METHOD FOR THE DETERMINATION OF THE RESULTS OF AGGLUTINATION REACTIONS

Abstract: Method for the measurement of the result of an agglutination reaction, in which method, after incubation, the reaction mixture is shaken so that the non-agglutinated portion is scattered homogeneously in the reaction mixture. Thereinafter, the absorbance of the agglutination remaining on the bottom of the reaction vessel is measured in an analyzer that measures vertically so that the beam of light or the reaction vessel is displaced in the horizontal plane and the absorbance is measured at several different points. The result of the agglutination reaction is obtained by comparison of the absorbance values.

Description: Method for the determination of the results of agglutination reactions The present invention is concerned with a method for the measurement of the results of aggluti¬ nation reactions in an analyzer of vertical measurement. Various agglutination tests are used, e.g., for the establishment of bacteria, viruses, antigen components of fungi or of their antibodies as well as of abnormal proteins (e.g. tumour-specific proteins) . Out of these tests should be mentioned hemagglutination (HA) , hemagglutination inhibition (HI) , indirect hem¬ agglutination, as well as latex agglutination. . The said tests are used in serological diagnostics, serotyping, as well as in various estab¬ lishments of antigens. For example, in epidemy studies as well as in the specific diagnostics of infection diseases, the said tests are highly usable. In blood group determinations, a reaction based on the agglutination of red blood cells is used. Depending on the test used, a positive result of agglutination may mean either a positive result or a negative result. Hemagglutination can be ascertained on the bottom of the reaction vessel as a diffuse covering. For example, in a HA-test, viruses and bacteria have an ability to agglutinate red blood cells under certain conditions on the bottom of the reaction vessel over a large area. If no hemagglutination takes place, the red blood cells are assembled in the reaction vessel usually as distinctly limited area, a so-called button. By now, the results of agglutination tests have been read visually. In different tests, the criteria for the interpretation of the results vary. When the results are read visually, a great number of different error factors are involved. Different persons see in different ways; the eye is easily tired and ' B U EA O PI the results may change, whereat the human error factor-r ¬ are large in the interpretation of the results. The agglutination tests may also be connected with a colour indicator, whose visual interpretation, in a negative or positive test, is, however, laborious. Tests of this type are used extensively in different determinations. What is also frequently con¬ cerned is tests carried out in massive numbers, such as syphilis and rheumatism tests, blood group determina- tions, and establishment of pregnancy. In such cases, the performance of the tests must be simple, automated, accurate, and reliably reproducible. So far, there has been no simple and reliable automated mode of performance of the said tests. When, e.g., blood groups are determined by means of the agglutination reaction by using available equipment (Groupamatic ) , difficulties are often encountered in particular in the interpretation of weak agglutination reactions, e.g., out of the following reasons: 1. The agglutinated reaction mixture is shaken before measurement so strongly that the agglutination mix is dispensed in the reaction mixture (GB Pat. No. 1,229,971) . 2. The reaction mixture is measured by means of two separate beams of measurement (GB Pat. No. 1,229,971 and US Pat. No. 3,883,308), the values yielded by them being compared with two separate threshold values. The formation, measurement and production of the result of agglutination reactions reliably and rapidly has, even as yet, not been resolved even though several inventions have been suggested (UK Pat. No. 1,532,057, US Pat. No. 3,707,354, and US Pat. No. 4,148,607) . BUREA O PI As a good solution is, however, to be con¬ sidered the arrangement in which the FP-9 analyzer system (Labsystems Oy, Finland) is used and which is described in a patent (US Pat. No. 4,290,997, Apparatus for Automatic Measurement of the Results of Aggluti¬ nation Tests) . The method in accordance with the present invent±on and with the said earlier invention permits, e.g., the measurement of the results of agglutination reactions, among other things, in a spectrophotometer, adsorption photometer, fluorometer, ' and nefelometer. The method in accordance with the present invention permits, e.g., the formation, measurement and production of the output of agglutination reactions reliably and rapidly