DE102010047060A1 - Diagnosis of dementia, preferably Alzheimer's disease, comprises supplying extracted components from a suction to an analysis unit and analyzing components in analysis unit for presence of e.g. paired helical filaments-tau plaques - Google Patents

Abstract

Diagnosis of dementia, preferably Alzheimer's disease, based on an eye surgery of removed components, comprises supplying the extracted components from a suction to an analysis unit and analyzing the components in the analysis unit for the presence and/or relative proportions of paired helical filaments (PHF)-tau plaques, beta -amyloid plaques and/or other disease-specific molecules. An independent claim is included for an apparatus for the diagnosis of dementia, comprising the suction for the extracted components and the analysis unit.

Description

The invention relates to a method and a device for diagnosing Alzheimer's disease.

Alzheimer's disease is a disease that typically occurs at a young age and causes an increasing loss of cognitive and non-cognitive abilities in the affected patients. In the brain of patients with Alzheimer's disease, β-amyloid plaques and pathologically altered nerve cell processes with intracellular fibrillar deposits of the τ-PHF protein are formed. In the course of the disease, the brain mass decreases greatly by death of the neurons, this is called a brain atrophy. The presence of β-amyloid plaques in the cerebral cortex is a recognized early pathological hallmark of Alzheimer's disease. Β-amyloid pathology begins in the cerebral cortex, whereas τ-PHF pathology first occurs in hippocampal formation. In addition to the brain, β-amyloid deposits are also found in the lens of the eye, the retina ( Parisi V. (2003) Correlation between morphological and functional retinal impairment in patients affected by ocular hypertension, glaucoma, demyelinating optic neuritis, and Alzheimer's disease. Semin Ophthalmol .; 18 (2): 50-7 ) and in blood vessels. Furthermore, there are indications of deposits of τ-PHF in the retina ( Gupta N, Fong J, Ang LC, Yücel YH. Retinal tau pathology in human glaucomas. Can J Ophthalmol. 2008 Feb; 43 (1): 53-60 ).

It is known that Alzheimer's disease patients increasingly suffer from eye diseases such as AMD and glaucoma ( Bayer AU, Ferrari F, Erb C. "High incidence of glaucoma among patients with Alzheimer's disease." Eur Neurol. 2002; 47 (3): 165-8; Vickers JC. (1997) The cellular mechanism underlying neuronal degeneration in glaucoma: parallels with Alzheimer's disease. Aust NZJ Ophthalmol. 105-9. Review ).

Due to age, people also suffer from cataracts per se.

Cataract or cataract is a clouding of the human eye lens. 90% of all cataracts are so-called old-age cataracts. The cause of cataract development is usually unknown. Nearly all people over the age of 65 have cataracts and around 50% notice visual disturbances if they are over 75 years old.

Each year, more than 650,000 cataract surgeries are performed in Germany in which the clouded lens is replaced by an artificial lens implant.

• • durch die intrakapsuläre Kataraktextraktion (ICCE) bei der über einen großen, oberen Hornhautschnitt die gesamte Linse in ihrer Kapsel mit einem Kältestift angefroren und entnommen wird

oder

• • durch die extrakapsuläre Kataraktextraktion (ECCE) bei der die vordere Kapsel mit einem kleinen Schnitt geöffnet wird und der Linsenkern und -rinde durch Phakoemulsifikation entfernt wird.

The removal of the eye lens is done either

• • the intracapsular cataract extraction (ICCE), in which a large, upper corneal incision is used to freeze the entire lens in its capsule with a cold stick

or

• • by extracapsular cataract extraction (ECCE), in which the anterior capsule is opened with a small incision and the lens nucleus and cortex are removed by phacoemulsification.

It is also possible to perform some steps of a cataract surgery with a femtosecond laser.

• • Glaskörperblutungen;
• • Traktive Netzhautablösungen;
• • Proliferative Vitreoretinopathie;
• • Entzündliche oder posttraumatische Glaskörperveränderungen.

Another common eye operation in old age is vitrectomy. In this case, the vitreous (corpus vitreum) is removed. Indications for a vitrectomy include

• • vitreous hemorrhages;
• • Tractive retinal detachments;
• • proliferative vitreoretinopathy;
• • Inflammatory or post-traumatic vitreous changes.

• • Faseroptik zur Beleuchtung;
• • eine Infusionskanüle, um den intraokular Druck konstant zu halten;
• • ein Vitrektom;

notwendig.In a vitrectomy, the vitreous is cut by successive piecewise cutting with a vitrectome, e.g. B. the phaco machine Visalis ^TM 100 from Carl Zeiss Meditec AG, removed. These are one

• • fiber optics for lighting;
• • an infusion cannula to keep the intraocular pressure constant;
• • a vitrectomy;

necessary.

