Apparatus and analysis for agglutination reaction
BACKGROUND OF THE INVENTION 
     The invention relates to a novel analysis process based on a simple or complex agglutination reaction, inter alia for blood analysis, more particularly but not exclusively for blood groupings, and to a novel apparatus for carrying the process into practice. 
     The known methods, most of which are either manual or part-automatic, use vessels (cavities) in which the reactions proceed. The vessels are either individual or grouped on a flat support. They are usually made of glass or plastics. 
     The vessels are usually of cylindrical shape and have either conical or spherical ends; they are of course open at the top so that they may be filled. Also, so that vessels grouped on a plate may be produced, the vessel axes must be parallel to one another. 
     In the case of grouped vessels, the same are disposed in rows in a perpendicular arrangement - i.e., they ae distributed in a rectangular or square shape on their support. 
     In endeavors to save on reagents, which are often costly, the tendency is to reduce vessel dimensions and therefore the dimensions of the support plates. 
     However, when a large number of vessels, even small vessels, are arranged on a support, it is impossible to arrange for centrifuging such that the axis of each vessel is completely coaxial with the direction of the centrifuging force, since the vessels are disposed in parallel relationship to one another. The only way of complying with such a condition is for the centrifuge to be of infinite radius. Consequently, agglutination after centrifuging for any given kind of reaction is not the same for each vessel. Some of the globules remain free in some vessels since such globules have been displaced on to the cone surface, and not its apex, by the centrifugal force. 
     As a check on the nature and quality of the reaction, the globules must be returned to the suspended state after centrifuging, to which end the plate is given a vibratory treatment for varying lengths of time. Interpretation of results is baaed on observation and estimation of the relative quantities of globules which have agglutinated and of globules which have remained in suspension. The estimation is made all the more difficult in that, when a large number of vessels are disposed on a support, the phenomenon of agglutination during centrifuging may be different in all of the vessels on the plate, for the reasons just mentioned. 
     SUMMARY OF THE INVENTION 
     This invention provides an analysis process and an apparatus for carrying the process into practice such that the difficulties mentioned are obviated because the direction of the centrifugal force is completely coaxial with the axis of each vessel. Also, the process and apparatus according to the invention make possible automatic reading of results which makes for reliable interpretation, based on very small amounts of specimen and reagents. 
     In the novel process which may involve simple or complex agglutination reactions, (a) a specimen for analysis is mixed with one or more reagents in a vessel of progressively decreasing cross-section; (b) the mixture is incubated; (c) the incubated mixture is then centrifuged at least one so as to hurl the particles onto the vessel base, the same being disposed substantially in the direction of the centrifugal force, with the result that the agglutinated particles stick to the vessel base whereas the unagglutinated elements remain free thereon, whereafter (d) centrifuging is stopped and, if necessary, operations (a), (b), (c) and (d) are repeated on the resulting product and a different reagent and the nature of the reaction which has occurred is determined by observing the presence or absence of the agglutinated or non-agglutinated elements, after a time interval sufficient for their separation. 
     The process can be carried into practice very advantageously with the use of a novel apparatus developed for the purpose. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     A detailed description will now be given of the process and apparatus with reference to the accompanying drawings, in which: 
     FIG. 1 is a section through a support in which a vessel is received; 
     FIG. 2 shows the vessel of FIG. 1 after charging: 
     FIG. 3 shows the same vessel after centrifuging; 
     FIG. 4 shows the vessel during reading of the reaction, the arrow A representing the direction of a light beam passing through the vessel; 
     FIGS. 5a and 5b represent the image of a positive reaction; 
     FIGS. 6a and 6b represent the image of a negative reaction; 
     FIG. 7 shows how the red globules and the plasma are separated in blood analysis; 
     FIG. 8 shows treatment of the blood specimen by an enzyme accelerating the agglutination or clumping reaction; 
     FIG. 9 shows a photoelectric measuring device for analyzing the results; 
     FIG. 10 is diagrammatic view of a vessel during vibration of the mixture; 
     FIG. 11 shows the vessel of FIG. 10 during the resuspension step by a stream; 
     FIG. 12 shows the constructional arrangement of the shape of the vessel; 
     FIG. 13 is a section on the line B--B of FIG. 12; 
     FIG. 14 is a section on the line A--A of FIG. 12; 
     FIG. 15 shows an alternative shape for the vessel; 
     FIG. 16 is a view showing the arrangement of a number of vessels on a support; 
     FIGS. 17 to 21 show alternative shapes for the support; 
     FIG. 22 is a simplified section through an automatic apparatus for performing the process; and 
     FIG. 23 is a plan view of the apparatus shown in FIG. 22. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT(S) 
     The main use for the purpose is in blood analysis. In blood analysis it is usually preferred to separate the blood specimen to be analyzed into globules and plasma before the process is carried into effect. Conveniently, the separation can be carried out by centrifuging, e.g. using a commercially available centrifuge. 
