Classifications

• • 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
  • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
  • G01N21/03—Cuvette constructions
  • G01N21/07—Centrifugal type cuvettes
• • 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

Definitions

• the present invention is concerned with a method for the measurement of properties of a liquid present in a vessel, for example for the measurement of the absorbance of a liquid, of the -properties of an agglutination, precipitate or any other reaction result placed on the bottom of a vessel, by means of radiation and of a detector receiving radiation while the vessel moves along with a rotor revolving around the vertical axis of a centrifuge, the beam of measurement coming from the source of radiation passing substantially horizontally and the intensity of the radiation passing through, or reflected from, the contents of the vessel being measured.
• the invention is also concerned with an apparatus for the implementation of the method, which apparatus comprises a rotor revolving around a vertical axis, to which rotor the vessel can be fitted, one or several detectors. receiving radiation and fitted so that the beam of measurement received by the detector(s) passes substantially horizontally, as well as an output unit.
• Various tests based on agglutinations are in common use, e.g., in the case of blood-group identifications, antibody determinations, and rheumatic-factor measurements.
• the agglutination of red blood cells is concerned, whereas, e.g., the rheumatic factor is commonly measured by means of the agglutination of latex particles.
• the objective of the method in accordance with the invention is to be able to ascertain the difference between agglutination and non-agglutination sufficiently clearly, reproducibly, and carefully.
• the principle of vertical measurement Saovaniemi, Osmo, "Performance and Properties of the Finnpipette Analyzer System", Proceedings of the Second National Meeting on Biophysics and Biotechnology in Finland, 183, 1976
• one beam of light does not produce a sufficient difference in absorbance between agglutination and non-agglutination.
• any uncertainty is eliminated by performing the measurement of the properties of the agglutination precipitate at several points once or several times so as to observe the formation of the precipitate as a function of time.
• the agglutinated precipitate formed on the bottom of the reaction vessel is, viz., structurally different from a non-agglutinated precipitate.
• the former is, e.g., unhomogeneous, at the middle part denser than at the sides, whereas the latter is even and relatively homogeneous.
• the method in accordance with the invention is characterized in that, for the purpose of the measurement of the properties of the liquid, after the vessel has pivoted for the measurement by the effect of centrifugal force so that its longitudinal axis is positioned substantially horizontally, a field of limited area is measured out of the bottom of the vessel while the vessel moves along with the rotor of the centrifuge through the space between the source of radiation and the detector, and the measurement result of the component field at each particular time under measurement is read constantly or at specified intervals for the purpose of processing and output of the information.
• the apparatus in accordance with the invention is characterized in that, at one time, either the source of radiation or the detector receiving radiation covers only a limited component field of the bottom of the vessel so that the source of radiation or the detector receiving radiation is a narrow, vertical slit placed outside the circle formed by the course of movement of the bottom of the reaction vessel or that the source of radiation or the detector receiving radiation is point- shaped and placed outside the circle formed by the course of movement of the bottom of the reaction vessel and displaceable in the vertical direction during the revolutions of the centrifuge or that the source of radiation or the detector receiving radiation is point- shaped and two or more of them have been fitted in a vertical line, and that, when several sources of light are used in a line, each source of light is in operation alternatingly.
• the measurement of the precipitate or any other type of reaction product produced is accomplished at several points in the following way.
• the reaction vessel is placed in a frame or holder which is fastened to the rotor of the centrifuge so that the vertical axis of the reaction vessel is during rotation of the rotor pivoted to the horizontal plane.
• the beam of measurement then passes in the direction of the vertical axis of the reaction vessel, which has been pivoted to the horizontal position.
• the source of light is placed, which is either pointshaped or of the shape of a vertical slit and whose light is directed at the centre of rotation of the rotor.
• the detector Inside the circle along which the mouth of the reaction vessel passes, the detector is placed so that the light coming from the said source of light falls on the detector.
• the reaction vessel When the rotor rotates, the reaction vessel by-passes the beam of measurement, whereby the various parts of the precipitate placed on the bottom of the reaction vessel pass at a high speed of revolution through the beam of measurement.
