Method For Diagnosing Alzheimer's Disease Spectrum Neurocognitive Disorders From Tear Sample, And Solution And Diagnostic Kit Useful In The Method
20230024661 · 2023-01-26
Assignee
Inventors
Cpc classification
International classification
G01N33/52
PHYSICS
Abstract
The invention relates to an aqueous solution of AuCl.sub.3×2H.sub.2O or HAuCl.sub.4×4H.sub.2O, ZnCl.sub.2 or ZnSO.sub.4, and AgNO.sub.3 having an Au.sup.3+ concentration of 0.8 mM-1.6 mM, a Zn.sup.2+ concentration of 15 μM-50 μM, and an Ag.sup.+ concentration of 5 μM-50 μM. The invention extends to a kit comprising a solution of the invention, a method of preparing said solution, and the use of a solution of the invention, a solution prepared by a method of the invention, or a kit of the invention in predicting and/or diagnosing and/or monitoring a neurocognitive disorder of the Alzheimer's disease spectrum. The invention extends to a method for predicting and/or diagnosing and/or monitoring a neurocognitive disorder of the Alzheimer's disease spectrum in a patient comprising contacting a tear sample from the patient normalised for protein concentration with a solution of the invention; applying an amount of the tear sample thus obtained to a surface; allowing the tear sample to dry; and drawing a conclusion based on the pattern of the thus obtained dried tear sample, whether the patient is at risk of or suffers from a neurocognitive disorder of the Alzheimer's disease spectrum.
Claims
1. An aqueous solution of AuCl.sub.3×2H.sub.2O or HAuCl.sub.4×4H.sub.2O, ZnCl.sub.2 or ZnSO.sub.4, and AgNO.sub.3, which comprises Au.sup.3+ at a concentration of 0.8 mM to 1.6 mM, Zn.sup.2+ at a concentration of 15 μM to 50 μM, and Ag.sup.+ at a concentration of 5 μM to 50 μM.
2. The solution according to claim 1, wherein the solution comprises the Au.sup.3+, Zn.sup.2+, and Ag.sup.+ ions in the following molar ratio: Au.sup.3+:Zn.sup.2+: Ag.sup.+=50:1:2.
3. The solution according to claim 1, wherein the solution is an aqueous solution of AuCl.sub.3×2H.sub.2O, ZnCl.sub.2 and AgNO.sub.3 comprising Au.sup.3+ at a concentration of about 1 mM, Zn.sup.2+ at a concentration of about 20 μM, and Ag.sup.+ at a concentration of about 40 μM.
4. A method of preparing a solution of claim 1, the method comprising: (a) dissolving AuCl.sub.3×2H.sub.2O or HAuCl.sub.4×4H.sub.2P in distilled or deionised water to obtain a solution with an Au.sup.3+ concentration of about 2 to 4 mM; (b) optionally freezing the solution at a temperature of −20° C. and then thawing it before step (c); (c) filtering the solution with an Au.sup.3+ concentration of about 2 to 4 mM through a bacterial filter of a maximum pore size of 0.45 μm, and then mixing the filtrate with distilled or deionised water to obtain a solution with an Au.sup.3+ concentration of about 0.8 to 1.6 mM; (d) adding a solution with a Zn.sup.2+ concentration of about 50 mM prepared using ZnCl.sub.2 or ZnSO.sub.4 and distilled or deionised water to the solution with an Au.sup.3+ concentration of about 0.8 to 1.6 mM to obtain a solution with a Zn.sup.2+ concentration of about 15 to 50 μM; (e) adding a solution with an Ag.sup.+ concentration of about 50 mM prepared using AgNO.sub.3 and distilled or deionised water to the solution with an Au.sup.3+ concentration of about 0.8 to 1.6 mM and Zn.sup.2+ concentration of about 15 to 50 μM, and, optionally, stirring the solution thus obtained.
5. A method of preparing a solution of claim 3, the method comprising: (a) dissolving AuCl.sub.3×2H.sub.2O in distilled or deionised water to obtain a solution with an Au.sup.3+ concentration of about 2 mM; (b) optionally freezing the solution to a temperature of −20° C., and then thawing it before step (c); (c) filtering the solution with an Au.sup.3+ concentration of about 2 mM through a bacterial filter with a maximum pore size of 0.45 μm, and then mixing it with distilled or deionised water to obtain a solution with an Au.sup.3+ concentration of about 1 mM; (d) adding a solution with a Zn.sup.2+ concentration of about 50 mM prepared using ZnCl.sub.2 and distilled or deionised water to the solution with an Au.sup.3+ concentration of about 1 mM to obtain a solution with a Zn.sup.2+ concentration of about 20 μM; (e) adding a solution with an Ag.sup.+ concentration of about 50 mM prepared using AgNO.sub.3 and distilled or deionised water to the solution with an Au.sup.3+ concentration of about 1 mM and Zn.sup.2+ concentration of about 20 μM, and, optionally, stirring the solution thus obtained.
6. (canceled)
7. A method for preparing a dried tear sample, the method comprising: (a) normalising a tear sample for protein concentration; (b) contacting the tear sample normalised for protein concentration with a solution of AuCl.sub.3×2H.sub.2O or HAuCl.sub.4×4H.sub.2O, ZnCl.sub.2 or ZnSO.sub.4, and AgNO.sub.3, which comprises Au.sup.3+ at a concentration of 0.8 mM to 1.6 mM, Zn.sup.2+ at a concentration of 15 μM to 50 μM, and Ag.sup.+ at a concentration of 5 μM to 50 μM; or with a solution prepared by a method of claim 4; (c) applying an amount, preferably an amount of about 1 μL, of the contacted normalised tear sample to a surface, preferably a hydrophobic plastic surface; (d) allowing the applied tear sample to dry.
8. A method for predicting and/or diagnosing and/or monitoring a neurocognitive disorder of the Alzheimer's disease spectrum in a patient, the method comprising: (a) normalising a tear sample from the patient for protein concentration; (b) contacting the normalised tear sample with a solution of AuCl.sub.3×2H.sub.2O or HAuCl.sub.4×4H.sub.2O, ZnCl.sub.2 or ZnSO.sub.4, and AgNO.sub.3, which comprises Au.sup.3+ at a concentration of 0.8 mM to 1.6 mM, Zn.sup.2+ at a concentration of 15 μM to 50 μM, and Ag.sup.+ at a concentration of 5 μM to 50 μM; or with a solution prepared according to the method of claim 4; (c) applying an amount of the contacted normalised tear sample, preferably an amount of 1 μL, to a surface, preferably a hydrophobic plastic surface; (d) allowing the applied tear sample to dry; (e) drawing a conclusion on the basis of the pattern of the dried tear sample thus obtained or of the pattern of an image of the dried tear sample thus obtained whether the patient is at risk of or suffers from a neurocognitive disorder of the Alzheimer's disease spectrum.