either automatically or partly non-automatically. In the method, agglutinations are promoted by appropriate centrifuging or shaking. Hereinafter the reaction mixture is incubated. After the incubation step, the shaking of the reaction mixture is performed so that even a weak agglutination mix is not scattered in the reaction mixture but remains on the bottom of the reaction vessel, but, however, strongly enough so that the non-agglutinated part can be placed homogene- ously in the reaction mixture. After a short standing time, the reaction mixture is measured. The absorbance of the agglutination produced on the bottom of the cuvette of the reaction mixture is measured (prefer¬ ably, e.g., by means of a vertical beam of measurement) so that the source of light or the reaction vessel is moved in the horizontal plane and, at the same time, the intensity of the light passing through is measured at several phases along the path of movement. The absorbance values measured by means of one beam of mea- surement at several different points are analyzed and compared in accordance with a program. The production of the output takes place automatically. OMPI The invention comes out in more detail from the following description and from the attached drawings, wherein Figure 1a shows an agglutination placed on the bottom of a cuvette for reaction mixture, the result being in this case positive, Figure 1b shows an agglutination placed on the bottom of a cuvette for reaction mixture, the result being in this case negative, Figure 2 schematically illustrates the mea¬ surement of a block of cuvettes for reaction mixture, Figure 3 is an exemplifying embodiment of a system for the determination of blood groups , Figure 4a illustrates the measurement of the bottom of a cuvette at several different points, and Figure 4b shows the corresponding measure¬ ment reaults. In the method described herein, and in the system in accordance with the said method,.the deter- mination of ABO-groups and of the Rh-factor is carried out, e.g., as follows: I. DETERMINATION OF ABO-GROUPS The red blood cells are ' examined by means of an agglu¬ tination test as follows: 1) A cell suspension of about 2.5 % is made of the red blood cells to be examined, in physio¬ logical salt solution. 2) Into the blood-group determination cuvettes, 50 μl of a - A, a - B, a - AB - serums are pipetted, as well as, into each cuvette, 50 yl of the red blood cell suspension prepared under section 1 ) . 3) Stirred in an agitator for 15". 4) Incubated at the room temperature for 10 '-15' . 5) Stirred as under section 3) . 6) Measured by means of a FP-blood group-analyzer at 540 nm. The apparatus is reset to zero. e.g., with 100 μl of corresponding anti-serum. Out of plasmas/serums, antibodies are examined likewise . by means of an agglutination test as follows: 1) A cell suspension of about 2.5 % is made of A- and B-test cells in physiological salt solution. 2) 50 μl of the plasma/serum to be examined as well as 50 μl of test cell suspensions are pipetted (see section 1 ) . 3) Stirred for 15". 4) The mixture is incubated at the room tempera¬ ture for 15' to 20 ' . 5) Stirred as under section 3) . 6) Measurement at 540 nm, the apparatus is reset to zero, e.g., by means of 100 μl of BSA-solution. II. DETERMINATION OF THE Rh-FACTOR 1) A suspension of about 2.5 % is made of the red blood cells to be examined, in physiological salt solution. 2) 50 μl of the suspension prepared under 1) as well as 50 μl of a - D - serum are pipetted into the FP-blood-group cuvettes. 3) Shaken or centrifuged for 15". 4) Incubated at the room temperature for 15'. 5) Stirred for 15". 6) Measured at the wave length of 540 nm, the apparatus is reset to zero by means of 100 μl of corresponding anti-serum. Rh-control BSA (22 %; pH 7.2) 50 μl Red blood cell suspension to be examined 50 μl Working procedure as in sections 1 to 6 The apparatus is reset to zero, e.g., by means of 100 μl of BSA. III. DETERMINATION OF D U -VARIANT 1 ) A cell suspension of 2 .5 % or 5 % is made of washed cells . 2) 100 μl of a - D - serum and 100 μl of 2.5-% or -—• 5-% cell suspension are pipetted into FP-blood- group cuvettes. 3) Stirred for 15". 4) The mixture is incubated at + 37 C for 15' . 5) The reed blood cells sensitized in this way are washed 3 x with physiological salt solution. 6) 100 μl of a - human - serum are added onto the washed cells . 7) Stirred for 15". 8) Shaken or centrifuged for 15". 