A confirmed diagnosis of Alzheimer's disease is currently only possible post mortem by histological examination of the brain. The current clinical diagnosis of living patients is very heterogeneous and is based on observations of relatives (external history), radiographic imaging (CT, MRI and PET), cognitive and memory tests and the exclusion of other neurodegenerative diseases in laboratory blood and cerebrospinal fluid examinations ( CSF).

In the US 2004-152068 It is proposed to detect β-amyloid in the eye lens by quasielastic light scattering (abbreviation: QLS) and fluorescence optical methods. Due to a large number of Alzheimer's disease patients and people in the risk group without their own eye lens (because of a Lensektomie in the context of a cataract surgery), the application of the method described limits. They are also in the remaining group Of patients with own eye lens many patients with cataract. A differential diagnosis of Alzheimer's disease and cataract is possible only conditionally with this method.

The invention has the task of improving and simplifying the diagnosis of Alzheimer's disease, especially in the elderly.

This object is achieved by a method for the diagnosis of dementias on the basis of an eye surgically removed components, wherein a suction feeds the removed components of an analysis unit and the components in the analysis unit on the presence and / or relative proportions of τ- PHF plaques and / or β-amyloid plaques are analyzed. It is advantageous if the removed components are lens material of the eye lens, which is taken during a cataract surgery and an in vitro analysis of the lens material on τ-PHF plaques and / or β-amyloid plaques takes place. It is further advantageous if the removed components are vitreous material which is taken during a vitrectomy and the in vitro analysis of τ-PHF plaques and / or β-amyloid plaques takes place.

The object is further achieved by a device for the diagnosis of dementias on the basis of an eye surgically removed components, which has an extraction for the extracted components and an analysis unit, wherein the analysis unit for analyzing the components for the presence and / or relative proportions of τ-PHF plaques and / or β-amyloid plaques. It is advantageous if the suction and the analysis unit are preferably connected to each other fluidly. It is also advantageous if the suction is connected to a transport device for the removed components, and the analysis unit for receiving the transport device and analysis of the components contained in the transport device is executed.

The method described here makes it possible to obtain a more exact diagnosis and better differential diagnosis than the above-described in vivo method by an in vitro (or ex vivo) analysis of the eye lens or vitreous body taken during a cataract or vitrectomy. In addition, multiple molecular markers can be applied in vitro and ex vivo without great effort, which improves the diagnostic information and the prognosis.

The lens removed during lensectomy is examined biochemically or histologically for the diagnosis of Alzheimer's disease.

• • intrakapsuläre Kataraktextraktion (ICCE)
• • extrakapsuläre Kataraktextraktion (ECCE)

A distinction is made between the two methods for removing the lens:

• • intracapsular cataract extraction (ICCE)
• • extracapsular cataract extraction (ECCE)

During intracapsular removal, the eye lens is removed as far as possible intact. This also allows the conduct of histological examinations.

After removal, the human lens is processed by appropriate histological procedures.

• • Thioflavin S & und deren Derivate (z. B. Pittsburgh Compound B) (IUPAC 4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline Chlorid);
• • Thioflavin T (IUPAC: 4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline chloride) und deren Derivate;
• • Kongo rot und dessen Derivate (IUPAC: 3,3'-(4,4' Biphenyldiylbisazo)bis-(4-amino-1-naphtalinsulfonsäure)-Dinatriumsalz);

sowie

• • Methlyen blau (Rember) (3,7-Bis(dimethylamino)-phenothiaziniumchlorid) und seine Derivate;
• • β-Amyloid-spezifische Antikörper, Antikörperfragmente, Peptide, Aptamere, RNA-Moleküle mit intrinsischen oder extrinsischen (z. B. durch Anhängen eines Fluroeszenz-Farbstoffes, enzymatischen Verfahrens wie Meerrettichperoxidase und Chromagen-3-Amino-9-Ehylcarboazol) Eigenschaften, welches sich zur optischen Darstellung eignen.