• the detector registers the absorption of light at different points of the precipitate as a function of time as the precipitate by-passes the source of light, and the electronics fitted into the apparatus decide whether the precipitate is agglutinated or not, based on the registered line of light absorbance values.
• the reaction vessel is placed on a pivoting holder of the rotor of the centrifuge, and it is pivoted so that, when the rotor rotates, the longitudinal axis of the vessel is horizontal and parallel to the radius of the rotor.
• the horizontal beam of measurement is directed towards, or away from, the centre point of the rotor in the direction of the radius of the rotor so that, when rotating, the reaction vessel by-passes the beam.
• the detector registers the absorption of light at subsequent points of the precipitate placed on the bottom of the reaction vessel rapidly and in the way determined by the control electronics.
• the apparatus may also be applied to the measurement of phenomena other than absorption of light, e.g., to the measurement of fluorescence, luminescence, etc.
• FIG. 2 is a schematical and partly sectional presentation of an apparatus in accordance with the invention
• Figure 3 schematically shows an alternative in which one slit-shaped source of light is used that can be shifted in relation to the reaction vessel.
• Figure 4 shows an absorbance curve obtained from the reaction vessel of Figure 1; underneath the curve, the reaction vessel of Fig. 1 is shown as viewed from the top (in the curves of the figures, the x-axis illustrates the location of the point of measurement on the path of movement and the y-axis illustrates the absorbance at each particular time or in each particular point),
• Figure 5 shows absorbance curves obtained by means of the source of light shown in Fig. o; underneath the curves the precipitate measured is shown as viewed from above,
• Figure 6 is a schematical- perspective view of a source of light that consists of several point-shaped sources of light
• Figure 7 shows absorbance curves, underneath of which the precipitate measured as a top view and the detector used are illustrated.
• Figure la there is a precipitate 4a on the bottom of the reaction vessel 2, the shape and density of the said precipitate being at different points different from those of the precipitate 4b shown in Figure 1b.
• the precipitate 4a illustrates an agglutinated situation
• Figure 4b represents a non-agglutinated precipi tate in the situation of measurement.
• Figure 2 shows the shaft 15 of rotation of the centrifuge, a rotor arm 16 connected to the shaft, as well as a holder 17, connected to the arm as pivotable around a horizontal shaft 18, the said holder being provided with two reaction vessels 2 in the horizontal position that is assumed by them when the rotor rotates.
• the light departing from the slit-shaped sources of light 1 passes through the reaction results, e.g. precipitate 4, placed on the bottom of the reaction vessels 2 and reaches the detectors 3, which, during rotation of the rotor of the centrifuge, register the absorption of light of the precipitate 4 at different points of the precipitate.
• Figure 3 schematically shows a slit-shaped source of light 1, a reaction vessel 2, and a detector 3.
• the length of the slit is preferably somewhat less than the diameter of the vessel bottom.
• a precipitate 4 On the bottom of the reaction vessel 2 there is a precipitate 4, which has layers of different types and thicknesses.
• the reaction vessel 2 passes across the stationary source of light 1 and detector 3 once in the transverse direction of the slit 1 so that the slit-shaped beam of light 5 passes, point by point, through the precipitate 4 on the bottom of the reaction vessel 2, the absorbance curve 6 shown in Fig. 4 is obtained, which curve may be continuous or consist of individual points.
• Figure 4 also shows a top view of the precipitate 4 on the bottom 2 of the reaction vessel.
• the measurement has been performed by at a time always measuring a stripe of the shape of the slit-shaped source of light out of the precipitate on the bottom of the reaction vessel and by producing the output of the measurement value of this stripe.
• the production of the output may take place for each stripe separately, whereby a stepwise curve or a curve consisting of individual points is obtained, or as a continuous measurement, whereby the detector measures constantly during the movement and a continuous curve is obtained as the result.
• Figure 5 shows a precipitate measured from the bottom of the reaction vessel so that the reaction vessel has passed across the point-shaped sources of light 8 to 10 shown in Figure 6 so that measurement has first been performed by means of the source of light 8 and the other beams 9 and 10 have been closed. During the next revolution of the rotor the measurement has been performed by means of the beam 9, and the beams 9 and 10 have been closed. Finally, the beams 8 and 9 have been closed, and the measurement has been performed by means of the beam 10.
• the beam 10 corresponds the scan 13 and the absorbance curve 16.
