exert an opposing force against said first element during said sliding, wherein said hollow portion is a circular portion.

2. The device according to claim 1, wherein said second element has a main body with substantially tubular shape open at both ends comprising said guiding means and said elastic means.

3. The device according to claim 2, wherein said second element further comprises a movable element configured to slide along said main body by means of said guiding means and wherein said elastic means comprises a plurality of springs inserted in said guiding means.

4. The device according to claim 3, further comprising a locking element of said movable element, in particular wherein said locking element has a circular shape and is fixed on said second element.

5. The device according to claim 3, wherein said movable element has a hollow portion for inserting said first element.

6. The device according to claim 5, wherein the hollow portion of said movable element is tilted by about 15 degrees.

7. The device according to claim 1, wherein the hollow portion of said first element has a circular cross section with a tilting of about 15 degrees.

8. The device according to claim 1, wherein said elastic means exerts an opposing force against said first element of maximum value equal to 2.14 N.

9. The device according to claim 1, wherein said first element and said second element are suitable to be reversibly connected therebetween before and after use.

10. The device according to claim 1, wherein said membrane comprises a millipore type, wherein said membrane of the millipore type is housed on said circular portion.

11. A kit comprising the device according to claim 1, and further comprising one or more of: one or more closing elements of a lower portion of said first element, and/or one or more solutions suitable for biochemical, confocal or molecular analysis of said ocular samples, and/or a supporting structure comprising one or more seats for said first element and/or one or more seats for said second element and/or one or more seats for said closing elements, and/or one or more of the membranes suitable for the ocular sampling.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0038] The figures of the enclosed drawings will be referred to, wherein:

[0039] FIG. 1, is a perspective and general representation of the device according to a preferred embodiment of the present invention;

[0040] FIG. 2 represents a side view of the device of FIG. 1;

[0041] FIG. 3 is an exploded representation of the second element of the device of FIGS. 1 and 2;

[0042] FIG. 4 is a perspective and general representation of the kit according to a preferred embodiment of the present invention;

[0043] FIG. 5 is a perspective representation of the supporting element of the kit of FIG. 4;

[0044] FIG. 6 is a side view of the first element of the device of FIG. 1 and of the closing element of said first element.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0045] By firstly referring to FIGS. 1 and 2, a device according to a preferred embodiment of the invention is designated as a whole with 1.

[0046] In the description sizes of the device are shown, not with limitative purposes, but established after the studies performed on the human eye morphology, so as to adapt advantageously to the eye without creating trouble.

[0047] The device comprises a first element 2 with substantially elongated shape having at one end a hollow portion 3 apt to house a membrane suitable for the ocular sampling.

[0048] The first element 2 (designated also as sub-system A or pickup stick) was devised for a double use mode: coupled with the second element 4 of the device (designated also as periocular base) for an assisted sampling, or in stand-alone mode for a manual sampling. The first element 2 advantageously will have an ergonomic shape, so as to be easy to be handled by the operator. The hollow portion 3 at one end thereof will be suitable to house a membrane for the ocular sampling, in particular it will be a circular cavity suitable to house the membrane of Millipore type.

[0049] The possibility of using said first element 2, even in stand-alone mode, allows performing an easy and safe sampling even in patients having pathologies therefor the eyeball has different morphological features than normal, it projects more or has smaller sizes, and which would prevent the correct positioning of the second element 4 of the device. The two elements (first and second element, respectively) are then suitable to be reversibly connected therebetween before and after use.

[0050] According to the embodiment represented in the figures the first element 2 has a cylindrical shape, in particular a solid cylindrical, slightly flared shape and at the lower end it has a hollow cylindrical section 3, with a circular base with an outer diameter of about 14 mm and an inner diameter of about 12 mm. This could be arranged for positioning the Millipore membrane with the support thereof, as already existing in the packaging itself.

[0051] The overall length of said first element is comprised between 80 and 90 mm, in particular 87 mm. The geometry and the sizes of said first element 2 were selected so that it could be handled easily by the operator during the sampling, even by increasing the distance of his/her hands from the patient's eye. An increase in the field of vision of the operator with respect to the membrane direct use follows and, consequently, a more controlled positioning of the membrane onto the ocular surface.