9) Stirred for 15", 10) Measured at 540 nm, the apparatus is reset to zero, e.g., by means of 100 μl of a - human - serum. Figure 1a shows an agglutination 2 on the bottom of the reaction-mixture cuvette 1 , the result being in this case positive. Figure 1b shows a negative ' result, in respect of agglutination, in cuvette.3, wherein the red blood cells are evenly dispensed in the reaction mixture 4. Figure 2 schematically shows how the block 5 of reactio -mixture cuvettes moves through the measure¬ ment beam 6 so that each reaction-mixture cuvette 7 in the cuvette block 5 is read by means of the vertical measurement beam 6 at different points. The vertical measurement beam 6 starts from an opening 8 of appro¬ priate size facing each reaction-mixture cuvette 7 and passes in the direction of the longitudinal axis of the reaction-mixture cuvette 7 to the corresponding detector 9. In this way, the agglutination 10 on the bottom of each reaction-mixture cuvette 7 can be mea¬ sured at several points by appropriately displacing the cuvette block 5 in the direction of the x-axis and/or, if required, also in the direction of the y-axis. Of course, the measurement beam 6 may also pass in the opposite direction, from the top downwards to a detector placed underneath the reaction-mixture cuvette 7, or the cuvette may also be stationary and the beam of light be displaced across the cuvette bottom. The beam of light may also move across the cuvette bottom at several points. Fig. 4a. Reference numerals 11, 12 and 13 denote different paths of move¬ ment. Figure 4b illustrates the corresponding measure¬ ment results. In Fig. 4b, the curve 15 corresponds, e.g to the measurement of point V2 as measured photometri- cally. Moreover, the equipment can be constructed so that the agitator is placed in connection with the photometric measurement head. In such a case, outputs in accordance with the above can be produced, e.g., as a function of shaking intensities, thereby finding out differences between the intensities of different agglutinations. For the calculation of ready results, the equipment is provided with appropriate computer equip- ment (Fig. 3 p) . Figure 3 illustrates a system as an exempli¬ fying embodiment of the method invention for the determination of blood groups: a. Whole-blood sample b. The blood-sample is centrifuged c. Plasma or serum and red blood cells separated in the tube d. The tube is placed in its position, indicated by the arrow e. 25 μl of red blood cells are taken out of the tube into a dosage-diluter (1) f. The dosage-diluter is emptied into an empty tube g. By means of a dosage-diluter, 1000 μl of reagent is added h. 50 μl of cell suspension is dosed by means of a dosage-diluter into four reaction-mixture cuvettes i. 200 μl of plasma are taken into a second dosage-diluter (2) j. Out of the dosage-diluter (2), 50 μl of plasma are dosed into each of the following four reaction-mixture cuvettes k. 50 Ul of reagent are added into the reaction- mixture cuvettes 1. The reaction-mixture cuvettes are incubated m. Centrifuging or shaking if required n. Shaking so that agglutinated mix is not dis¬ pensed in the reaction mixture. During the shaking, non-agglutinated red blood cells are dispensed in the reaction mixture o. After a certain period of time, the shaking is followed by automatic measurement by means of a vertical measurement beam at 5 to 10 different points through the bottom of each reaction-mixture cuvette p. Storage, calculation, analysis, and output of the measurement result. As an application of the method of the inven¬ tion, systems can be constructed, e.g., with different degrees of automation o ' r with different mechanical embodi¬ ments. Such systems may comprise one apparatus or several separate apparatuses. In the calculation and analyzing of the results, the measurement results may be compared, e.g., with standard values, with each other, and/or with positive or negative results, or with both. - UREA OMPI

Claims: WHAT IS CLAIMED IS: 1. Method for the determination of the result of an agglutination reaction by measuring the intensity of the light passed from a source of light through the bottom of the reaction vessel, c h a r ¬ a c t e r i z e d in that the source of light or the reaction vessel is displaced in the horizontal plane and, at the same time, the intensity of the light passing through is measured at several different points along the path of movement. 2. Method as claimed in claim 1, c h a r - a c t e r i z e d in that the intensity is being measured constantly during the movement. 3. Method as claimed in claim 1 or 2, c h a r a c t e r i z e d in that the reaction vessel is displaced and the source of light is kept stationary. 4. Method as claimed in any of claims 1 to 3 , c h a r a c t e r i z e d in that the reaction vessel is displaced during the measurement relative the source of light along several parallel straight lines. _ .. OMP!

Inventors: EKHOLM PERTTI, KAUKANEN ESKO, PARTANEN PAUL, SUOVANIEMI OSMO, EKHOLM, PERTTI, KAUKANEN, ESKO, PARTANEN, PAUL, SUOVANIEMI, OSMO
Applicants: LABSYSTEMS OY
Application year: 1983
Main classification: G01N33
IPC codes: G01N21, G01N33