     It has also been found advantageous if, before being placed in the vessel in which the mixing step (a) is performed, the blood specimen has added to it an enzyme, such as bromelin, accelerating the agglutination or clumping reaction. 
     Since the volume of the blood specimen is very reduced, being e.g. 5 μl per vessel, it has been found that the vessel walls have appreciable hydrophilic or hydrophobic effects. It is therefore advisable to cover the vessel walls before use with a thin layer of proteins to ensure that the mixing step (a) is performed correctly. There can be used for this purpose e.g. ox-blood serum albumins. For the rest, the protein coating can be given, as required, to either the entire wall surface or just to some of the wall surface, and the proteins used can be all of the same or of various kinds. The use of various kinds of protein instead of or concurrently with the variant of partial coating can be considered when different hydrophilic or hydrophobic effects are required on different portions of the vessel walls. 
     The steps hereinbefore described, although useful for the practice of the process, are optional. Consequently, either after one or the other of the steps just outlined or directly, the blood specimen to be analyzed is prepared (by dilution). One possible practical procedure is the following: 
     One or more dilution tubes or &#34;hoods&#34; is or are added to the specimen tube and they are introduced into a conveyor system which can be a chain system or a rotating system or any other appropriate system. The conveyor moves each system embodied by a specimen tube and a hood past one or more dilution stations. The same preferably comprise a probe, the same sampling the red globules in the specimen tube, and an element for transferring the red globules into the or each hood associated with the specimen tube. Simultaneously with probe movement, one or more pumps deliver or delivers to the or each hood a physiological solution which may or may not contain an enzyme such as bromelin. The time taken to prepare and to dilute the specimen in this way is approximately twelve minutes. 
     It is is required to use the apparatus for blood groupings by the bromelin method and by the Coombs method simultaneously, two hoods are required, one containing the physiological solution and the bromelin, while the other contains only the physiological solution. 
     The process can then be carried into effect, preferably by means of the apparatus to be described in greater detail hereinafter. Basically, such apparatus comprises a number of vessels 1 (e.g. FIG. 1) secured to a support 2. Preferably, the support resembles in shape a ring or a disc. Each vessel 1 is formed with a top aperture 3 for the introduction of the specimen for analysis and of the reagents, each vessel having a place or site 4 for receiving the specimen and reagents. In the embodiment shown for example in FIG. 1, the site 4 takes the form of a recess or hollow of a kind such that the specimen and reagents collect therein, provided that they are subject only to the force of gravity. 
     As can be seen in FIGS. 12 to 14, the vessel cross-section decreases continuously towards the outside in its terminal portion 5 -- i.e., the portion remote from the centrifuging axis Z. Also, in that part of the site 4 which is remote from the centrifuging axis Z, the vessel has a surface 6 which inclines upwardly towards the outside as far as a location 7 of the terminal portion of the vessel where the cross-section thereof is at the narrowest dimension. 