[0052] The second element 4, represented in FIG. 1 acts as support for the first element 2, which is inserted in properly sized housing and it can slide on guides 5 until reaching the ocular surface. Elastic means 6 comprising 3 compression springs inserted in the linear guides 5 allow a passive adjustment of the forces interacting with the tissues, by drastically increasing the interaction safety and reducing considerably the risk of damages. The joint use of the two elements of the device then allows an assisted sampling, with increase in the level of accuracy, repeatability and safety.

[0053] The device then allows a targeted and reproducible sampling; it is able to filter tremor or unskilled motions by the operator and to increase safety in the interaction with the ocular surface of the patient, by avoiding risks of damages to the tissues.

[0054] According to the embodiment represented in FIGS. 1-5 the second element 4 has a hollow cylindrical geometry, preferably with a side curvature, so as to allow greater ergonomicity. Said second element 4 comprises a main body 10 preferably with an outer diameter of 42 mm and an inner diameter of 30 mm so as to obtain a complete winding of the orbit and not to obscure the vision of the patient. The outer diameter preferably will have slightly larger sizes than those of the orbit to allow a stable positioning around the eye, to limit the motion of the eyelids and to not create discomfort or constriction in the patient. Said main body will have a base with curve profile, with radius equal to about 2 mm, in order to allow a more comfortable, safer and less troublesome rest onto the patient's face, the height of said main body preferably will be about 30 mm.

[0055] Said second element 4, in the main body thereof 10 has guiding means 5, in particular three linear guides arranged at 120 one with respect to the other one for housing a mobile disc 8 sliding along the hollow cylinder of the main body of the element. The elastic means 6 is positioned in the linear guides, in particular three compression springs having the following features: inner diameter equal to 2.55 mm, outer diameter equal to 3.05 mm, 9 active turns, free length (if not subjected to load) equal to 20.57 mm, length at compaction equal to 7.75 mm. The three springs have a stiffness, K, equal to 0.16 N/mm and they result to resist to a maximum force equal to 2.14 N, measured experimentally during imprinting procedures performed in laboratory. The presence of the mobile disc 8 on linear guides allows guiding the operator in performing the cytological pick-up, by compensating possible tremor or involuntary motions. The use of the properly sized springs 6 guarantees the control of the maximum forces applicable by the operator's hand during imprinting, thus increasing safety in the interaction with the ocular surface and avoiding tears of tissues. The material of the springs is stainless steel with a working temperature between 200 C. and +280 C.

[0056] The mobile element 8 shaped like a disc has a hollow portion, designated in figures with numeral 9, advantageously tilted by about 15 degrees coherently with the natural curvature of the human eye. Said hollow portion 9 (or hole) will be suitable for inserting the first element 2 by allowing the sampling in different points of the ocular surface by simply rotating the second element 4. The discharge of the mobile disc from the second element will be advantageously avoided by the presence of a circular element 7 fastened to the upper end of the periocular support. This for example could have the sizes of 2 mm in thickness, 41 mm of outer diameter and 38 mm of inner diameter and be fastened, for example by interlocking in the upper portion of said second element.

[0057] The second element thus was planned for allowing the insertion of the first element in the hollow portion 9 tilted by 15 degrees. In fact the inner disc facilitates the sampling of the involved portion by addressing the operator's hand in pre-established areas for the sampling, by avoiding possible errors or inaccuracies by the operator. In this way the analysis of the side areas results to be possible without modifying the membrane and without the risk of damaging the ocular wall.

[0058] According to an embodiment the complete device could have a weight not exceeding 250 grams excluding the springs. The used material, excluding the springs, for creating the device could be for example polycarbonate or PVC, both materials guarantee the possibility of sterilizing in autoclave (as Tg=150 C.), or by means of gamma rays.

[0059] A subject of the present invention is also a kit for the ocular sampling, in particular for the sampling and analysis of the outer layers of the ocular surface by impression cytology.

[0060] By making reference to FIG. 4, this shows a preferred embodiment of the kit designated as a whole with 15. The kit could include one or more devices according to the present invention and/or one or more membranes for the ocular sampling, preferably of the Millipore type.