• the beams 9 and 8 correspond the scans 12 and 11 and the absorbance curves 15 and 14. It is also natural that the point-shaped source of light may move along the slit shown in Figure 6, being alternatingly in positions 8, 9 and 10.
• a detector placed outside the rotor may consist of several sub-detectors 1 to 10 mounted in a vertical line, the said sub-detectors registering the light coming from the source of light placed inside the rotor and through the precipitate placed on the bottom of the vessel, each of them at a height of its own. In this way highly abundant information is obtained from the precipitate placed on the bottom of the reaction vessel.
• each detector may also consist of several separate sub-detectors positioned in a matrix form, whereby a complete topography is obtained from the reaction during each revolution. It is also natural that the evolution of the topographic result may be observed as a function of time, whereby different topographies in relation to time mean different final results.
• the source of light and the detector may change places, whereby the detector is placed outside the circle of the rotor and the source of light inside. In such a case the detector must be correspondingly given the shape of a point or of a vertical slit.
• this method there may be several reaction vessels on the circumference of the same rotor, in which case the results are registered subsequently from one vessel after the other in the way described above and the apparatus is provided with electronics capable of sorting and arranging the readings.
• the holders for reaction vessels on the rotors so that a matrix of vessels consisting of several individual reaction vessels can be placed on each holder, e.g. a matrix of the type used as a .cuvette set in the FP-9 photometer (U.S. Patent 4,144,030).
• the apparatus may be constructed as a multi-channel apparatus so that it is provided with several beams of measurement of the type described above, one beam above the other, at the heights corresponding the reaction vessels.
• a point-shaped source of light or detector it is possible to allow it to move in the vertical direction so that, during the movement of rotation, the beam of measurement passes through the precipitate on the bottom of the reaction vessel at several points, during each subsequent revolution of the rotor slightly higher or lower. In this way it is possible to assemble abundant information on the shape and structure of the precipitate, and thereby to decide more certainly whether it is agglutinated or not.
• the measurementit is possible to assemble information on the evolution of the precipitate as a function of time, on its location, shape, and/or on the density of its various points.
• the method of measurement may be based on photometry or multiphotometry, the latter meaning a photometer which comprises several channels so that each sample has a source of light and a detector of its own.
• the method of measurement may, of course, being a single-channel or multi-channel method, be additionally based, e.g., on turbidometry, fluorometry, or, e.g., on the use of a source of radiation and a receiver for luminescence, laser beam, ultrasound, etc. phenomena.
• the positioning of the reaction vessels or equivalent, of sources of measurement beams, of detectors, etc. auxiliary equipment may be performed in the way most appropriate in each particular case.
• the equipment may also involve various degrees of automation, e.g., in the pipetting of the samples and reagents, in the shifting of the beams of measurement, and in the processing of the results. It is natural that, in stead of one method of measurement, the reaction vessels may be measured simultaneously or subsequently by means of two or more wave lengths or methods of measurement (e.g., photometry and fluorometry), the final result being based on the information thereby obtained.
• the reaction vessels may be measured simultaneously or subsequently by means of two or more wave lengths or methods of measurement (e.g., photometry and fluorometry), the final result being based on the information thereby obtained.
• the invention permits, e.g., enzyme and end-point measurements in the reaction vessel.
• enzyme measurements it is possible to observe the progress of the reaction at certain time intervals, and the final result is determined from the measurement values in this way obtained.

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• Physics & Mathematics (AREA)
• Biochemistry (AREA)
• General Physics & Mathematics (AREA)
• Life Sciences & Earth Sciences (AREA)
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• Analytical Chemistry (AREA)
• Pathology (AREA)
• General Health & Medical Sciences (AREA)
• Health & Medical Sciences (AREA)
• Immunology (AREA)
• Spectroscopy & Molecular Physics (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)
• Automatic Analysis And Handling Materials Therefor (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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• 1981
  • 1981-07-24 JP JP56502659A patent/JPS57501248A/ja active Pending
  • 1981-07-24 WO PCT/FI1981/000058 patent/WO1982000360A1/en active IP Right Grant
  • 1981-07-24 EP EP81902326A patent/EP0056058B1/en not_active Expired

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