9. The method according to claim 8, wherein the conclusion based on the pattern is a conclusion based on the average particle size and/or particle density of the particles constituting the pattern.
10. The method according to claim 9, wherein an optical microscope, and optionally, one or more computer software, is used for the determination of the average particle size and/or particle density.
11. The method according to claim 10, wherein an image is taken of the dried tear sample, and the drawing a conclusion on the basis of the pattern of the image comprises (i) calculating the ratio between the sample area covered by particles and the number of particles, that is, the area/particle ratio; (ii) deriving a diagnostic value (DV) from the area/particle ratio using the following formula:
DV[%]=P.sub.(area/particle ratio)/Control.sub.(area/particle ratio))×100, wherein P.sub.(area/particle ratio) is the value of the area/particle ratio determined from the dried tear sample, Control.sub.(area/particle ratio) is the arithmetic average of area/particle ratio values determined from dried tear samples of a group of individuals with known diagnosis of not having neurocognitive disorder of the AD spectrum, wherein said dried tear samples are prepared by a method comprising: (a) normalising the tear samples for protein concentration; (b) contacting the tear samples normalised for protein concentration with a solution of AuCl.sub.3×2H.sub.2O or HAuCl.sub.4×4H.sub.2O, ZnCl.sub.2 or ZnSO.sub.4, and AgNO.sub.3, which comprises Au.sup.3+ at a concentration of 0.8 mM to 1.6 mM, Zn.sup.2+ at a concentration of 15 μM to 50 μM, and Ag.sup.+ at a concentration of 5 μM to 50 μM; (c) applying an amount, preferably an amount of about 1 μL, of each of the contacted normalised tear samples to a surface, preferably a hydrophobic plastic surface; (d) allowing the applied tear samples to dry; (iii) comparing the DV with one or more reference values
12. The method according to claim 11, wherein the method further comprises one or more image transformation steps facilitating image processing.
13. The method according to claim 8, wherein the neurocognitive disorder of the Alzheimer's disease spectrum is Alzheimer's disease.
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. The method according to claim 7, wherein the tear sample of step (a) is fresh tear, stored tear, frozen and thawed tear, dried and reconstituted tear, or a supernatant obtained from any of the foregoing by centrifugation.
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. A solution formed by the method of claim 4, wherein the solution comprises the Au.sup.3+, Zn.sup.2+, and Ag.sup.+ ions in the following molar ratio: Au.sup.3+:Zn.sup.2+:Ag.sup.+=50:1:2.
24. A solution formed by the method of claim 4 wherein the solution is an aqueous solution of AuCl.sub.3×2H.sub.2O, ZnCl.sub.2 and AgNO.sub.3 comprising Au.sup.3+ at a concentration of about 1 mM, Zn.sup.2+ at a concentration of about 20 μM, and Ag.sup.+ at a concentration of about 40 μM.
25. A method for preparing a dried tear sample, the method comprising: (a) normalising a tear sample for protein concentration; (b) contacting the tear sample normalised for protein concentration with a solution of AuCl.sub.3×2H.sub.2O, ZnCl.sub.2, and AgNO.sub.3, which comprises Au.sup.3+ at a concentration of about 1 mM, Zn.sup.2+ at a concentration of about 20 μM, and Ag.sup.+ at a concentration of about 40 μM; or with a solution prepared by a method of claim 5; (c) applying an amount, preferably an amount of about 1 μL, of the contacted normalised tear sample to a surface, preferably a hydrophobic plastic surface; (d) allowing the applied tear sample to dry.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
DETAILED DESCRIPTION OF THE INVENTION
[0055] The aqueous solution defined in the appended claims is a solution of the components defined in said claims prepared using distilled or deionised water. The aqueous solution is prepared with the exclusive use of the listed salts (that is, AuCl.sub.3×2H.sub.2O or HAuCl.sub.4×4H.sub.2O, preferably AuCl.sub.3×2H.sub.2O; ZnCl.sub.2 or ZnSO.sub.4, preferably ZnCl.sub.2; and AgNO.sub.3).
[0056] Thus, the method, solution and kit of the invention are useful in the prediction and/or diagnosis and/or monitoring of a neurocognitive disorder of the Alzheimer's disease spectrum [e.g. Alzheimer's disease or mild cognitive disorder (MCD)], on the basis of a sample of human tear. The prediction, diagnosis or monitoring of the neurocognitive disorder of the AD spectrum refers—in relation to a neurocognitive disorder of the Alzheimer's disease spectrum—to predicting, diagnosing, or monitoring by the use of the L-AD method as an independent method, as well as to predicting, diagnosing, or monitoring by the use of the L-AD method as a method supporting other diagnostic methods.
[0057] In the description and the claims, the term “tear sample” also covers a supernatant obtained by centrifuging tear taken from a patient. Therefore, as the tear sample of the invention, one may use the original tear sample, optionally upon freezing and thawing, or the supernatant obtained by centrifuging the original, optionally frozen and thawed tear sample. In the latter case, the supernatant or a part thereof is normalised for protein concentration, and is treated with a solution of the invention. In another embodiment, the original tear sample from the patient is dried, for example, for the purposes of transport and/or storage, and is later reconstituted before use (testing). Thus, the tear sample used according to the invention may be fresh tear, stored tear, frozen and thawed tear, dried and reconstituted tear, or a supernatant obtained by centrifuging any of these. All of the above can be referred to as a native tear sample as long as they are not treated with L-AD solution or other salt solution.
[0058] The solution of the invention (also referred to as “L-AD solution”) is preferably prepared by dissolving AuCl.sub.3×2H.sub.2O with a minimum Au.sup.3+ content of 57% and with a 40% compensation based on MW, in distilled water to obtain a solution with an Au.sup.3+ concentration of 2 mM. Optionally, the solution thus obtained is frozen at a temperature of −20° C., and then thawed before further steps. The solution thus obtained is filtered through a filter with a pore size of 0.45 μm, and to 500 μL of this filtered solution 499.1 μL of distilled water is added, followed by the addition of 0.4 μL of a ZnCl.sub.2 stock solution with a Zn.sup.2+ concentration of 50 mM prepared in distilled water, then 0.8 μL of an AgNO.sub.3 stock solution with an Ag.sup.+ concentration of 50 mM prepared in distilled water, and finally the solution thus obtained is stirred by vortexing.
