WO2008003312A2 - Procédé et dispositif d'étude du comportement d'adhérence des thrombocytes - Google Patents

Procédé et dispositif d'étude du comportement d'adhérence des thrombocytes Download PDF

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Publication number
WO2008003312A2
WO2008003312A2 PCT/DE2007/001211 DE2007001211W WO2008003312A2 WO 2008003312 A2 WO2008003312 A2 WO 2008003312A2 DE 2007001211 W DE2007001211 W DE 2007001211W WO 2008003312 A2 WO2008003312 A2 WO 2008003312A2
Authority
WO
WIPO (PCT)
Prior art keywords
platelets
measuring chamber
sample carrier
wall
parts
Prior art date
Application number
PCT/DE2007/001211
Other languages
German (de)
English (en)
Other versions
WO2008003312A3 (fr
Inventor
Pedro Mestres-Ventura
Lutz Weber
Original Assignee
Universität des Saarlandes
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universität des Saarlandes filed Critical Universität des Saarlandes
Priority to EP07785609A priority Critical patent/EP2041547A2/fr
Publication of WO2008003312A2 publication Critical patent/WO2008003312A2/fr
Publication of WO2008003312A3 publication Critical patent/WO2008003312A3/fr

Links

Classifications

    • G01N15/1433—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00138—Slides

Definitions

  • the invention relates to a method and a device for investigating the adhesion behavior of platelets according to the preamble of claim 1 or claim 4.
  • platelets play an essential role in blood clotting. They adhere to the walls of damaged vessels, thereby activating further platelets to aggregate to form a platelet plug which seals off the injured site.
  • the extent to which the platelets present in the blood are able to adhere to a substrate by adhesion is an important characteristic of the state of the hemostasis system.
  • the invention has for its object to reduce the effort required to investigate the adhesion behavior of platelets.
  • This object is achieved by a method in which a suspension of a blood sample of separated platelets is passed into a measuring chamber and which after a predetermined incubation period on the wall of the measuring chamber by adhesion adhering platelets are observed by means of a microscope.
  • a device with a sample carrier which has a measuring chamber for receiving a suspension of a blood sample of separated platelets, wherein the sample carrier is transparent in the region of the measuring chambers from at least one side to the wall To observe the measuring chamber by adhering adherent platelets with the help of a microscope.
  • platelets are extracted from a given amount of blood sample while avoiding additional activation, and the platelet fraction is diluted to form a suspension.
  • the suspension conducted into the measuring chamber is incubated, for example at 37 °, for a predetermined period of time. Thereafter, the adhering to the chamber wall platelets can be observed by means of a microscope.
  • the number of adhering platelets per unit area can be determined as a characteristic variable.
  • the various forms of the platelets which adjust depending on the activation state. For counting the platelets and evaluating the forms, automatic image evaluation methods are also possible.
  • test is designed to aid in the diagnosis of hereditary or acquired disorders of thrombocyte function.
  • test is suitable to investigate the effect of drugs on hemostasis, in particular the platelets. Further applications arise in quality control in blood banks and in transfusion medicine. In addition to applications in the evaluation of new substances, the test can also be important in the quality assurance of already established drugs.
  • the suspension is injected into a microchannel comprising the measuring chamber and passing through the sample carrier.
  • Complete filling of the microchannel with the suspension ensures that the sample volume from which platelets can settle on the measuring chamber wall is always the same. It does not require an exact dimension of a sample amount.
  • staining of the platelets may be carried out, e.g. through a tetrazolium-based substrate.
  • a fluoromarker e.g. Rhodamine G6
  • labeled platelets can be observed in a fluorescence microscope.
  • the microchannel containing the measuring chamber extends between two adjoining parts forming the sample carrier, which are expediently plate-shaped or comprise a plate-shaped section.
  • a Einl ⁇ ssk ⁇ n ⁇ l ⁇ bêt and / or a Ausl ⁇ ssk ⁇ n ⁇ l ⁇ bêt can / can be in fluid communication with a protruding from one of the parts of the pipe socket, to which, for example, attach a pipette or connect a pump.
  • the inlet channel section may be in fluid communication with a receptacle formed on the sample vessel for receiving the suspension, in particular for receiving a sample total.
  • the suspension contained in the vessel can be drawn into the microchannel via a suction device that can be connected to the tube attachment at the outlet channel section.
  • the tube attachment connected to the outlet channel section has an overflow channel at the free end, which leads to a collecting vessel formed on the sample carrier. Excess sample material thus remains on the sample carrier and does not escape into the environment.
  • a series arrangement of a plurality of measuring chambers and on the sample carrier a plurality, each comprising at least one measuring chamber comprising microchannels may be formed in a parallel arrangement.
  • the contour of the sample carrier in plan view corresponds to the contour of an ordinary microscope slide.
  • the sample carrier can then easily be used in the microscope like such a slide.
  • the Messkam- merwand a, preferably flat, wall portion whose Adphaseshunt with respect to platelets relative to the wall portion adjacent to the parts of the measuring chamber, preferably with respect to the entire remaining measuring chamber wall is increased.
  • the Messkam- merwand a, preferably flat, wall portion whose Adphaseshunt with respect to platelets relative to the wall portion adjacent to the parts of the measuring chamber, preferably with respect to the entire remaining measuring chamber wall is increased.
  • the sample carrier may be made of different materials with different adhesiveness with respect to platelets.
