Abstract:
A sample handling method, in which the information on the sample to be examined is transferred throughout the entire measuring process by transferring the sample cup. The sample cups are handled in two independently operating drums, in which case at least two different handling phases can be carried out simultaneously in the measuring device. The diagnostic measuring device comprises a cassette drum, on the circumference of which the sample cup cassettes containing sample cups have been placed. The sample cup is fed to the first handling drum, which is simultaneously an incubation drum. After that the sample cup is transferred to a second handling drum, where washing, drying, measuring and discharge of the sample cup are carried out. The drums have been placed near each other, overlapping so that the transfers of the sample cup can be performed directly from one drum to another by means of transfer plungers.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a diagnostic method and device. 
     An object of the invention is a method for handling samples, in which method a sample is brought to a measuring device, steps relating to the measuring process are carried out on the sample, and the sample is measured. 
     Diagnostic assay measurements are carried out in laboratories and hospitals, for example. Such analyses usually concern handling a patient&#39;s blood samples. The measuring methods and measuring devices that are used vary to a great extent. For example laboratories usually handle large amounts of samples. Particularly in screening tests, assay measurements are carried out so that the samples are brought together to efficient measuring devices, in which a large number of samples are measured at a time in one batch measurement. However, there is also a need for single measurements in hospitals, because the blood sample of a hospitalised patient should be analysed as quickly as possible. Screening test methods are not suitable for this. 
     Another object of the invention is a diagnostic measuring device. In particular, the object of the invention is a measuring device that enables patients&#39; blood samples in hospitals to be tested as quickly as possible. One test result that is required quickly in hospitals is an analysis of whether there are signs of a recent myocardial infarction in the patient&#39;s blood sample. From the treatment point of view, it is of crucial importance that a diagnosis can be made at the earliest stage possible. In such case it is not possible to use the diagnostic analysis equipment used in screening tests, since they are used to measure a large number of samples at a time. It is not possible to use them for measuring single samples. 
     As the need for rapid measurement of single samples is known, attempts have been made to develop sample handling methods and measuring equipment in which the measurement of a single sample can be performed rapidly. However, a sample handling method or measuring device of this kind must also be such as to enable the addition of new samples to be measured throughout the measuring process without disturbing the steps for measuring the previous samples that have already been started. 
     2. Description of Related Art 
     One known diagnostic measuring device is presented in U.S. Pat. No. 5,635,364. In it, test tubes containing samples can be loaded on a rotating carousel-type drum around which there is a drum containing reagents. The samples are transferred to a separate analysis drum with a transfer pipette. However, the device is very large and elaborate. 
     Another automated random access analyser is presented in U.S. Pat. No. 5,679,309. This measuring device features three carousel-type drums. The drum in the middle is an analysis drum, on both sides of which there is a sample drum and a reactance drum. The samples and the reactants are transferred to the analysis drum by means of pipette-type transfer equipment. 
     The disadvantage of known sample handling methods and measuring devices is their elaborateness. It is possible in various methods and the equipment used in them to carry out random access to fetch a certain sample for measuring. They do not, however, comprise arrangements for effectively accelerating measurement. The slowness of sample handling methods and measuring devices is often caused by the fact that the steps relating to the measuring are carried out alternately in the process. Slow steps carried out in succession substantially increase the throughput time of the measuring process. 
     One problem in the known sample handling methods and measuring devices is the fact that the samples are usually transferred within the measuring process by means of pipettes. Such sample transferring members within a device are slow and elaborate. In addition, they are difficult to keep clean. 
     The purpose of this invention to obtain a quick and efficient sample handling method, most advantageously for hospital use. Another purpose of the invention is to obtain a simple and efficient measuring device without the disadvantages of the known devices presented above. 
     BRIEF SUMMARY OF THE INVENTION 
     It is characteristic of a sample handling method relating to the invention 
     that the sample to be examined is transferred to the sample receptacle of the measuring device most advantageously by pipetting, 
     that the information on the sample to be examined is transferred in the measuring device throughout the entire measuring process by transferring the said sample receptacle, 
     and that the sample receptacle is handled in the measuring device in at least two independently operating handling members in such a way that at least two different handling phases are carried out simultaneously in the measuring device. 