[0059] The L-AD solution has an antiseptic effect preventing it from becoming infected; it is also not light-sensitive, and can be stored at room temperature.
[0060] When using a solution of the invention, by the use of an L-AD solution or a kit, wherein the L-AD solution is preferably an aqueous solution of AuCl.sub.3×2H.sub.2O, ZnCl.sub.2 and AgNO.sub.3, and preferably the concentration of Au.sup.3+ is 1 mM, the concentration of Zn.sup.2+ is 20 μM, and the concentration of Ag.sup.+ is 40 μM, and the kit preferably comprises such an L-AD solution, a dried tear sample of the invention is prepared using a method comprising normalising a tear sample from a patient for protein concentration (1.5 mg/mL to 15 mg/mL, preferably 1.5 mg/mL), and then contacting the normalised tear sample with L-AD solution, the ratio between the volume of the sample and the volume of L-AD solution in the contacting step is preferably (0.9-1.1):(6-7), preferably 1:6.5, and then applying an amount, preferably an amount of about 1 μL, of the contacted and normalised tear sample to a surface; preferably a hydrophobic plastic surface, and the tear sample amount is allowed to dry.
[0061] The use of a hydrophobic plastic surface (e.g. polystyrene) during the preparation of the dried tear sample, that is, the application of the tear sample to a hydrophobic plastic surface showed the unexpected benefit of ringless stagograms. At the same time, native (i.e. not treated with L-AD solution), dried tear samples do not produce ringless stagograms even on hydrophobic plastic surfaces.
[0062] The method of preparing a dried tear sample of the invention preferably comprises the steps of
[0063] a) centrifuging, preferably at 300×G for 3 minutes, a native tear sample from a patient, optionally after freezing and thawing, wherein freezing is carried out preferably at −20° C.;
[0064] b) homogenising the supernatant of the sample thus obtained;
[0065] c) optionally freezing the homogenised supernatant to a temperature at −75° C., and then thawing it;
[0066] d) performing protein assay, preferably BisANS protein assay with an amount of the supernatant, preferably an amount of about 0.5 μL;
[0067] e) normalising an amount, preferably an amount of about 1.4 μL of the homogenised supernatant to a pre-determined concentration, preferably 1.5 mg/mL, preferably using distilled water for the normalisation;
[0068] f) adding an amount, preferably an amount of about 6.5 μL, of L-AD solution to an amount, preferably to an amount of about 1 μL, of the normalised supernatant, preferably while stirring with a pipette;
[0069] g) applying, preferably pipetting, an amount, preferably an amount of about 1-μL, of the tear sample treated with the L-AD solution to a surface, preferably a hydrophobic plastic surface, more preferably a Petri dish made from such plastic;
[0070] h) allowing the supernatant applied to the surface to dry, preferably for 20 minutes, preferably at a relative humidity of about 50% to obtain a dried tear sample of the invention;
[0071] i) optionally archiving the dried tear sample of the invention thus obtained, optionally comprising sealing the Petri dish, preferably with parafilm, in case the dried tear sample of the invention is in the preferably used Petri dish.
[0072] Thus, typically, the dried tear sample is obtained by drying 1 μL of original normalised tear sample treated with L-AD solution or of normalised supernatant treated with L-AD solution, wherein the supernatant is obtained by centrifuging the original tear sample. The conditions under which the tear samples are dried are preferably as follows:
[0073] relative humidity: 40% to 55%; temperature: room temperature, e.g. 24° C., with passive evaporation; drying time: at least 20 minutes, preferably at least 30 minutes.
[0074] When using a solution of the invention, i.e. the solution of various “reactive” ions (preferably in the ratio of Au.sup.3+:Zn.sup.2+:Ag.sup.+=50:1:2), due to the properties of the components, a complex between the ion components of the multi-component solution and the tear proteins is formed, thereby creating a special structure depending on the number (amount) and position of the affected amino acids (cysteine, methionine, histidine, arginine). When preparing an L-AD solution—on the basis of the different standard potentials of the metal ions—, a fragile chemical balance is created. Since both the quantities and qualities of the given protein species are different (despite normalising the total protein amount), the pattern induced by the L-AD solution will also be individual and specific. The components of the L-AD solution bind to amino acids and form cross-bridges, and because different proteins have different and limited numbers of target amino acids, the patterns will also be different in the two groups. The number of epitopes affects the 3D structure as a differentiating factor. In a sample with a given protein content, the number of bonds created by the above-mentioned metal ions will be unique (first-degree specificity). Reactive ions unable to avoid molecular binding force proteins into unique spatial conformations depending on other native properties of the proteins (second-degree specificity). The quantitative and qualitative parameters of a given protein are not significantly different in the control and AD groups (groups of patients characterisable with a neurocognitive disorder of the Alzheimer's disease spectrum) (mass spectrometry results), but an additive measurement thereof already shows different patterns (third-degree specificity). In the world of molecules, crystallisation is among the most sensitive processes. Minimal disturbances (e.g. an extra component or the lack thereof) may also generate absolute differences in each property (shape, size, angles and number of edges, etc.) of the forming crystals. The molecular differences between the samples direct the above phase transition into various directions (fourth-degree specificity). This method detects the joint manifestation in the tear of many changes brought by a neurodegeneration of the brain. Above all, our purpose was to predict neurodegenerative diseases (neurocognitive disorders of the Alzheimer's disease spectrum, especially Alzheimer's disease), and/or quickly and accessibly detect them for those affected.
[0075] On the basis of the results of the L-AD test, the sample profile of the patients without neurocognitive disorder of the Alzheimer's disease spectrum and that of the patients characterisable with a neurocognitive disorder of the Alzheimer's disease spectrum can be distinguished to a significant extent. In the case of tear samples treated with L-AD solution and dried, the particles constituting the pattern of the dried sample, the crystals, crystal structures formed as a single block show different patterns for patients without neurocognitive disorder of the AD spectrum and for patients characterisable with a neurocognitive disorder of the AD spectrum. On the contrary, native dried samples do not show differences of diagnostic relevance between patients without neurocognitive disorder of the AD spectrum and patients characterisable with a neurocognitive disorder of the AD spectrum. Column A of
[0076] However, the dried tear samples of the invention show—also by microscopy (e.g. optical microscope or electron microscope)—that in the case of patients characterisable with a neurocognitive disorder of the Alzheimer's disease spectrum the dried tear samples of the invention are paler in terms of density and its particle range is smaller as compared to the dried tear samples of the invention for patients showing no cognitive deficits. Columns B to D of
[0077] The observable particles (peptide or protein agglomerates, and the images thereof) are larger in size and more densely dispersed in the images of Line I, and are smaller in size and more sparsely dispersed in Line II. Based on the above differences,
[0078] a) a conclusion can be made as to whether a patient is at risk of or suffers from a neurocognitive disorder of the Alzheimer's disease spectrum (that is, a prediction or diagnosis can be provided in relation to the neurocognitive disorder of the Alzheimer's disease spectrum); and/or
[0079] b) the status of a patient can be monitored in relation to the risk of a neurocognitive disorder of the Alzheimer's disease spectrum, or in relation to a neurocognitive disorder of the Alzheimer's disease spectrum.