     The vessels are filled with the red globule specimens of the blood to be analyzed and the test sera and with the specimens of the corresponding plasma and the test globules. In routine blood grouping, there may be the following distribution of specimens and reagents relatively to one another: 
     
         ______________________________________                                    
Vessel  specimen         reagent                                          
______________________________________                                    
1       reference substance                                               
                         (for color intensity)                            
2       red globules     anti serum A                                     
3       red globules     anti serum B                                     
4       red globules     anti serum A+B                                   
5       plasma           globules A.sub.1                                 
6       &#34;                globules A.sub.2                                 
7       &#34;                globules B                                       
8       &#34;                globules 0                                       
to which are usually added in case of determination of                    
the rhesus factor                                                         
9       red globules     anti serum D no. 1                               
                         (or anti D)                                      
10      red globules     anti serum D no. 2                               
                         (or anti CDE)                                    
______________________________________                                    
 
    
     Also, and either concurrently with the determinations just described or in a subsequent step, the determination (on the red globules) of antigens other than A and B, e.g. Kell&#39;s antigen, or of sub-groups of A or of AB and, on the plasma, of abnormal agglutinimes and of syphilis can be arranged for. The control to which the sensitivity of the reading device is adjusted can be inter alia a suspension of globulin or a suspension of red globules in albumin. 
     Step (a) of mixing the specimens for analysis with the reagents can be carried out quite simply by vibrating the vessel horizontally or vertically or both simultaneously before and/or during the incubation step (b) and on a periodic or continuous basis. Mixing occurs in that part of the vessel which has the reference 4 in FIG. 1 (See e.g. FIG. 2). 
     The reaction takes some time to develop its full effect. The mixture must therefore be left for a sufficiently long incubation time (b); if required, incubation can be speeded up or completed by the vessel being vibrated as hereinbefore described. 
     The incubated mixture is then given the centrifuging step (c) so that the mixture is hurled against the vessel base 7 (FIG. 3). Centrifuging is by rotation of the vessel around the axis Z along which it is secured and it can be repeated or given in consecutive steps at various speeds. 
     Upon the cessation of centrifuging (step [d]), for example after a few minutes, one of the two following cases may be observed: 
     I. If the reaction is positive, agglutination is complete and the agglutinates remain stuck to the vessel base portion 7; 
     II. If the reaction is completely or partly negative, the non-agglutinated elements, e.g. the non-agglutinated red globules, slide down from the vessel base 7 along the inclined wall 6 (FIG. 1). 
     The slide-down does not start immediately upon the cessation of centrifuging but usually starts after a short wait. Also, the rate of sliding may vary according to the nature and intensity of the reaction occurring, and so measurement of the rate of sliding gives further information on the extent of negative reactions. 
     Reading of the reaction (FIG. 9) -- i.e., observation of the presence or absence of unagglutinated elements on the inclined wall -- can be accomplished for example by means of a visual microscope arrangement. Preferably, however, reading is automatic and can be provided for example by a facility comprising inter alia a microscope and a photoelectric cell. The microscope can also have one or more filters making it possible to read the result of the reaction at various wavelengths. The reaction can also be read by measuring the varying extents of transparency of the agglutinated elements remaining on the vessel base after a particular time. If the specimen or the reagent used is radioactive or contains a radioactive addition substance, the nature of the reaction is determined automatically by means of a facility comprising a radioactivity counter. 
     The process is also of use for blood groupings by the Coombs&#39; test or method. In this event the product arising after step (d) (cessation of centrifuging) is treated by further steps for faciliting agglutination, including washing and returning the reaction product to the suspended state. For washing, an appropriate washing liquid, such as a physiological solution or an anti-globulin solution, in a predetermined quantity and directed towards the vessel base can be used, the stream being produced by pressure injection. The vessel can also be vibrated vigorously horizontally and/or vertically. 
     After these further steps, the cycle of step (a), (b) (c) and (d) is repeated on the washed and re-suspended product, to which a new reagent has been added, and the resulting reaction is read in the manner hereinbefore described. 
     An embodiment of the apparatus for carrying the process into effect will now be described. 
     As previously described, the supports have a number of vessels or receptacles or the like 1 formed with an aperture 3, a place or site 4 for receiving the specimen and reagents, and an inclined surface 6. 
     Surface 6 is at an angle α to the horizontal, such angle being between 30° and 60°. In a special variant the angle is from 45° to 50°. The optimum angle depends, of course, upon the particular reagents used, the centrifuging speed and the nature of the specimens for analysis. The base of the inclined surface 6 can be plane but is preferably concave. 
     Referring to FIG. 3, between the location 4 and the surface 6 there is an intermediate or transition zone 8 which is inclined but which could be horizontal, the inclination angle β of zone 8 is, in FIG. 3, a fraction of the inclination angle α (FIG. 2) of surface 6. Satisfactory results were obtained with an angle β of from 8° to 20°, but the value can be higher. FIG. 15 shows a variant in which the angle β of the zone 8 coincides with the angle of the surface 6. 