     According to one preferred embodiment of the sample handling method relating to the invention 
     the sample receptacle used for measuring in the measuring device is transferred from the sample receptacle store into the actual measuring device, 
     within the measuring device, the sample receptacle is handled in at least two independently operating handling members, 
     and all the various phases of the measuring process are divided to be performed by the said independently operating handling members. 
     According to a second preferred embodiment of the sample handling method relating to the invention, in the measuring device, at least two phases of the measuring process are carried out simultaneously in the same independently operating handling member. 
     According to a third preferred embodiment of the sample handling method relating to the invention 
     in the measuring device, the sample receptacle to be used for measuring is selected from the independently operating sample receptacle store for the first independent handling device of the actual measuring device, in which handling device at least the dispensing and incubation of the sample are carried out, 
     and the sample receptacle is transferred from the first handling device to the second independently operating handling device in which the sample is at least measured and most advantageously the sample receptacle is also washed, dried and discharged. 
     It is characteristic of the measuring device relating to the invention, 
     that the measuring device comprises members for transferring the sample receptacle within the measuring device, 
     that the measuring device comprises at least two independently operating handling members and members for transferring the sample receptacle from one handling member to the second handling member, 
     and that sample receptacle handling devices have been placed in conjunction with at least two independently operating handling members, with the aid of which handling devices at least two different handling phases can be carried out simultaneously on at least two sample receptacles. 
     According to one preferred embodiment of the measuring device relating to the invention 
     the measuring device comprises an independently operating sample receptacle store and at least two independently operating handling members, 
     the sample receptacle store has members for transferring a measuring receptacle of the desired type to the handling member of the actual measuring device, 
     that the measuring device comprises at least two of the following sample receptacle handling stations: a sample dispensing station, an incubation station, a sample receptacle washing station, a sample receptacle drying station, a sample measuring station and a sample receptacle discharge station, 
     and the said handling stations have been divided so as to be in conjunction with at least two independently operating handling members so that at least two different handling stations can be made to operate simultaneously. 
     According to a second preferred embodiment of the measuring device relating to the invention 
     the sample receptacles are sample cups and the sample receptacle store is a cassette drum on the circumference of which the sample cup cassettes containing sample cups have been placed, 
     the independently operating handling members are rotating drums, 
     and the cassette drum, the first sample cup handling drum and the second sample cup handling drum have been placed in conjunction with each other partly on top of each other so that the sample cup can be transferred directly from one drum to another. 
     According to a third preferred embodiment of the measuring device relating to the invention 
     the first sample cup handling drum is an incubation station, in conjunction with which a sample cup feeding station and a sample dispensing station have been placed, 
     a sample receptacle washing station, a sample receptacle drying station, a sample measuring station and a sample receptacle discharge station have been placed in conjunction with the second sample cup handling drum, 
     and both handling drums operate independently, so that at least one handling station of the first handling drum can be made to operate simultaneously with at least one handling station of the second handling drum. 
     According to a fourth preferred embodiment of the measuring device relating to the invention, at least two of the different handling stations that have been placed in conjunction with the second sample cup handling drum can be made to operate simultaneously. 
     According to a fifth preferred embodiment of the measuring device relating to the invention 
     the axles of the cassette drum and sample cup handling drums of the measuring device are parallel, 
     and the measuring device comprises a sample cup feeding device and, between the first and the second handling drum, a sample cup transfer device, which devices have transfer plungers parallel to the axles of the said drums. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     In the following, the invention is described by way of examples with reference to the accompanying drawings, in which 
     FIG. 1 shows a section of a sample cup cassette, seen from the side. 
     FIG. 2 shows the placing of a sample cup cassette in the cassette drum, seen from the side. 
     FIG. 3 shows a cassette drum, seen from above. 
     FIG. 4 shows a section of a diagrammatic view of the equipment arrangement, seen from the side. 
     FIG. 5 shows a diagrammatic view of the equipment arrangement, seen from above. 
     FIG. 6 shows a diagrammatic view of the sample cup handling drum, seen from above. 
     FIG. 7 corresponds to FIG.  6  and shows the sample cup handling drum in another position. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     FIG. 1 shows a tubular sample cup cassette  31 , in which single cup-like sample cups  32  have been placed one after another. The sample cup cassette  31  is placed in the cassette drum of the measuring device in an upright position so that the sample cups are in an upright position with the right side up. Depending on the method used in the measuring, the sample cups  32  can be completely empty cups or the walls of the sample cups can be coated with marker substances required in the measuring. 