[0080] Hence, the method of the invention (alternative references: “L-AD diagnostic test”, or “LacrimAD test”, or “L-AD test”) can successfully differentiate patients characterisable with a neurocognitive disorder of the AD spectrum, especially patients with Alzheimer's disease, from control individuals adjusted for elderly age and for gender. The L-AD diagnostic test is simple, quick (sample processing and evaluation takes only 30 minutes), cost-effective (with an estimated total cost of HUF 10000/person/sample), and non-invasive. Upon interacting with the tear sample normalised for protein amount, the reagent of unique composition according to the invention (L-AD solution) is able to detect differences of diagnostic power between healthy individuals and patients characterisable with a neurocognitive disorder of the AD spectrum, especially Alzheimer's disease, at a sufficiently high specificity and sensitivity.
[0081] Thus, the invention also relates to a method for predicting and/or diagnosing and/or monitoring a neurocognitive disorder of the Alzheimer's disease spectrum in a patient, the method comprising:
[0082] (a) normalising a tear sample from the patient for protein concentration;
[0083] (b) contacting, preferably mixing, the normalised tear sample with a solution of the invention;
[0084] (c) applying an amount of the contacted normalised tear sample, preferably an amount of about 1 μL, to a surface, preferably a hydrophobic plastic surface;
[0085] (d) allowing the applied tear sample to dry;
[0086] (e) drawing a conclusion on the basis of the pattern of the dried tear sample or on the basis of the pattern of an image of the dried tear sample as to whether the patient is at risk of or suffers from a neurocognitive disorder of the Alzheimer's disease spectrum.
[0087] The conclusion based on the pattern is typically a conclusion based on the average particle size and/or particle density of the particles constituting the pattern of the dried tear sample, and the average particle size and particle density are typically examined using microscope, at room temperature, e.g. at a temperature of 24° C. to 25° C. The dried tear sample is round-shaped and, when 1 μL of the contacted normalised tear sample is applied to the surface, has a typical diameter of about 0.9 to 1.3 mm, optimally about 1.1 mm.
[0088] In an embodiment of the invention, the method for predicting or diagnosing or monitoring a neurocognitive disorder of the Alzheimer's disease spectrum comprises taking an image, e.g. a digital photograph, of one or more tear samples contacted with an L-AD solution and dried (i.e. dried tear samples according to the invention), optionally using a microscope, preferably an optical microscope.
[0089] In an embodiment of the invention, an overview image of the dried tear sample, preferably of a rectangular shape, is also made, e.g. using a magnification of 64×, and/or a high-resolution image, e.g. using a magnification of 320×. Optionally, the overview image is taken of the visual field of the microscope, and the high-resolution image is preferably taken of an area of the overview image or of the visual field of the microscope where the pattern shows the highest reproducibility and the lowest diversity. In a preferred embodiment of the invention, in the case of an overview image or microscope visual field with a shape of a circular disk, the high-resolution image is taken at half of the radius of the circular disk. If a carrier with a hydrophobic plastic surface is used when drying the tear sample, then the stagogram will be ringless and completely homogeneous enabling making an image of any part thereof within the limits of the stagogram.
[0090] Optionally, one or more image transformation steps enabling image processing, e.g. binary conversation, is also carried out. Optionally, the image is subjected to graphical conversion, e.g. a two-colour graphical conversion, e.g. black-and-white graphical conversion. “Colour” refers to dense colours and/or patterns. “Two-colour graphical conversion” refers to converting the relevant information of the image into two colours. The skilled person may carry out the graphical conversion using a computer software program, a number of options are available, e.g. Photoshop.
[0091] In a preferred embodiment of the invention, one or more values are calculated for the dried tear sample on the basis of the image obtained by optical microscope, optionally on the basis of the image generated (obtained) by image transformation step(s), e.g. graphical conversion, via analysis (e.g. manually; using an image analysing computer software program, e.g. ImageJ), said one or more values are selected from:
[0092] (i) the number of particles in the sample area, that is, particle count;
[0093] (ii) the sample area covered by particles expressed, for example, in μm.sup.2; and
[0094] (iii) the average size of the particles expressed, for example, in μm.sup.2, which is the ratio between value (ii) and value (i) (i.e. the sample area covered by particles divided by the number of particles in the sample area), or in other words (and hereinafter), the area/particle ratio (or area/particle count ratio);
[0095] wherein the term “sample area” refers to the examined part of the visual field of the microscope or to the examined part of the image taken (made) of the visual field of the microscope.
[0096] In the context of the images, “particle” refers to the notion according to the general understanding in the field, that is, an image-part that can be considered as one unit definable by its contour and/or shade is considered as one particle. Typically, particles are of varied shape. Thus, each particle in the image corresponds to one particle contributing to the pattern of the dried tear sample.
[0097] In a preferred embodiment of the invention, one or more of (i) the particle count, (ii) the value of the sample area covered by particles, and (iii) the area/particle count ratio [the ratio of (ii) to (i)] is compared with the corresponding reference value(s), and on the basis of such comparison
[0098] a) a conclusion is made as to whether the patient is at risk of or suffers from a neurocognitive disorder of the Alzheimer's disease spectrum (that is, a prediction or diagnosis is made in relation to a neurocognitive disorder of the Alzheimer's disease spectrum); and/or
[0099] b) the status of the patient is monitored in relation to the risk of a neurocognitive disorder of the Alzheimer's disease spectrum, or in relation to a neurocognitive disorder of the Alzheimer's disease spectrum.