  • the two parts of the sample carrier may be different in material, so that adhering to only one of the parts, which forms one of the two parallel to the plate plane wall sections of the measuring chamber, platelets.
  • Said wall portion may be subjected to an adhesion-enhancing surface treatment and, conversely, the adhesiveness of the wall portions of the measuring chamber adjoining said wall portion may be reduced by a corresponding surface treatment.
  • the latter surface treatment is particularly suitable for sections of the microchannel lying outside the measuring chambers in order to prevent the adhesion of platelets there.
  • a combination of plastic / glass is also possible.
  • One of the two parts could consist of a plastic film.
  • the surface treatment may also be carried out in a coating, e.g. based on silica compounds, by plasma polymerization.
  • a coating e.g. based on silica compounds
  • fluoropolymer-based coatings are applied.
  • a significant advantage of the invention over the prior art is also that after counting or / and qualitative assessment of the adhering platelets located in the channel system liquid medium can be exchanged for a there solidifying fixation without triggering the adherent platelets.
  • the analysis result is thus "frozen”, can be archived and is available for later checks and inspections.
  • FIG. 1 shows a sample carrier according to the invention in a perspective view
  • FIG. 2 shows the sample carrier of FIG. 1 in a sectional view
  • FIG 3 is a sectional view of a sample carrier according to a second embodiment of the present invention
  • 4 shows a partial view of a sample carrier according to the invention, with a measuring chamber and a grid provided on the measuring chamber.
  • a sample carrier 1 made of plastic consists of a first part 2 with a plate-shaped portion 3 and a second, formed in the form of a plate part 4, which is connected to the plate portion 3 of the first part 2.
  • the two parts 2 and 4 are each made in one piece by injection molding of different plastic materials. Both materials are transparent. Part 4 could also be made from a film.
  • microchannels 20 each having three measuring chambers 5, an inlet channel section 6, an outlet channel section 7 and intermediate sections 8 connecting the measuring chambers 5, two depressions are formed in the plate section 3 of the first plastic part 2, which are covered in a fluid-tight manner by the plate-shaped part 4.
  • the measuring chamber 5 facing away from the end of the inlet channel section 6 is in communication with a protruding from the plate section 3 pipe extension 9.
  • the measuring chambers 5 facing away from the end of the outlet channel 8 is connected to such a pipe neck 10.
  • an overflow channel 11 At the free end of the pipe socket 10 is an overflow channel 11, which leads to a collecting vessel 12.
  • the plastic material of the plate-shaped part 4 has a thrombocyte to large adhesion.
  • the part 2, however, consists of a plastic material with low adhesiveness.
  • a suspension from a blood sample separated platelets is filled as shown in arrow 13 via the tube 9.
  • the inner diameter of the pipe socket 9 is dimensioned so that a pipette can be used for this purpose.
  • the suspension passes through the inlet channel section 6 into the first measuring chamber 5 and then via the channel intermediate sections 8 into the further measuring chambers and finally into the outlet channel section 7. Then it ascends in the tube extension 10.
  • the rising can be supported by a connectable to the pipe socket 10 suction device, such as a pipette.
  • Excess sample material runs according to arrow 15 through the overflow channel 11 into the collecting vessel 12. By the collecting vessel 12 ensures that the entire sample remains on the sample carrier and does not enter the environment.
  • platelets adhere only to a wall section 16 of the measuring chambers 5 which is parallel to the plane of the plate and formed by the plate-shaped part 4.
  • a dye is introduced via the tube approach 9, which displaces the sample suspension and stains the adhering to the wall portion 16 of the measuring chambers 5 platelets so that they are visible under the microscope.
  • the sample carrier 1 shown in Rg. 1 and 2 has in plan view the dimensions of a microscope slide and can therefore be inserted like such a carrier in a microscope, under which then adhered to the wall portion 16 by adhesion platelets can be observed.
  • the wall section 16 it is easy to determine the number of adhering platelets per unit area.
  • the platelets can be classified and the frequencies of certain shape characteristics exhibiting platelets can be determined.
  • An exemplary embodiment of a sample carrier 1a shown in FIG. 3 has, instead of tube attachments 9, vessels 17 for filling a sample total.
  • a suction pump which ensures that a microchannel 20a comprising measuring chambers 5a, is quickly completely filled with sample liquid, can be connected to a pipe socket 10a connected to an outlet channel 7a.
  • a square in cross-section measuring chamber 5b is formed.
  • a plate-shaped part 4b On its side facing away from the measuring chamber, a plate-shaped part 4b has a grid 19, which facilitates the counting in a microscope of platelets adhering to a wall section 16b of the measuring chamber 5b.
  • the channel wall formed by the part 4 can be treated such that the adhesion of platelets is avoided. Errors by counting within the channel sections of adherent platelets are excluded.
  • the embodiments described above have two plate-shaped components which are firmly connected to each other. It would also be possible to connect both components in a fluid-tight but yet detachable manner, e.g. with the help of a clamping device.
  • a glass slide could form a stable base and have a top clamped thereto having the channels and measuring chambers.
  • the glass surface could be modified, for which purpose wet-chemical and physical processes are suitable.
  • the top has some ductility and may be e.g. made of an elastomer or silicone.
  • the channels and measuring chambers can be easily sealed fluid-tight by the glass plate.
  • a hardened fixing medium advantageously remains after loosening the clamp connection on the glass slide, which can then be archived like other microscopy samples. The upper part with the measuring chambers and channels is available for further analysis.