     The mouth at the lower end of the sample cup cassette  31  in FIG. 1 is slightly tapered so that single sample cups  32  do not come out of the cassette  31  on their own. Discharge of the sample cups  32  from the cassette one at a time is effected by using a thrust plunger. FIG. 1 shows a diagrammatic view of the thrust plunger  33 , which in the measuring device is situated in conjunction with the sample cassette drum, as presented in more detail in the subsequent figures. 
     On the outer surface of the sample cup cassette  31  there has been formed a tapered point  35   35 , below which, at the lower end of the cassette  31 , its outside diameter is smaller. The tapering has been made in order to enable the sample cup cassette  31  to be placed with the narrow end first in the correspondingly dimensioned holder of the sample cassette drum. This structure is presented in more detail in the next figure. 
     FIG. 2 shows diagrammatically the placing of the sample cup cassette  31  containing single sample cups  32  in the cassette drum  30  of the measuring device. FIG. 2 only shows that part of the cassette drum  30  that forms the holder of the sample cup cassette  31 . The sample cup cassette  31  is put into its place in such a way that the narrower part below the tapered point  35  is pushed into the opening between the limiters  36   a  and  36   b.  The opening is suitably wide, so that the sample cup cassette  31  can also be pushed into place in a slightly inclined position, whereby the cassette  31  remains with its tapered point  35  resting on the limiters  36   a  and  36   b.  When the cassette  31  is pushed into an upright position, it is locked against the sill  37  of the holder, which sill is at the upper end of the cassette. 
     It can be seen in FIG. 2 that the sample cup cassette  31  is provided with a bar code  34 , which enables the sample cups  32  in the cassette to be identified. To enable the bar code  34  to be read, an opening  38  has been made in the holder of the cassette drum  30  for the bar code reader  39 , which is at the centre of the drum. Identification of the sample cups is necessary when there are sample cups that have been provided with different coatings in the cassette drum  30 . This being the case, one sample cup cassette  31  always only contains sample cups  32  that are coated in the same way. Therefore, the sample cup  32  that is to be used is always identified by the bar code  34  of the sample cup cassette  31 . The sample cups  32  themselves do not bear any identifying marks. However, empty and uncoated sample cups can also be used in the measuring device, in which case it is naturally not necessary to identify them. 
     FIG. 3 shows the cassette drum  30  from above. It can be seen in the figure that the sample cup cassettes  31  have been placed on the circumference of the drum  30  in such a way that their bar codes  34  are facing the bar code reader  39  at the centre of the drum  30 . In this way, the bar code reader  39  can read through the openings  38  the sample cup cassettes  31  that are on the drum  30 . When the type of the next assay measurement to be carried out is known, the computer controlling the measuring device is instructed to fetch for use a sample cup  32  corresponding to the measuring method. Then the cassette drum  30  is rotated so that the sample cup cassette  32  provided with the corresponding identification moves to the feeding station of the cassette drum  30 . After that a sample cup suitable for the measuring method used is taken from this sample cassette  31 . 
     FIG. 4 shows a diagrammatic view of the structure of the measuring device  20  and a diagrammatic view of the passage of a single sample cup  32  in the measuring device  20  by way of one example. For the sake of clarity, the measuring device  20  has therefore been simplified, and neither do the sections of the various drums  30 ,  40  and  50  of the measuring device  20  represent a real sectional plane. The passage of a sample cup  32  in the measuring device  20  may, however, vary considerably, since the movements and operation of the drums  30 ,  40  and  50  can be changed. In this case the passage of the sample cup  32  in the measuring device  20  differs from that presented in FIG.  4 . 
     FIG. 4 shows on the right the cassette drum  30  of the measuring device  20 , in which cassette drum the sample cup cassettes  31  are placed. To measure a new sample, the cassette drum  30  is rotated so that the desired sample cup cassette  31  moves to the feeding point for the sample cups  32 . The bar code reader  39  checks the bar code of the sample cup cassette  31  to ensure that the cassette that is at the feeding point contains sample cups  32  meant precisely for measuring this sample. After that a new sample cup  32   a  is taken for use from the sample cup cassette  31  in such a way that the plunger  33 , which is in conjunction with the drum  30 , presses the sample cups in the cassette  31  downwards. Then the sample cup  32   a  that is lowest in the cassette  31  is fed into the sample cup station of the first handling drum  40  of the measuring device  20 . 