[0061] Said kit could further comprise even one or more closing elements 13 of said first element. The closing elements, designated in FIG. 6 with numeral 13, are suitable to preserve the sample by avoiding the deterioration thereof. These closing elements have a specular geometry with respect to the first element so as to allow the perfect housing of the lower portion of the first element inside thereof. The upper portion thereof preferably is tilted by 15 degrees, in order to allow a perfect coupling with the first element, and it has a portion including a liquid quantity for preserving the membrane after sampling, for example up to a maximum of 300 l. The kit then could further comprise even solutions suitable for the biochemical, confocal or molecular analysis of the collected samples.

[0062] The kit could further include a supporting structure, such as for example that represented in FIG. 5 and designated as a whole with the numeral 11, comprising one or more seats 11 for said first element, one or more seats 11 for said second element and one or more seats 11 for said closing elements.

[0063] Detailed description of the method for using the device and the kit according to the preferred embodiment of the present invention represented in FIGS. 1-6:

[0064] Both for the manual sampling (with stand-alone use of the first element 2) and for the assisted sampling (with joint use of the first and second element 4), the procedure for assembling the device to the membrane used for the sampling results to be the same.

[0065] At first the plastic support of the membrane have to be removed, by using sterile tweezers, by making attention to remove each residue in order to obtain a smooth surface which does not run the risk of scratching the ocular epithelium.

[0066] The passages are schematized here below: [0067] insertion of the membrane, comprising the plastic support thereof, in the lower portion of the first element. [0068] insertion of the first element in the hollow portion 9 (hole) of the second element [0069] detection of the area of interest and widening of the eyelid area of the patient; [0070] positioning of the second element on the ocular orbit with orientation of the hole according to the area of interest; [0071] by means of grasping the second element 4 with the thumb and middle finger, applying a slight pressure onto the upper end of the first element 2 with forefinger, until reaching the ocular surface; [0072] keeping the pressure for 4-5 seconds and then removing the device from the eye; [0073] positioning the device on a flat surface and extracting the first element from the second element; [0074] inserting the first element into one of the closing elements 13 supplied with the kit 15, based upon the analyses to be performed; [0075] possible preparation of the documents accompanying the shipment/delivery to the laboratory which will perform the analysis, in line with the existing rules relating shipment of biological sample for research/diagnosis.

[0076] The device of the present invention results easy to be used, effective and extremely interesting as instrument for predicting the alteration of the ocular surface. The possibility of performing a biochemical analysis, in addition to the molecular one, would allow to identify protein markers which, with respect to the molecular markers, would allow to overcome post-transcriptional or post-translational locks and/or modifications. At the same time, a quick and safe outpatient screening would allow a prompt pharmacological response and a quicker therapeutic application, an essential requirement in the Ophthalmological practice. The flexibility of this device allows the the quick transfer of the impression cytology to the ophthalmological sanitary service and subsequently to the other, sanitary and not (for example veterinary). [0077] The use of the device in the basic research and translational field allows a quick and standardized sampling, independently from the operator's experience, by offering a real support for clinical trials and national and International multicentre studies. All this assisted by a quick shipment to the reference centres for the analyses or analyses on site. [0078] The use in sanitary field (outpatient, primary care physician, first aid) would allow: a timely base diagnosis of predictive type and/or therapeutic monitoring; a support to the clinic by evaluating markers validated for diagnosis, prognosis and/or therapeutic monitoring (differential analysis in case of bacterial and/or viral infections or over-infections as well as evaluation of the effectiveness of a therapy or pharmacological damage, through cytotoxicity tests).

[0079] Examples of application areas of the device and of the kit according to the present invention are: [0080] medical field (Outpatient: Ophthalmology; Dermatology; Dentology); [0081] veterinary field (Outpatient for analyses of infections); [0082] agro-feeding field (onsite sampling for analyses of adulterations).

[0083] The present invention has been sofar described by referring to some preferred embodiments. It is to be meant that other embodiments belonging to the same inventive core may exist, as defined by the protective scope of the herebelow reported claims.

