WO1982000355A1

WO1982000355A1 - Method and apparatus for the measurement of the properties of an agglutination

Info

Publication number: WO1982000355A1
Application number: PCT/FI1981/000059
Authority: WO
Inventors: Oy Labsystems; O Suovaniemi; P Ekholm; J Jaernefelt; P Partanen; E Kaukanen
Original assignee: Labsystems Oy
Current assignee: Thermo Fisher Scientific Oy
Priority date: 1980-07-24
Filing date: 1981-07-24
Publication date: 1982-02-04
Anticipated expiration: 1983-01-24
Also published as: EP0056414A1

Abstract

Procede et appareil de mesure des proprietes d'un agglomerat, d'un precipite (34), ou d'un produit de reaction places au fond d'un recipient (32) a l'aide d'une radiation et d'un detecteur (35) qui recoit cette radiation. Le rayon de mesure provenant de la source de radiation (37) passe sensiblement dans le sens de l'axe vertical du recipient, et l'intensite de la radiation passant au travers du precipite (34) se trouvant au fond (33) du recipient, ou reflechie par le precipite est mesuree. La mesure de la formation, la position, et la forme du precipite place au fond du recipient et/ou la densite ou autres proprietes de differents points du precipite s'effectuent par rapport a des champs de surface limites, le resultat de la mesure etant lu pour chaque champ. La sortie est produite sur la base du groupe de valeurs ainsi obtenues.Method and apparatus for measuring the properties of an agglomerate, a precipitate (34), or a reaction product placed at the bottom of a container (32) using radiation and a detector (35) who receives this cancellation. The measuring radius from the radiation source (37) passes substantially in the direction of the vertical axis of the container, and the intensity of the radiation passing through the precipitate (34) located at the bottom (33) of the container , or reflected by the precipitate is measured. The measurement of the formation, the position, and the shape of the precipitate placed at the bottom of the container and / or the density or other properties of different points of the precipitate are made with respect to limited surface fields, the result of the measurement being read for each field. The output is produced based on the group of values thus obtained.

Description

Method and apparatus for the measurement of the properties of an agglutination.

The present invention is concerned with a metho for the measurement of the properties of an agglutination, a precipitate, or of a corresponding reaction result place on the bottom of a vessel by means of radiation and of a detector that receives radiation, whereat the beam of measurement coming from the source of radiation passes substantially in the direction of the vertical axis of the vessel, and the intensity of the radiation passing through, or reflected from, the precipitate on the bottom of the vessel is measured. The invention is also con¬ cerned with an apparatus for the implementation of the method, which apparatus comprises a detector receiving radiation, the said detector being located so that the measurement beam received by it passes substantially in the direction of the vertical axis of the vessel, an output unit, and possibly a source of radiation.

Various tests based on agglutinations are in common use, e.g., in the case of blood-group identifi¬ cations, antibody determinations, and rheumatic-factor measurements. In blood-group analytics and in antibody determinations 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. It has been customary to read the results of agglutination reactions visually. An experienced rea_der is also quite skilful in distinguishing between aggluti¬ nation and non-agglutination. Such a mode of output is, however, quite subjective, and this is why the result is not always completely reliable. In clear situations with strong agglutination, visual reading is certainly no problem, but weak reactions of agglutination are often problematic. Among agglutination reactions, most diffi¬ cult to interpret is, e.g., a weak Rh-positive result o^btained in blood-group identifications. In such situ¬ ations it is of essential importance to obtain a reliable output, because the safety of the patient is concerned.

The objective of the method in accordance with the invention (observation of, e.g., agglutination reactions and reading of the final results) is to be able to ascertain the difference between agglutination and non-agglutination sufficiently clearly, reproducibly, and carefully. By means of the principle of vertical mea¬ surement (Suovaniemi, Os o, "Performance and Properties of the Finnpipette Analyzer System", Proceedings of the Second National Meeting on Biophysics and Biotechnology in Finland, 183, 1976) it is possible to measure agglu¬ tination reactions. However, in the case of very weak agglutinations, one beam of light does not produce a suf¬ ficient difference in absorbance between agglutination and non-agglutination. In the method in accordance with the invention 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. By performing, e.g., the measurement of absorption of light at several different points of the precipitate obtained, it is possible to distinguish between agglutinated precipitate and non-agglutinated precipitate reliably.