     In this exemplary embodiment, the first handling drum  40  is called an incubator drum, since only the sample dispensing and the incubation and shaking phases are carried out there. However, it is also possible that the tasks to be performed are distributed in a completely different way between the various handling drums  40  and  50  of the measuring device  20 . 
     After a new sample cup  32   a  has been fed to the first handling drum  40 , i.e. to the incubator drum  40 , the drum  40  rotates so that the sample cup  32   a  moves to the dispensing station, to the sample dispensing device  41 . In FIG. 4, the sample cup that has moved to the dispensing device  41  is marked with reference number  32   b.  When the dispenser probe  42  of the dispensing device  41  has dispensed the liquid sample solution into the sample cup  32   b,  the reciprocating vibratory motion of the incubator drum  40  is started in order to incubate the sample. Thus the incubator drum  40  is simultaneously a vibrator. In the incubation phase the sample is shaken, for example, for 15 minutes at a temperature of 36° C. 
     After the incubation of the sample has been carried out, the vibratory motion of the incubator drum  40  is stopped. After that the drum  40  will rotate to a position where the sample cup containing the incubated sample solution is transferred to the second handling drum  50  of the measuring device  20 . FIG. 4 shows the plunger  43  situated at this point of the measuring device  20 , which plunger lifts the sample cup upwards to the handling drum  50 . The sample cup that has been transferred to the handling drum  50  is marked with reference number  32   c.    
     In this exemplary embodiment, all the other preparatory phases required for measuring, such as washing and drying of the sample cup  32 , sample measuring and discharging of the sample cup  32 , have been programmed to be carried out by the second handling drum  50 . However, the tasks can also be divided in a different way between the handling drums  40  and  50 . It is advantageous to divide the most time-consuming phases relating to measuring to different drums  40  and  50 . Because different work phases can be carried out independently and simultaneously on both handling drums  40  and  50 , the entire measuring process can be carried out in the shortest time possible. Incubation is the step that usually takes the most time, so the division of tasks presented in this example is advantageous. This is also the reason why the first handling drum  40  can take approximately three times as many sample cups  32  to be incubated as the second handling drum  50 , where the work phases to be carried out take considerably less time. 
     After this, in FIG. 4, the second handling drum  50  rotates so that the sample cup moves to the washing station  51 . The sample cup that is at the washing station  51  is marked with reference number  32   d.  At the washing station  51  the sample solution is discharged from the sample cup  32   d,  and the sample cup  32   d  is washed for example, for  30  seconds. After that the sample cup  32   d  is emptied and dried. At the washing station  51 , the emptying of the sample cup  32   d  and the feeding and discharge of detergent are carried out by means of pipettes  52 . In practice, the feeding and discharge pipettes are most advantageously separate, but for the sake of simplicity, FIG. 4 shows only one pipette  52  of the washing station  51 . The sample cup drying station is also most advantageously a separate station, as is shown in the next figure, but it is not shown in FIG.  4 . After the sample cup  32   d  has been washed and dried, measurement is carried out. 
     In the example shown in FIG. 4 the measuring is carried out on the coating layer on the inside of a dry sample cup. Alternatively however, sample cups that contain fluid can also be measured in the measuring device shown in FIG.  4 . Then the reagent solution is added to the sample cup before measuring. The reagent adding station can then be for example, at or next to the drying station marked with reference number  59  in (see FIG.  6 ). 
     For measuring, the handling drum  50  of the measuring device  20  rotates so that the sample cup moves to the measuring station  53 . The sample cup at the measuring station  53  is marked with reference number  32   e.  During measuring, the measuring head  54  of the measuring station  53  is above the sample cup  32   e  and the light shield  55  of the measuring head  54  is pressed down against the surface of the handling drum  50 . FIG. 4 also shows that to ensure impermeability to light, an annular groove  56  has been formed on the surface of the handling drum  50  at each sample cup. 