[0100] According to a preferred embodiment of the invention, in relation to the patient giving the tear sample, a diagnostic value (DV) is derived from the area/particle ratio using the following formula:
DV[%]=(P.sub.(area/particle ratio)/Control.sub.(area/particle ratio))×100
[0101] wherein
[0102] P.sub.(area/particle ratio) is the value of the area/particle ratio determined from a dried tear sample according to the invention of the patient,
[0103] Control.sub.(area/particle ratio) is the arithmetic average of area/particle ratio values determined from dried tear samples according to the invention of patients with a known diagnosis of not having (not showing) neurocognitive disorder of the AD spectrum,
[0104] and then the DV value thus obtained is compared with one or more reference values, and on the basis of said comparison of the DV value with the one or more reference values
[0105] a) a conclusion is made as to whether the patient is at risk of or suffers from a neurocognitive disorder of the Alzheimer's disease spectrum (that is, a prediction or diagnosis is made in relation to the neurocognitive disorder of the Alzheimer's disease spectrum); and/or
[0106] b) the status (condition) of the patient is monitored in relation to the risk of a neurocognitive disorder of the Alzheimer's disease spectrum, or in relation to a neurocognitive disorder of the Alzheimer's disease spectrum.
[0107] When carrying out the invention, the Control.sub.(area/particle ratio) value can be determined on the basis of the present description, or a value optionally already available can be used. Whichever option is used, the conditions of determining P.sub.(area/particle ratio) and Control.sub.(area/particle ratio) must be such as to enable comparison. Thus, the determination of P.sub.(area/particle ratio) and Control.sub.(area/particle ratio) is carried out using tear samples normalised for the same protein concentration, and using L-AD solutions of the same composition, furthermore the ratio between the volume of the normalised tear sample and the volume of the solution of the invention contacted (preferably mixed) with the former is constant, as are the volume of the contacted normalised tear sample applied to the surface and the wettability of the carrier applied. Furthermore, preferably identical microscope and camera systems are used but it is not necessary. In case of using a new detection system (microscope and camera), new photos of the control (reference) stagograms must be taken as reference samples. In case only digital comparison is carried out, the pixel numbers of the images must be increased or reduced according to the ratio between the magnification of the new microscope objective and the magnification of the old microscope objective (in line with the new requirements). The image analysis software programs must use the same algorithms for digital image scaling, graphical conversion, pixel colour scale range (e.g. 8- or 16-bit), and black-and-white pixel identification to ensure interchangeability. Several programs comply with the international standards for these parameters (e.g. PhotoShop, ViewNX-Nikon, ImageJ etc.).
[0108] The reference value is determined using patients with a known diagnosis of not having (showing) neurocognitive disorder of the AD spectrum and their data, and patients known to have a neurocognitive disorder of the Alzheimer's disease spectrum (e.g. MCD patients, AD patients) and their data, and considering medical ethics.
[0109] In a preferred embodiment of the invention, the DV [%] value is compared with an RV [%] reference value. The RV [%] value can be determined as follows.
[0110] The values of the area/particle ratio are determined using the L-AD method for patients characterisable with a neurocognitive disorder of the AD spectrum and for patients showing no neurocognitive disorder of the AD spectrum. A value for each patient is calculated from the value of the area/particle ratio of the patient thus obtained, using the following formula, which is analogous to the above formula:
V[%]=(P.sub.(area/particle ratio)/Control.sub.(area/particle ratio))×100
[0111] wherein P.sub.(area/particle ratio) is the value of the area/particle ratio determined from a dried tear sample according to the invention of a patient identified as a patient characterisable with a neurocognitive disorder of the Alzheimer's disease spectrum using a known diagnostic method,
[0112] Control.sub.(area/particle ratio) is the arithmetic average of area/particle ratio values determined from dried tear samples according to the invention of a group of individuals with a known diagnosis of showing no neurocognitive disorder of the AD spectrum.
[0113] The above formula is fittingly applied when the goal is to determine the value of V and RV for patients showing any neurocognitive disorder of the AD spectrum. For example, if the goal is to determine the value of RV for Alzheimer's disease, then the value of V is calculated by substituting the numerator of the fraction in the formula of V with the value of the area/particle ratio determined from a dried tear sample according to the invention of a patient identified as being a patient characterisable with Alzheimer's disease using a known diagnostic method (that is, other than the method of the invention).
[0114] In line with the above, and in compliance with medical considerations and medical ethics, the V values obtained for the individual patients are used to determine the numerical value of RV [%] for a neurocognitive disorder of the Alzheimer's disease spectrum, e.g. Alzheimer's disease or MCD.
[0115] In a preferred embodiment of the invention, the RV [%] value for the presence of MCD or for the risk of Alzheimer's disease is 50% or is less than 50%. In a preferred embodiment of the invention, the RV [%] value for Alzheimer's disease is 5% or is less than 5%.
[0116] In a preferred embodiment of the invention, a DV [%] value of not higher than 50% and higher than 5% for a patient leads to the conclusion that the patient is having a mild cognitive disorder (MCD) or is at risk of Alzheimer's disease. In a preferred embodiment of the invention, a DV [%] value of 5% or lower than 5% for a patient leads to the conclusion that the patient has Alzheimer's disease.
[0117] When carrying out the invention, the RV [%] value can be determined on the basis of the above, or a value optionally already available can also be used. Whichever option is used, when the values of DV [%] and RV [%] are determined, the conditions must be such as to enable comparison. The determination of the values of DV [%] and RV [%] is carried out using tear samples normalised for the same protein concentration, and using L-AD solutions of the same composition, furthermore the ratio between the volume of the normalised tear sample and the volume of the solution of the invention contacted (preferably mixed) with the former is constant, as are the volume of the contacted normalised tear sample applied to the surface and the wettability of the carrier applied. Furthermore, preferably identical microscope and camera systems are used but it is not necessary. In case of using a new detection system (microscope and camera), new photos of the control (reference) stagograms must be taken as reference samples. In case only digital comparison is carried out, the pixel numbers of the images must be increased or reduced according to the ratio between the magnification of the new microscope objective and the magnification of the old microscope objective (in line with the new requirements). The image analysis software programs must use the same algorithms for digital image scaling, graphical conversion, pixel colour scale range (e.g. 8- or 16-bit), and black-and-white pixel identification to ensure interchangeability. Several programs comply with the international standards for these parameters (e.g. PhotoShop, ViewNX-Nikon, ImageJ etc.).
[0118] In an embodiment of the invention, standard statistical methods (one-way ANOVA Bonferroni, p***<0.001) are used to compare the area/particle ratio of the gender- and age-adjusted group of patients without cognitive deficits (control group) and the area/particle ratio of the group of patients with a neurocognitive disorder of the Alzheimer's disease spectrum.