Abstract

L'invention concerne un procédé destiné à étudier le comportement d'adhérence des thrombocytes. L'invention est caractérisée en ce qu'une suspension de thrombocytes séparés d'un échantillon sanguin est amenée dans une chambre de mesure, et en ce que les thrombocytes accrochés par adhérence à la paroi de la chambre de mesure sont, après un temps d'incubation, observés au microscope. Le dispositif selon l'invention est caractérisé en ce qu'il comprend un porte-échantillon (1) présentant une chambre de mesure (5) pour la réception d'une suspension de thrombocytes séparés à partir d'un échantillon sanguin, et en ce que le porte-échantillon est transparent afin de pouvoir observer au microscope les thrombocytes accrochés par adhérence à la paroi de la chambre de mesure.
PCT/DE2007/001211 2006-07-07 2007-07-05 Procédé et dispositif d'étude du comportement d'adhérence des thrombocytes WO2008003312A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP07785609A EP2041547A2 (fr) 2006-07-07 2007-07-05 Procédé et dispositif d'étude du comportement d'adhérence des thrombocytes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006031475.1 2006-07-07
DE102006031475A DE102006031475A1 (de) 2006-07-07 2006-07-07 Verfahren und Vorrichtung zur Untersuchung des Adhäsionsverhaltens von Thrombozyten