     After the measurement has been carried out, the handling drum  50  of the measuring device  20  rotates again. Now the measured sample cup comes to the discharge station. The sample cup to be discharged is marked in FIG. 4 with reference number  32   f.  It can be seen in FIG. 4 that there is an opening in the sample cup discharge station, in the bottom plate  57  under the handling drum  50 , whereby the sample cup  32   f  is able to fall downwards. The downward movement of the plunger  58  ensures that the sample cup  32   f  has with certainty been discharged from the measuring device  20 . 
     FIG. 5 shows the equipment arrangement of the measuring device  20 , seen from above. The sample receptacle containing the sample to be measured, which sample receptacle is usually a test tube, is brought into the pipetting unit  21  and placed in the sample station  22 , which in this example is called a test tube station. In the pipetting unit  21 , beside the test tube station  22 , there is a sample diluting station  23 , and at the opposite side of the test tube station  22 , a pipette washing point  24 . The pipette  42  of the dispensing device  41  of the pipetting unit  21  is used to perform dispensing of the sample into the sample cup, and possibly dilution. 
     Before starting measurement, the identification code or number of the test tube that contains the sample to be measured is given to the measuring device  20 . The identification can also be read from the bar code of the test tube. When the test tube has been placed in the test tube station  22 , the measuring method is selected. If more than one test is to be carried out, the control device is instructed to fetch a corresponding number of sample cups for the measurements. 
     When the measurement is started, the cassette drum  30  is turned to a position where a sample cup cassette  31  containing sample cups  32  suited precisely for this measurement at the sample cup feeding station. After that the sample cup  32  is fed to the first handling rum  40 , i.e. to the incubator drum. Immediately after that the incubator drum  40  rotates and moves this sample cup to the dispensing station  25 . If various sample cups have been taken onto the drum  40 , they move one by one to the dispensing station  25 . 
     In FIG. 5 the various work phases of measurement proceed so that the dispensing pipette  42  of the dispensing device  41  takes sample solution from the test tube  22  and dispenses 10 μl of the solution and 20 μl of the buffer solution into the first sample cup in the dispensing station  25 . Before dispensing, the pipetting tip already contains the buffer, which is separated from the sample by a small air gap in the pipetting tip. Correspondingly, sample solution and buffer solution are also dispensed into the other sample cups which are brought in their turn into the dispensing station  25 . 
     After dispensing, the reciprocating vibratory motion of the incubator drum  40  is started in order to incubate the samples. In the incubation phase, the samples are shaken for example, at a temperature of 36° C. for 15 minutes. After incubation, the incubation drum  40  rotates to a position where the first sample cup moves to the elevator station  26 , where the sample cup is lifted to the handling drum  50 . Other sample cups can also be lifted to the handling drum  50  in a similar manner already before washing, drying and measuring are started. 
     After the sample cups have been lifted to the second handling drum  50 , the handling drum  50  is rotated so that the first sample cup moves to the washing station  51 . The sample cup is washed for 30 seconds, for example. After washing, the handling drum  50  rotates again so that the washed sample cup moves to the drying station  59  and the next sample cup to the washing station  51 . The washing of the first sample cup and the drying of the second sample cup can then be carried out simultaneously, and they both take 30 seconds in this case. 
     After drying, the second handling drum  50  rotates again so that the dried sample cup moves to the measuring head  54  of the measuring station  53 . Correspondingly, the second sample cup that has been in the washing station  51  moves into the drying station  59  and the third sample cup into the washing station  51 . If the sample cup measurement can be carried out in the same time as washing and drying, the handling drum  50  can be rotated so that the sample cups proceed at regular intervals from one handling phase to another. Then the measuring of the first sample cup, the drying of the second sample cup and the washing of the third sample cup occur simultaneously. In this way each sample cup is washed, dried and measured in turn and discharged from the discharge station  60  after measurement. The concentration and measuring results of each sample cup are shown on the display and also printed on paper. 