Comparative Experiments

[0084] PAS staining on impression cytologies performed with EYEPRIM device and comparison with sampling method with Millicell membrane (Millipore) currently used in the most part of the basic and translational research studies.

[0085] The comparison between the two devices was performed by evaluating the pre-Analytical (during sampling) and Analytical (laboratory survey) attitudes.

1. Pre-Analytical: Comparison by Pick-Up Type

[0086] In case of EYEPRIM (device described in US2011/319789) the pick-up results to be partial and it seems not so easy to be implemented as the device is not ergonomic and therefore it does not replace in a satisfying way the classic method with Millicell support (Millipore membrane). The reluctance would lie in the fact that the syringe-like device results to be uncomfortable at the time of the ocular sampling. To this purpose, the device according to the present invention brings an improvement both in the classical outpatient sampling (Millicell) and compared to the EYEPRIM method. This results from the analysis of questionnaires developed specifically for the device analysis. In the specific case, the device according to the present invention was analysed for physical and mechanical features as sub-system 1 (A), sub-system 2 (B) and sub-system 1 assisted by the sub-system 2 (A+B). The voluntary subject (operator) expressed his/her opinion relating the used methods and his/her sensations, having also tested EYEPRIM. [0087] 85% of consulted operators consider the sub-system A light, ergonomic and easy to be handled, they have no comments relating the shape and features and they do not show interest in using it. [0088] 60% of consulted operators, instead, show little interest in the sub-system B by considering it bulky or with not ideal geometry for the use thereof; however, they recognize a potential improvement in the sampling procedure and a higher effectiveness and simplicity. To this purpose the device was made lighter and refined in the shapes.

[0089] On the whole, about 70% of operators consider to be satisfied by the proposed device and evaluate it simple to be used, by giving a good/optimum overall judgment and they prefer it to EYEPRIM.

[0090] Ultimately, from this study it resulted that the device according to the present invention sub-system 1 is more ergonomic, lighter and more flexible than the EYEPRIM device. The device resulted to be easy to be used as, since it is not in the syringe-like type, the operator has not to impart a pressure which, above all in the least skilled people, could destabilize the hand at the moment of impact onto the ocular surface. The spring has no buffer systems. Such limitation is compensated in the device sub-system 1 assisted by the sub-system 2 which allows not only a targeted sampling but even a calibration of the membrane-ocular surface contact force (protection effect).

[0091] Furthermore the device has a supported environmental impact as if one wishes it only the membrane can be replaced. The device comes not directly in contact with the patient and, if wished, the specialist can bring it always with him/her for the in office procedures. The cap, by offering the fastening possibility at time of the pick-up, has a convenience in the shipment to the analysis laboratory.

2. Analytical: comparison of the membranes by means of histological coloration according to PAS method and acquisition in transmitted light digital microscopy.

[0092] The samplings reported hereinafter were performed by an Ophthalmologist specialist on volunteers (four samplings on three healthy volunteers, in outpatient seat) and the process was performed in laboratory by a specialized operator (Biologist). The membranes were removed from the support (technique of detachment by means of surgical knife) and subjected to PAS coloration (Periodic Acid Schiff Hotchkiss-MCManus #04-130802, Bioptica), a differential coloration which is performed directly onto the prefixed membrane (citofix; BioOptica). The coloration passages were: 1. hydration in distilled water (3 min); 2. Treatment with 1% Periodic Acid (5 min) and washing in distilled water (1 min); 3. Coloration with Schiff base reagent (2 min) and washing in distilled water (3 min); 4. nuclear countercoloration with Mayer Hematoxylin (2 min) and differentiation in spring water (5 min); 5. balancing in phosphate buffer and positioning of the coloured membrane on cover glass for closing with cover glass (mountant based upon glycerol/gelatin in phosphate buffer).

[0093] The membrane of the device EYEPRIM results to be opaque to the passage of the light beam (direct microscope), by preventing a clear acquisition of the transmitted light image. On the contrary, the device according to the present invention, as it does not change the type of perfectly tested membrane, allows a clear and distinct acquisition of the images by means of optical microscopy and in some cases immunofluorescence.