Publications (2)

Publication Number Publication Date
WO2008003312A2 true WO2008003312A2 (fr) 2008-01-10
WO2008003312A3 WO2008003312A3 (fr) 2008-03-06

Family

ID=38695522

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2007/001211 WO2008003312A2 (fr) 2006-07-07 2007-07-05 Procédé et dispositif d'étude du comportement d'adhérence des thrombocytes

Country Status (3)

Country Link
EP (1) EP2041547A2 (fr)
DE (1) DE102006031475A1 (fr)
WO (1) WO2008003312A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9662650B2 (en) 2013-07-29 2017-05-30 Atlas Genetics Limited Fluidic cartridge and method for processing a liquid sample
US9816135B2 (en) 2013-07-29 2017-11-14 Atlas Genetics Limited Fluidic cartridge for nucleic acid amplification and detection
US9908114B2 (en) 2013-07-29 2018-03-06 Atlas Genetics Limited Cartridge, cartridge reader and method for preventing reuse of the cartridge
US9993818B2 (en) 2013-07-29 2018-06-12 Atlas Genetics Limited Valve which depressurises, and a valve system
US9999883B2 (en) 2013-07-29 2018-06-19 Atlas Genetics Limited System and method for processing fluid in a fluidic cartridge

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3237180A1 (de) * 1981-10-08 1983-04-28 Fuji Photo Film Co., Ltd., Minami-Ashigara, Kanagawa Film und verfahren fuer die analyse der blutplaettchen-haftfaehigkeit
EP0089240A1 (fr) * 1982-03-16 1983-09-21 The University Of Newcastle Research Associates Limited Méthode pour la détermination précoce de la grossesse
US20040142408A1 (en) * 2000-11-08 2004-07-22 Kirk Gregory L. Device and method for monitoring leukocyte migration
WO2006065739A2 (fr) * 2004-12-14 2006-06-22 Millennium Pharmaceuticals, Inc Dispositif permettant d'agreger, d'imager et d'analyser des thrombi, et procede d'utilisation associe

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2234857C1 (ru) * 2003-04-21 2004-08-27 Брилль Григорий Ефимович Способ количественной оценки эффективности сдвиговой регуляции функции тромбоцитов

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3237180A1 (de) * 1981-10-08 1983-04-28 Fuji Photo Film Co., Ltd., Minami-Ashigara, Kanagawa Film und verfahren fuer die analyse der blutplaettchen-haftfaehigkeit
EP0089240A1 (fr) * 1982-03-16 1983-09-21 The University Of Newcastle Research Associates Limited Méthode pour la détermination précoce de la grossesse
US20040142408A1 (en) * 2000-11-08 2004-07-22 Kirk Gregory L. Device and method for monitoring leukocyte migration
WO2006065739A2 (fr) * 2004-12-14 2006-06-22 Millennium Pharmaceuticals, Inc Dispositif permettant d'agreger, d'imager et d'analyser des thrombi, et procede d'utilisation associe

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 200464 Derwent Publications Ltd., London, GB; AN 2004-660258 XP002460086 & RU 2 234 857 C1 (BRILL G E) 27. August 2004 (2004-08-27) *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9662650B2 (en) 2013-07-29 2017-05-30 Atlas Genetics Limited Fluidic cartridge and method for processing a liquid sample
US9816135B2 (en) 2013-07-29 2017-11-14 Atlas Genetics Limited Fluidic cartridge for nucleic acid amplification and detection
US9908114B2 (en) 2013-07-29 2018-03-06 Atlas Genetics Limited Cartridge, cartridge reader and method for preventing reuse of the cartridge
US9993818B2 (en) 2013-07-29 2018-06-12 Atlas Genetics Limited Valve which depressurises, and a valve system
US9999883B2 (en) 2013-07-29 2018-06-19 Atlas Genetics Limited System and method for processing fluid in a fluidic cartridge

Also Published As

Publication number Publication date
EP2041547A2 (fr) 2009-04-01
WO2008003312A3 (fr) 2008-03-06
DE102006031475A1 (de) 2008-01-10

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