The β-amyloid plaques can be visualized inter alia by the following molecular markers:

• Thioflavin S and its derivatives (eg Pittsburgh Compound B) (IUPAC 4- (3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl) -N, N-dimethylaniline chloride);
• Thioflavine T (IUPAC: 4- (3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl) -N, N-dimethylaniline chloride) and derivatives thereof;
• Congo red and its derivatives (IUPAC: 3,3 '- (4,4'-biphenyldiylbisazo) bis (4-amino-1-naphthalenesulfonic acid) disodium salt);

such as

• • Methlyene blue (Rember) (3,7-bis (dimethylamino) -phenothiazinium chloride) and its derivatives;
• Β-amyloid-specific antibodies, antibody fragments, peptides, aptamers, RNA molecules with intrinsic or extrinsic (eg by attaching a fluorescence dye, enzymatic process such as horseradish peroxidase and chromagen-3-amino-9-ethylcarboazole) properties, which are suitable for optical representation.

• • Thiazohydrazid und dessen Derivate;
• • τ-PHF-spezifische Antikörper, Antikörperfragmente, Peptide, Aptamere, RNA-Moleküle mit intrinsischen oder extrinsischen (z. B. durch Anhängen eines Fluroeszenz-Farbstoffes, enzymatischen Verfahrens, wie Meerrettichperoxidase und Chromagen-3-Amino-9-Ehylcarboazol) Eigenschaften, welches sich zur optischen Darstellung eignen.

The τ-PHF plaques can be made visible, inter alia, by the following molecular markers:

• Thiazohydrazide and its derivatives;
• • τ-PHF-specific antibodies, antibody fragments, peptides, aptamers, RNA molecules with intrinsic or extrinsic (eg, by attaching a fluorescent dye, enzymatic method, such as horseradish peroxidase and chromagen-3-amino-9-ethylcarboazole) properties , which are suitable for optical representation.

In the ECCE the lens cover is opened by a small cut (Kapsulorhexis). By ultrasound (phacoemulsification), a water jet or a laser, the lens is shattered and the lens fragments are removed by a suction-irrigation approach (aspiration-irrigation maneuver). This procedure is done with a phaco machine carried out, for. B. with the phaco machine Visalis ^TM 100 from Carl Zeiss Meditec AG.

The invention described here provides that the rinsing solution used u. a. due to suitable buffer conditions (eg pH), the β-amyloids or τ-PHF are not altered.

In addition, in the context of the aspiration process the flushed fragments are not discarded in contrast to the state of the phaco technique but directed into a special vessel for storage of the lens fragments and possibly for direct diagnosis. This vessel may be located at the end of the aspiration tube in or on the phaco machine.

The vessel, in one possible embodiment, is a disposable disposable article (a separate vessel is used for each patient) and may be provided with a patient-specific code (e.g., bar code, matrix code).

In a particular embodiment of the invention, the rinsing liquid can be separated from the lens fragments by filtration or centrifugation to reduce the volume.

• • Enzymgekoppelter Immunadsorptionstest (EIA) bzw. Enzyme Linked Immunosorbent Assay (ELISA) oder allgemein enzymatische Assays;
• • Immunochromatographische Tests;
• • Radioimmunoassay (RIA);
• • Western-Blot;
• • Fluoreszenz-basierte Assays;

The lens fragments in the vessel are then further treated as necessary for further analysis with appropriate buffer solutions. The detection of β-amyloid or τ-PHF can be carried out, inter alia, by the following methods:

• • Enzyme-linked immunosorbent assay (EIA) or Enzyme Linked Immunosorbent Assay (ELISA) or general enzymatic assays;
• • immunochromatographic tests;
• • Radioimmunoassay (RIA);
• • Western blot;
• Fluorescence-based assays;

In this case, the markers mentioned above, but also other β-amyloid and τ-PHF-specific markers can be used.

• • Massenspektroskopischer Analyse (MALDI-TOF, SELDI);
• • Biochip-basierter Assays;

vorteilhaft.In addition, the use of

• Mass spectroscopic analysis (MALDI-TOF, SELDI);
• Biochip-based assays;

advantageous.

• • separat in einem Labor oder beigestellten Analysegerät;
• • mittels Teststreifen, welche in die Lösung im Gefäß eingeführt werden,
• • „online” in oder an der Phako-Maschine

statt.Depending on the analysis method, the analysis takes place

• • separately in a laboratory or provided analyzer;
• • by means of test strips, which are introduced into the solution in the vessel,
• • "online" in or on the phaco machine

instead of.

Depending on the design and degree of automation, the combination with "liquid handling" and microfluidic device may be advantageous.

It is also advantageous to examine the lens body (corpus vitreum) taken during a vitrectomy for the existence of β-amyloid and τ-PHF. It is expected that β-amyloid plaques and τ-PHF will also be in the gelatinous vitreous body.