     It can be noticed from the description of the measuring process presented above that the sample cup  32  proceeds through the entire measuring process, but all the different work phases are not by any means carried out successively in turn, as in known measuring devices. According to the invention, the measuring process can be carried out substantially faster when, besides the simultaneous work phases carried out at different handling drums  40  and  50 , different work phases are also carried out simultaneously at the same handling drum  50 . In other words, in the example presented, the division of tasks is such that in the measuring device  20  relating to the invention, all of the subsequent work phases are carried out simultaneously. Firstly, incubation is carried out on some of the sample cups  32  at the first handling drum  40 , and sample cups  32  which are further on in the process, at different phases, are washed, dried, measured and discharged simultaneously at the second handling drum  50 . It is clear that this procedure substantially accelerates the measuring process and the throughput of the sample cup  32  in the measuring device  20 . 
     However, it is not always possible to use the second handling drum  50  in the way presented above. For example, there may be some reasons concerning the handling of the sample, which dictate that only one sample cup can be taken at a time from the incubator drum  40  to the handling drum  50 , for example. Therefore the order of the various operations can be modified as necessary. The number of sample cups that can be taken to the second handling drum  50  at a time is also limited. For example, in FIG. 5, the handling drum  50  can clearly take fewer sample cups than the incubator drum  40 . 
     FIG. 5 also shows a detail presented in the previous figure. On the surface of the handling drum  50 , around the sample cups  32  annular grooves  56  have been formed, into which the light shield that is in conjunction with the measuring head  54  is pressed for the duration of the measurement thus ensuring that the space between the measuring head  54  and the sample cup  32  is impermeable to light. 
     FIG. 6 shows the second handling drum  50 , seen from above. The sample cups are lifted to the handling drum  50  at the elevator station  26 . FIG. 6 also shows the location of the washing station  51 , the drying station  59 , the measuring head  54  and the discharge station  60  in relation to the handling drum  50 . In the example in FIG. 6, the handling drum  50  has eight places for sample cups, the places being grouped in a regular manner. This means that the angle of rotation of the handling drum  50  is always 45° as the sample cup proceeds from one handling phase to another. 
     However, the other handling stations  61 ,  62  and  63 , which have been placed between the sample cup handling stations, can also be seen in FIG.  6 . These handling stations are the washing head washing station  61 , the reference well  62  and the opening  63  for the probe. They are meant to be used as necessary between the handling phases described above, as shown in the next figure. 
     It can be seen in FIG. 7 that the handling drum  50  has been turned by an angle of /2, i.e. by 22.5°, in which case the washing head washing station  61  is at the washing station  51 , the reference well  62  is at the measuring head  54  of the measuring station  53  and the opening  63  is at the probe of the discharge station  60 . This intermediate position of the handling drum  50  can be used at any time to wash the washing heads of the washing station  51  or to verify the reference standard. The opening  63  is included in the embodiment presented in this example because the downward movement of the probe of the discharge station  60  occurs simultaneously with the movement of the measuring head  54  towards the sample cup. 
     It is essential for the measuring device  20  presented above that the cassette drum  30  containing sample cups, the incubator drum  40  provided with a motion, and the sample handling drum  50  can each be rotated separately in either direction. This means that each of these drums  30 ,  40  and  50  can operate independently and each perform their own task irrespective of one another. This enables the first samples to be measured and the next samples to be incubated simultaneously, for example. However, when the sample cup  32  moves from one drum to another, the movement of these drums must naturally be co-ordinated and stopped for a while in a position where the sample cup can be moved. 
     In the embodiment of the measuring device  20  presented above the members used to transfer and handle the sample cups  32  are drums  30 ,  40  and  50 . These members can, however, be made so that they also move in any direction in the xy-coordinate system i.e. in the xy-plane. In this way, too, the movements of the sample cup handling members can be co-ordinated so that the sample cup can move from the cassette store to the incubator and from there further to the handling member and to the measuring station. In this case, too, it is essential that the handling members can operate independently and carry out different operations simultaneously. 
     The measuring device  20  relating to the invention can be made to be completely computer-controlled and thus automatic. The user of the device only needs to place a sample receptacle containing a sample in the sample station  22 . In this example the sample is in a test tube that is brought to the test tube station  22 , but the sample may also be in a different receptacle. The control devices of the measuring device  20  are not presented in the drawings. The measuring device  20  can also be made to be independent and continuously operating in such a way that a sample feeding device is connected to it, which device feeds test tubes containing samples or other sample receptacles to the sample station  22  of the measuring device. 
     It is obvious to a person skilled in the art that the different embodiments of the invention may vary within the scope of the claims presented below.