     As shown in FIGS. 12 to 14, the terminal cross-section 5 of vessel 1 is bounded by the surface 6 at the bottom and by a tunnel-shaped passage at the top. The section A--A in FIG. 14 shows the progressively decreasing cross-section of the vessel, such cross-section reaching its lowest value at the location 7. 
     Since there are advantages in using a photoelectric system to study the reaction, the vessel is embodied, at least in the part near the inclined surface 6, by a transparent substance, preferably plastics (see e.g. FIG. 9). 
     To facilitate the performance of the process according to the invention, a number of vessels 1 are arranged in the shape of an open or closed circle on the support 2 (e.g. FIG. 17), the main axes of the vessels being disposed along radii R  which all converge towards the axis Z (e.g. FIG. 12 or 16) of the support 2. Consequently, all the vessels are at the same distance from the axis Z. However, a number of concentric rows of vessels 1 could be placed on a single support 2. 
     Of the many possible ways of distributing the vessels 1 on a support, those shown in FIGS. 17 to 21 have been selected. Referring to FIG. 17, the vessels 1 are arranged on a support 10 in the shape of a closed circle. However, it may be advantageous if there is an aperture 12 in a substantially circular support 11, in the manner shown in FIG. 18, so that the support 11 can be slid laterally around a spindle or placed on an appropriately shaped arm. 
     The support 13 shown in FIG. 19 can take the form of a closed disc which, as shown in FIG. 20, can also be formed with a recess 15. 
     FIG. 21 is a view to an enlarged scale showing vessels 1 arranged in an open circle 10 and a closed circle 11. 
     Measurement of whether the reactions are positive or negative, in accordance with FIGS. 5a-5b and 6a-6b, are made in satisfactory conditions by means of a photoelectric facility diagrammatically shown in FIG. 9. A light beam from a light source 27 is made parallel by a condenser 32, passes through prisms 33, 34 and goes through a final lens 35 to reach cell 30; the same has wires 29 connected to an electrical measuring instrument (not shown). 
     A complete automatic system, e.g. for blood group analysis, is shown diagrammatically in FIG. 22. Rising from a base 16 is a column 17 having secured to it for example three arms 18-20 (see also FIG. 23). Column 17 is arranged so that it cannot rotate around its axis in bearing 21 of base 16, nor can the arms 18-20 rotate around column 17. Consequently, the various stations of the apparatus can be arranged in a vertically stepped or staged system around a main vertical axis which is the centrifuging axis Z. 
     A cylindrical sleeve 22 is secured to the two top arms 19, 20 and guides the supports 11 (e.g. of FIG. 18) and allows the same to slide downwardly. The supports 11 as shown in FIG. 18 and because of the recess in them, can be fitted on the arm 20; they are engaged by a system (not shown) which lowers them stepwise along the axis Z as embodied by the sleeve 22. After the specimen and reagents have been introduced at 23, the mixture has sufficient time for incubation as its travels downwards along sleeve 22. 
     After passing along the incubation path the vessel-carrying supports 11 are placed automatically by the step-by-step system in a centrifuge 24 which at this time is stationary. The mixture which has been incubated and which is present at location 4 in vessel 1 is hurled during centrifuging along the intermediate zone and the inclined surface or wall 6 as far as the vessel base location 7. Upon the completion of centrifuging the supports 11 move stepwise along a guidesleeve 25 retained by an arm 18 long enough for the mixture of specimen and reagents to separate out into an agglutinated part remaining on the vessel base 7 and an unagglutinated part which descends along the inclined wall 6, so that the nature of the reaction can be defined in accordance with FIGS. 5a-5b or FIGS. 6a-6b. 
     When a particular support 11 reaches the bottom position, the nature of the reaction can be measured by means of a microscope arrangement 26 (see also FIG. 9) comprising a light source 27, an optical system 32-35 and a photoelectric cell 30 connected to an indicating instrument. 
     The system for conveying the supports along the equipment is considered to be known and so has been neither described nor shown. Similar considerations apply to the elements for loading and unloading the supports.