• • Faseroptik zur Beleuchtung;
• • eine Infusionskanüle um den intraokular Druck konstant zu halten;
• • ein Virektom;

notwendig.In a virectomy, the vitreous is cut by successive, piecewise excision with a virectome, z. B. the phaco machine Visalis ^TM 100 from Carl Zeiss Meditec AG, removed. These are one

• • fiber optics for lighting;
• • an infusion cannula to keep the intraocular pressure constant;
• • a virectome;

necessary.

Analogous to the human lens ICCE, the aspirated vitreous fragments are not discarded here, but are directed into a special vessel for storage at the end of the virectome. All subsequent steps are similar to the ICCE procedure described above.

• • β-Amyloid und τ-PHF
• • verschiedene Subformen von β-Amyloid (β-A39, β-A40, β-A41, β-A42).

To improve the quality of the diagnosis and prognostic purposes, it may be advantageous to detect several, disease-specific target molecules in the samples, eg. B.

• Β-amyloid and τ-PHF
• Various subforms of β-amyloid (β-A39, β-A40, β-A41, β-A42).

The invention will be explained in more detail below with reference to FIG.

The figure shows schematically an eye 1 consisting of the lens 2 , the capsule bag 3 and the cornea 4 , In phacoemulsification becomes a cannula 5 through an opening of the cornea 4 and capsule bag inserted in these. There is the lens 2 destroyed by suitable means (ultrasound, laser, water jet) and disassembled into fragments so small that these by means of negative pressure, which from a suction unit 6 via an aspiration tube 7 to the cannula 5 is guided, can be sucked. Usually, a rinsing solution is used for this, which is not shown here means in the capsular bag 3 is introduced. Such a device is for example in the DE 198 52 574 described in the applicant, the entire contents of which is hereby incorporated by reference. Between the cannula 5 and the suction unit 6 is a catcher 8th arranged for the extracted fragments. The with the Fragments added rinse solution 9 will be in the catcher 8th collected. The analysis of the fragments for β-amyloid and τ-PHF can in a first variant with suitable, not shown, means directly in the catcher 8th be performed. Alternatively, the collecting device 8th via a separating device 10 with which help the container with rinsing solution with fragments of an eye lens 9 can be removed and transported to a separate analysis device. In this then, as described above, the analysis is carried out on β-amyloid and τ-PHF.

Both imaging techniques, such as those in the DE 10 2007 061 987 the applicant, the entire contents of which is hereby incorporated, as well as known measuring devices are used for in vitro diagnosis.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 2004-152068 [0012]
• DE 19852574 [0038]
• DE 102007061987 [0039]

Cited non-patent literature

• Parisi V. (2003) Correlation between morphological and functional retinal impairment in patients affected by ocular hypertension, glaucoma, demyelinating optic neuritis, and Alzheimer's disease. Semin Ophthalmol .; 18 (2): 50-7 [0002]
• Gupta N, Fong J, Ang LC, Yücel YH. Retinal tau pathology in human glaucomas. Can J Ophthalmol. 2008 Feb; 43 (1): 53-60 [0002]
• Bayer AU, Ferrari F, Erb C. "High incidence of glaucoma among patients with Alzheimer's disease." Eur Neurol. 2002; 47 (3): 165-8; Vickers JC. (1997) The cellular mechanism underlying neuronal degeneration in glaucoma: parallels with Alzheimer's disease. Aust NZJ Ophthalmol. 105-9. Review [0003]

Claims (6)

Method for the diagnosis of dementias, in particular Alzheimer's disease, based on an eye surgically removed components, wherein a suction feeds the removed components of an analysis unit and the components in the analysis unit on the presence and / or relative proportions of τ-PHF Plaques and / or β-amyloid plaques and / or other disease-specific molecules. The method of claim 1, wherein the withdrawn components are lens material of the crystalline lens taken during cataract surgery and in vitro analysis of components on τ-PHF plaques and / or β-amyloid plaques and / or other disease-specific molecules The method of claim 1, wherein the withdrawn components are the vitreous material taken during a vitrectomy and in vitro analysis of τ-PHF plaques and / or β-amyloid plaques and / or other disease-specific molecules Device for the diagnosis of dementias, in particular Alzheimer's disease, based on an eye surgically removed components, which has a suction for the removed components and an analysis unit, wherein the analysis unit for analyzing the components on the presence and / or relative proportions of τ -PHF plaques and / or β-amyloid plaques and / or other disease-specific molecules is executed. Apparatus according to claim 4, wherein the suction and the analysis unit are preferably fluidically connected to each other. Apparatus according to claim 4, wherein the suction is connected to a transport device for the removed components, and the analysis unit for receiving the transport device and analysis of the components contained in the transport device is executed.

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