[0119] Preferably, the diagnostic method of the invention is used in a patient without none of the following conditions:
[0120] post-operative condition after an eye surgery; post-operative condition after an intervention directly affecting the flow and/or metabolism of the tear; post-operative condition after a surgery affecting lacrimal canal or lacrimal gland; a disease of the lacrimal canal;
[0121] those inflammatory and immunological processes, autoimmune diseases, viral infections and bacterial infections which primarily affect the eyes and the lacrimal glands and influence the sensitivity and validity of the L-AD test.
[0122] Of course, the diagnostic method of the invention cannot and does not aim to replace the complex medical task that is a synthesis of the results of the clinical anamnesis, laboratory tests, neurological, psychiatric and physical examinations, cognitive tests, and brain imaging (CT, MR) examinations. However, the use of the method of the invention can facilitate the medical diagnostic work and can replace other expensive or painful examinations (e.g. lumbar puncture, brain PET) as it is simple, quick, easily available, painless, and cost-effective. Similarly to the above-mentioned examinations, the method of the invention, i.e. LacrimAD laboratory diagnostic method developed by us can help clinicians in making a probability clinical diagnosis of a neurocognitive disorder of the Alzheimer's disease spectrum, especially Alzheimer's disease, and to make a more accurate diagnosis of MCD than before.
[0123] Table 1 compares the characteristics of the tests used in the diagnostics of neurocognitive disorders of the AD spectrum such as Alzheimer's disease with the characteristics of the method of the invention.
TABLE-US-00001 TABLE 1 characteristics of the tests used in the diagnostics of neurocognitive disorders of the AD spectrum such as Alzheimer's disease Cost/capital Test Invasivity Pain Sensitivity (HUF) Comment Cranial CT — + + 43000 Used in routine Cranial MR + +++ 84000 diagnostics Brain — + ++ 51000 SPECT FDG PET — + ++++ 240000 No health insurance coverage in dementia Amyloid — + +++++ 900000 Only used in PET studies but not in routine diagnostics Tau PET — + +++++ 900000 scientific clinical Liquor +++ +++ ++++ 20000 Use in routine biomarkers diagnostics is limited at the moment Plasma ++ + ++++ 18000 Validation in biomarkers progress LacrimAD — — at least 10000 Validation in tear test ++++ progress
[0124] Such novel analysis of the tear is useful in the diagnostic screening of a very large population, and in helping the clinical diagnosis of neurocognitive disorders of the Alzheimer's disease spectrum (AD spectrum neurocognitive disorders) including the genetic forms of Alzheimer's disease, Down's syndrome depending on age, isolated and combined cerebral amyloidopathies, tauopathies, the combined manifestation of Alzheimer's disease with type II diabetes mellitus, and the manifestation of mild cognitive disorder (MCD) related to Alzheimer's disease, especially Alzheimer's disease. From the viewpoint of the patient, one great advantage is that the method is non-invasive and painless.
EXAMPLES
Example 1
[0125] Preparation of the LacrimAD solution
[0126] AuCl.sub.3×2H.sub.2O with an Au.sup.3+ content of 57% and with a 40% compensation based on MW was dissolved in distilled water to obtain a solution with an Au.sup.3+ concentration of 2 mM (9.5 mg salt/10 mL). Part of the solution was divided into 50-mL aliquots, which were frozen at a temperature of −20° C., then thawed, and the preparation of the solution proceeded according to Example 1A. The remainder of the solution was not frozen, and the preparation of the solution proceeded according to Example 1B.
Example 1A
[0127] The frozen solution according to Example 1 was thawed, and then filtered through a filter with a pore size of 0.45 μm. To 500 μL of the solution thus obtained, 499.1 μL of distilled water was added. To the solution thus obtained, 0.4 μL of a ZnCl.sub.2 stock solution (50 mM; 6.815 mg salt/mL) was added, followed by the addition of 0.8 μL of a AgNO.sub.3 stock solution (50 mM; 8.494 mg salt/mL), and the solution thus obtained was finally vortexed (300 rpm/30 sec.), and stored at room temperature.
[0128] Ion concentrations of the solution thus obtained:
[0129] Au.sup.3+ concentration: 1 mM,
[0130] Zn.sup.2+ concentration: 20 μM,
[0131] Ag.sup.+ concentration: 40 μM.
[0132] The solution was vortexed before diagnostic use.
Example 1B
[0133] The non-frozen solution according to Example 1 was filtered through a filter with a pore size of 0.45 μm, and to 500 μL of the solution thus obtained, 499.1 μL of distilled water was added. To the solution thus obtained, 0.4 μL of a ZnCl.sub.2 stock solution (50 mM; 6.815 mg/mL), followed by the addition of 0.8 μL of a AgNO.sub.3 stock solution (50 mM; 8.494 mg/mL), and the solution thus obtained was finally vortexed (300 rpm/30 sec.), and stored at room temperature. Ion concentrations of the solution thus obtained:
[0134] Au.sup.3+ concentration: 1 mM,
[0135] Zn.sup.2+ concentration: 20 μM,
[0136] Ag.sup.+ concentration: 40 μM.
[0137] The solution was vortexed before diagnostic use.
Example 2
[0138] At the Memory Ambulance of the Psychiatric Clinic of the Faculty of General Medicine of the University of Szeged (SZTE), tear samples from 159 patients (n=159) with an AD diagnosis made on the basis of the DSM-5 [American Psychiatric Association, (2013) and NINCDS-ADRDA (McKhann, 1984] criteria (AD group) were taken upon written consents (Ethics Approval Nos.: SZTE, 3543/2015; 93/2018-SZTE/4263; 152/2018-SZTE/4356). 10 patients characterisable with a mild cognitive disorder were also enrolled in the study; these patients had an MCD diagnosis made on the basis of the test used for patients with Alzheimer's disease (MCD group, n=10). As controls (n=80), gender- and age-adjusted individuals not showing cognitive deficits were invited to participate in the study (control group). To ensure specificity, individuals with Down's syndrome without Alzheimer's disease (n=8) (Down group) and individuals with Parkinson's disease (n=25) (Parkinson group) were also enrolled. Individuals with a neurocognitive disorder not associated with AD, as well as those with an eye surgery during a 10-year period preceding the study and those affected by a diagnosed ophthalmological pathology or a pathology affecting the surface of the eyes were excluded from the study.
[0139] The tests were conducted using both fresh and frozen tear samples. It was concluded that freezing (at −20° C.) does not affect the sensitivity indicators of the test. Upon determining the protein content, thawed samples were treated and measured at a standardized protein concentration.