The method in accordance with the invention is characterized in that the measurement of the formation, location, and form of the precipitate placed on the bottom of the vessel and/or of the density or other properties of different points of the precipitate is performed in respect of component fields of limited area separately, -the measurement result being read for each

OMPI component field and the output being produced on the basis of the group of values in this way obtained. The apparatus in accordance with the invention is charac¬ terized in that the detector consists of several sub- detectors placed in the same plane close to one another. The employment of a detector matrix may take place, e.g., in the following two ways:

1) The performance of the measurement at several points is performed by employing a detector matrix composed of small sub-detectors. The detector matrix can be positioned directly underneath a reaction vessel which is open at the top or placed under a trans¬ parent cover, whereby the reaction vessel is illuminated from above by means of homogeneous light. Each sub- detector registers the light absorption of the portion of the precipitate placed directly above the said sub- detector, and the electronics of the apparatus processes the results and decides whether the precipitate is agglu¬ tinated or not. In the method it is possible to use a source or radiation built in the apparatus, or it is possible to use the daylight, the general illumination of the room, or any other source of light that gives uniform light. Stability of the source of light is in this case not critically necessary, because the evaluation of the result is simultaneously based on a comparison of the signals received from the different sub-detectors, which comparison can be performed electronically.

2) The measurement at several points can also be accompished so that the reaction vessel is illuminated from below by means of homogeneous light and that the image of the precipitate placed on the bottom of the vesse is focused by means of a lens onto the detector matrix. The light falling onto the detectors is registered in the way described in section 1. ^' Some of the features typical of the apparatus based on the method described above are as follows:

1) Therein the principle of vertical easure¬ ment is applied, such as in the "FP-9" photometer (U.S. Patent 4,144,030).

2) In stead of one detector, a detector matrix consisting of several sub-detectors is used for each reaction vessel, which sub-detectors of the matrix register the light-absorbing or light-emitting property of the precipitate or any other reaction result on the bottom of the vessel only from a certain point, each sub-detector from an individual point of its own. 3) The precipitate to be examined is illumi¬ nated appropriately, e.g., by means of homogeneous light. It is possible to use an external, sufficiently homo¬ geneous light coming primarily from above, such as, e.g., daylight, the general illumination of the room, or any other available source of radiation.

4) The signals received from the sub-detectors are compared with each other and/or with blank, reference, and/or standard values, e.g., electronically, and it is decided whether the result equals, e.g., agglutination or not.

5) The apparatus may be provided with a permanently programmed output unit, which directly indi¬ cates whether the precipitate in the vessel is aggluti¬ nated or not, e.g. as + and - display. . 6) The apparatus may be a multi-channel appa¬ ratus, in which case it has a detector matrix of its own for each channel. On the other hand, the channels may be arranged as a matrix corresponding the extensively used and widely spread, e.g., so-called pit plates, cuvette sets, etc.

The invention will be described in more detail below with reference to the attached drawings, wherein

Figures la and lb illustrate two different cases in a reaction vessel, Figure 2 is a schematical presentation of one embodiment of the apparatus in accordance with the invention, Figure 3 illustrates absorbance readings obtai¬ ned by means of the apparatus shown in Fig. 2, and under¬ neath the graphical presentation the measured precipitate is shown as viewed from above, Figure 4 is a schematical presentation of a second embodiment of the apparatus in accordance with the invention, and

Figure 5 is a partly sectional view of the apparatus in accordance with the invention, wherein it is possible to measure the precipitates of several vessels simultaneously.

In Figure la there is a precipitate 34a on the bottom of the reaction vessel 32, the shape and density of the said precipitate being at different points differ- ent from those of the precipitate 34b shown in Figure lb.

The precipitate 3^a illustrates an agglutinated situation, whereas Figure 34b represents a non-agglutinated precipi¬ tate in the situation of measurement.

Figure 2 shows a reaction vessel 32 open at the top, which reaction vessel contains the reaction mixture as well as the precipitate 3 on the bottom 33. On the detector 35 placed underneath the vessel, sub-detectors 36 are seen which register the light coming from the source of radiation 37 and passing through the precipitate 34. The sub-detectors are arranged in a line and/or in a matrix consisting of rows. It is evident that, in stead of the square sub-detector matrix shown in the figure, it is also possible to use matrixes of other shapes, such as, e.g., of the shape of a line, circle, etc., and it is possible to apply detectors consisting, e.g., of sectors and rings placed one inside the other.