[0140] During the study, tear samples treated with the L-AD solution of Example 1A and Example 1B, resp. were used.
[0141] During the preliminary experiments conducted in preparation of the study, two types of teardrops (tear samples) applied to and dried on special hydrophobic plastic surfaces were examined (
[0142] Tests were conducted using an optical microscope and a DSLR camera. The graphical conversion was carried out by the consecutive use of various digital image analysis programs (ViewNX2, Photoshop, and ImageJ). These programs are commercially available, and are suitable for the present purposes. Image processing involved the following steps: [0143] a. Technical background of the photograph taken [0144] i. Microscope objectives: Leitz Wetzlar [0145] 1. 160/-, EP, 132/0.40 PHACQ1 [0146] 2. 160/-, PL, FLUOTAR, 6.3/0.20; about 90-95 grid/mm resolution [0147] ii. Spacer (light path): PERIPLAN 10×18, TL160 mm [0148] iii. magnification of 320×, rectangle [0149] b. Primary (raw) photograph: RAW/MC/8 bit/minimum 16 MP (Nikon NEF); ISO 100; 1/160 shutter speeds; ˜40.000 Lux [0150] c. WewNX2 (Nikon) [0151] i. Opened using the program ViewNX2 [0152] ii. Edit.fwdarw.Sharpness 1.fwdarw.Contrast 1.fwdarw.D-Lightening HS 1.fwdarw.Nature.fwdarw.Save [0153] iii. Convert file.fwdarw.TIFF (8 bit).fwdarw.LZV compression [0154] d. Photoshop CS3 (or CC) [0155] i. TIFF opened using the program Photoshop CS3 (or CC) [0156] ii. Colour mode (image).fwdarw.devignetting (correction: midtone, exp. 15%, paintbrush mode: 1400).fwdarw.grayscale [0157] iii. Image.fwdarw.Corrections.fwdarw.Exposure.fwdarw.parameters:
TABLE-US-00002 Exposure +1 Offset −0.2 Gamma correction 0.75 (.fwdarw.) [0158] iv. Filter.fwdarw.Sharpening.fwdarw.Blur mode.fwdarw.parameters:
TABLE-US-00003 Amount 150 Radius 0.5 Threshold 0 [0159] v. Foreground colour: white (#ffffff) (L100/RGB:255) [0160] vi. Background colour: white (#ffffff) (L100/RGB:255) [0161] vii. Filter.fwdarw.Artistic.fwdarw.Stamp (sketch).fwdarw.parameters:
TABLE-US-00004 Light/dark balance 10 Smoothness 1 [0162] viii. Saving done (TIFF, interlacing, LZW) [0163] e. ImageJ [0164] i. The graphical TIFF generated as a result of step d. was opened in ImageJ. [0165] ii. Analyze.fwdarw.set scale: [0166] Settings (see
[0173] Quantitative data from the digital photographs (optionally 16 megapixel in the present case) were also analysed.
[0174] During the preliminary experiments conducted in preparation of the study, the analyses covered, among others, the ratio between the areas of coverage of the two colours in the black-and-white pattern of the graphical conversion, and the particle count and particle size data.
[0175] The analyses also covered the data for the particle count, for the sample area covered by particles, and for the area/particle count.
[0176] In order to prepare the % graduation of the diagnostic scale, the value of RV was determined as follows:
[0177] (1) For AD patients:
[0178] with the use of the formula V=(P.sub.(area/particle ratio)/Control.sub.(area/particle ratio)×100, a value was calculated for each patient known to have AD and enrolled in the study via substituting the numerator of the fraction in the formula with the value of the area/particle ratio of the given AD patient, and substituting the denominator of said fraction with the arithmetic average of the values of the area/particle ratios of the patients in the control group. (See
[0179] (2) For MCD patients:
[0180] with the use of the formula V=(P.sub.(area/particle ratio)/Control.sub.(area/particle ratio))×100, a value was calculated for each patient known to have MCD and enrolled in the study via substituting the numerator of the fraction in the formula with the value of the area/particle ratio of the given MCD patient, and substituting the denominator of said fraction with the arithmetic average of the values of the area/particle ratios of the patients in the control group.
[0181] The Control.sub.(area/particle ratio) data according to the number of cases processed so far 109.264+/−1.8879 (SEM), wherein SEM=SD/√n (the quotient of the standard deviation and the square root of the case number).
[0182] Next, a statistical analysis was carried out (one-way ANOVA Bonferroni, p***<0.001).
[0183] On the basis of the case numbers processed so far, the values of RV for AD and MVD were 5% and 50%, respectively.
[0184] In order to provide a diagnostic classification of the individual patients according to L-AD test, the value calculated above for each patient was compared with the RV value. The status of the patient was classified as follows:
[0185] when V≤5%, it was concluded that the L-AD test classifies the patient as a patient with Alzheimer's disease,
[0186] when 5%<V≤50%, it was concluded that the L-AD test classifies the patient as a patient with MCD,
[0187] when V>50%, it was concluded that the L-AD test classifies the patient as a patient showing neither Alzheimer's disease nor MCD.
[0188] The results thus obtained were compared with the previously made diagnoses of the patients. In case the result of the L-AD test was in agreement with the known diagnosis, the result of the L-AD test of the patient was qualified as a true positive (in case of AD patient) or a true negative (in case of patient not showing cognitive deficits). In case the result of the L-AD test classified a patient known to be not showing cognitive deficits as a patient with AD, then the result of the L-AD test was qualified as a false positive. In case the result of the L-AD test classified a patient known to have AD as a patient without Alzheimer's disease or MCD, then the result of the L-AD test was qualified as a false negative. The results are presented in a tabulated form (see Table 2).
[0189] On the basis of the results of the L-AD test, it was found that the sample profile of the control group and that of the AD group are distinguishable to a significant degree. Under an optical microscope, the AD samples are paler in terms of density, and their particle size ranges are smaller than those of the controls. The data were also examined using scanning electron microscope to qualitatively and visually differentiate the structuredness of the complexes formed by the components of the L-AD solution and of the teardrop (
[0190] The quantitative analysis of the results of the L-AD test was carried out upon normalisation for age and gender.
[0191] In order to determine specificity and sensitivity, our results were also compared with other diseases, such as with the L-AD-specific stagograms of individuals with Parkinson's disease and of individuals with Down's syndrome without Alzheimer's disease.