Figure 3 shows how, out of the measurement arrangement shown in Fig. 2, each sub-detector (1 to 25) yields the corresponding absorbance values (1 to 25). The x-axis of the system of coordinates illustrates the location of the line to which the sub-detector belongs, and the y-axis illustrates the absorbance. Figure 4 shows a reaction vessel 32, which contains the reaction mixture 31 and, on the bottom, the precipitate 34, and a source of light 37 underneath the vessel. Above the vessel there is a matrix detector 35 and between the vessel and the detector a lens 38. By means of the lens 38, an exact image of the precipitate 34 is formed on the matrix detector 35_, whereby each sub- detector 36 receives light in accordance with what the precipitate 34 has allowed to pass through at its different points.

The quantity of radiation falling onto each detector from the incoming radiation depends on the absorption, transmission, scattering, or any other pheno¬ menon of the precipitate 34 at the different points of the precipitate.

The invention is not confined to the above embodiments only, but it may show even considerable variation within the scope of the patent claims.

Figure 5 shows how it is possible, for example, to construct an apparatus in accordance with this prin¬ ciple wherein it is possible simultaneously to measure the precipitates occurring in all the reaction vessels of a so-called^" microtiter-disk. Underneath each reaction vessel 32 there is a detector matrix 35 of its own. An electronic computer unit built in the construction of the apparatus decides whether the precipitate is agglutinated (+) or not (-) and indicates the result on the monitor screen 39.

The method of measurement may be based on photo- metry 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 turbidometr , fluorometry, or, e.g., on the use of a source of radiation and a receiver for lumi¬ nescence, 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 simul- taneously or subsequently by means of t\ιo or more wave lengths or methods of measurement (e.g., photometry and luorometry), the final result being based on the infor¬ mation thereby obtained.

It is evident that, by placing several detector matrixes side by side, it is simultaneously possible to measure precipitates or other reaction results of a certain structure and position placed in several reaction vessels.

Thus, as a source of radiation, it is possible to use either a source of radiation placed in the apparatus, a source of radiation placed apart from the apparatus, or the general illumination of the room, e.g. the daylight.

Claims

WEAT IS CLAIMED IS:

1. A method for the measurement of the pro¬ perties of an agglutination, a precipitate (34), or of a corresponding reaction result placed on the bottom of a vessel (32) by means of radiation and of a detector (35) that receives radiation, whereat the beam of measurement coming from the source of radiation (37) passes substan¬ tially in the direction of the vertical axis of the vessel, and the intensity of the radiation passing through, or reflected from, the precipitate (34) on the bottom (33) of the vessel is measured, c h a r a c ¬ t e r i z e d in that the measurement of the formation, location, and form of the precipitate placed on the bottom of the vessel and/or of the density or other properties of different points of the precipitate is performed in respect of component fields of limited area separately, the measurement result being read for each component field and the output being produced on the basis of the group of values in this way obtained.

2. A method as claimed in claim 1, c h a ¬ a c t e r i z e d in that a detector divided into several parts (36) is used as the detector (35) receiving radiation, the signals given by the various parts, i.e. sub-detectors, being compared with each other and/or with blank, reference and/or standard values.

3- A method as claimed in claim 2, c h a r ¬ a c t e r i z e d in that as the source of radiation is used a homogeneous radiation (3D outside the apparatus and that the detector (35) receiving radiation is placed directly underneath the vessel.

4. An apparatus for the implementation of the method as claimed in claim 1, which apparatus comprises a detector (35) receiving radiation, the said detector being located so that the measurement beam received by it passes substantially in the direction of the vertical axis of the vessel (32), an output unit, and possibly a source of radiation (37), c h a r a c t e r i z e d in that the detector (35) consists of several sub-detectors (36) placed in the same plane close to one another.

5. An apparatus as claimed in claim 4, c h a r a c t e r i z e d in that the sub-detectors (36) are fitted directly underneath the vessel (32).

6. An apparatus as claimed in claim 4, c h a r a c t e r i z e d in that a lens (38) is fitted between the detectors (36) and the vessel (32), the said lens focusing the radiation coming through the bottom (33) of the vessel onto the detectors.

7. An apparatus as claimed in any of claims 4 to 6, c h a r a c t e r i z e d in that a unit consisting of several vessels can be fitted to the apparatus, whereby underneath each vessel there is a detector (35) consisting of several sub-detectors (36) and the measurement can be performed simultaneously or subsequently from several different vessels.