TABLE-US-00005 TABLE 2 Assessment of the diagnostic specificity and sensitivity of LacrimAD in AD and in other neuropsychiatric diseases True True False False positive negative positive negative Specificity Sensitivity EC — 62 18 — 77.5% — PD — 18 7 — 72% DS — 5 3 — 62.5% AD 138 — — 21 — 88%
[0192] EC=elderly control; PD=Parkinson's disease; DS=Down's syndrome without Alzheimers disease condition; AD=Alzheimers disease; True positive=agreement between the L-AD test and the AD diagnosis; True negative=control individuals showed control values also in the L-AD test; False positive=a control, PD, or DS patient showing a pattern corresponding to AD patient; False negative=AD patient showing a pattern corresponding to control.
[0193] Below is a description of the detailed conditions of the experiments and assessments pertaining to the above.
TABLE-US-00006 TABLE 3 Laboratory consumables and chemicals used Manufacturer/ Description Catalogue No. Distributor Gold chloride (AuCl.sub.3 * .sub.2H.sub.2O) A-260 (57% Reanal (MW = 339.36 g/mol) Au) Zinc chloride (MW = 136.3 g/mol) 96468-50g FLUKA/BioChemica Silver nitrate (MW = 169.88 g/mol) 03090-101-160 Molar Chemicals Kft. Puradisc 25 AS25 0.45pm 514-8021 VWR International Kft. sterile filter Glass capillary, 1.2 mm OD, 300044 CEBIO 0.69 mm ID, 100 mm Petri dish (90 × 14.2 mm) 90-190 BIOLAB Zrt. UltraPure Dnase/Rnase-free 10977035 Life Technologies Inc. distilled water 0.1-10 μL pipette tips of Biosphere 70.1130.200 Sarstedt Kft. plus quality, neutral, boxed 20-200 μL pipette tips of Biosphere 70.1130.202 Sarstedt Kft. plus quality, neutral, boxed Parafilm WS-5000-10 Izinta 96-well plate half area CLS3695- Biocenter Kft. 100EA 4,4′-Dianilino-1,1′-binaphthyl-5,5′- D4162-5MG Sigma Aldrich © disulfonic acid dipotassium salt SmartPak ® DQ3 Purification Pack SPR00SIA1 MERCK Centrifuge tube with inner plug cap 734-1862 VWR International Kft. (15 mL) 2 mL PP Eppendorf 3-204-85-0 Biocenter 0.2 mL PCR tube AB-0620 Biocenter Essential oil mixture for air Aromax Natural purification Products Ethanol 96% 02911-469-430 Molar Chemicals Kft.
[0194] Laboratory Devices Used:
[0195] 1. Tabletop centrifuge (300 g);
[0196] 2. Manual pipettes (0.1 to 3 μL; 0.5 to 10 μL, 2 to 20 μL, 20 to 200 μL);
[0197] 3. Fluorescent plate reader (e.g. BMG NOVOStar);
[0198] 4. Normal transmission microscope;
[0199] 5. DSLR camera (min. 16 MP, DX);
[0200] Preparation of the Applied L-AD Solution
[0201] 1. AuCl.sub.3×2H.sub.2O, minimum Au.sup.3+ content: 57%, compensation: 40% (based on the MW);
[0202] 2. This was dissolved in distilled water to obtain a solution of 2 mM.
[0203] 3. The Au solutions were divided into aliquots of 50 mL each and were frozen at −20° C.;
[0204] 4. Composition of the L-AD solution, and dissolution order
[0205] a. 500 μL Au.sup.3+ solution (2 mM)
[0206] b. +499.1 μL distilled water (final Au.sup.3+ concentration: 1 mM)
[0207] c. +0.4 μL ZnCl.sub.2 solution (stock solution: 6.815 mg/50 mM/mL), final concentration: 20 μM;
[0208] d. 5-minute incubation+vortexing twice;
[0209] e. +0.8 μL AgNO.sub.3 solution (stock solution: 8.494 mg/50 mM/mL), final concentration: 40 μM;
[0210] f. The quantities not in current use were stored at room temperature, and were vortexed before use.
[0211] Practical Steps of the L-AD Process
[0212] 1. Sample Preparation and Storage
[0213] a. Samples thawed after freezing (−20° C.) were centrifuged (300×G/3 min)
[0214] b. 2/3 of the supernatant was carefully pipetted into a new PCR tube, and then after homogenisation 1×2.2 μL was taken out of it, and later an aliquot of 1.1 μL was taken out.
[0215] c. The aliquots were frozen (−75° C.), and a BioBank documentation was compiled.
[0216] 2. Sample Processing:
[0217] a. From the 2.2-μL sample (thawed after freezing), a volume of 1.4 μL was taken out;
[0218] b. Furthermore, 0.5 μL was used for protein determination (BisANS protein assay);
[0219] c. Based on the measured protein concentration, it was normalised to a concentration of 1.5 mg/mL using distilled water;
[0220] d. (1) During the preliminary experiment native teardrops (2×1 μL) from the given diluted sample were applied to the hydrophobic plastic surface (Petri), and an aliquot of 1 μL of the remainder was pipetted into a new PCR tube. Next, 6.5 μL of the L-AD solution was added while stirring with a pipette. The final protein concentration of the mixture thus obtained: 0.2 mg/mL. The treated sample was also pipetted into the Petri dish (min. 3×1-μL drops);
[0221] d. (2) During the test, 1 μL of the given diluted sample was pipetted into a new PCR tube. Next, 6.5 μL of the L-AD solution was added while stirring with a pipette. The final protein concentration of the mixture thus obtained: 0.2 mg/mL.
[0222] The treated sample was pipetted to the hydrophobic plastic (Petri) surface (a drop of 1 μL);
[0223] e. About 20 minutes of drying was required for the L-AD stagograms to develop (relative moisture content: about 50%);
[0224] f. After taking the photographs, the Petri dishes were encoded and archived (completely wrapped with parafilm to prevent moisture from entering).
[0225] Taking Photographs of the Samples
[0226] a. (1) During the preliminary experiment a minimum of 2 native normalised drops were photographed from one sample (depending on the available amount of sample with a protein concentration of 1.5 mg/mL), and a minimum of 3 overall photographs were taken of the normalised drops treated with the L-AD solution (circular disk-shaped) (see
[0227] a. (2) During the test, from the dried samples of each individual for the analysis of the area/particle ratio a minimum of 1 high-resolution photograph (rectangular shaped) (see
[0228] b. The high-resolution photograph was taken at half of the radius of the drop (see
[0229] c. All RAW/NEF photos were archived. After the RAW-TIFF conversion, a graphical image was generated.