|Publication number||US3923463 A|
|Publication date||Dec 2, 1975|
|Filing date||Oct 9, 1973|
|Priority date||Oct 9, 1972|
|Also published as||DE2349901A1, DE2349901B2, DE2349901C3, USRE30627|
|Publication number||US 3923463 A, US 3923463A, US-A-3923463, US3923463 A, US3923463A|
|Inventors||Bagshawe Kenneth Dawson, Kemble James Edward|
|Original Assignee||Bagshawe Kenneth Dawson, Kemble J E|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (9), Referenced by (28), Classifications (6)|
|External Links: USPTO, USPTO Assignment, Espacenet|
United States Patent Bagshawe et al. I 51 Dec. 2, 1975 APPARATUS FOR PERFORMING 3.607090 9/1971 Maxon 23/259 x CHEMICAL AND BIOLOGICAL ANALYSIS 3,620,678 ll/l97l Gulgan 1 .1 23/259 X 3,749,916 7/1973 Thomas ct al. 250/328 X lm'vnwrsi Kenneth Dawson Bagshawe, 115 3.784,826 1/1974 Bagshawe et 111. 1 250 322;
George St., Marble Arch, London WC2; James Edward Kembl 80 Primary Examiner-Robert M. Reese P rk R t Burg s Hill, SUSSQX, Attorney, Agent, or FirmR0se & Edell both of England 221 Filed: Oct. 9, 1973  ABSTRACT A system for the automated analysis of large numbers ] App! 9101404320 of liquid samples, in which a multiplicity of sample tubes are loaded in racks into a cassette and the 30 Foreign Application p i Data loaded cassette is transferred from station to station, Oct 9 972 United Kingdom 4652M with operations of sample insertion. dilution, reagent addition and withdrawal for filtering being performed 52 U.S. c1. 23/253 R; 250/328 2 51 1m. 01. G01N 31/00;G01T 1/00 Procesbmg apte to e  Field of Search 23/253 R, 230 R, 259; Semi each ,module mcludmg appflmus 250/328 for performing one of the abovementioned operations on cach individual sample tube when it is located at a  References Cited parctiiclularl opterationalbloczl tionhitgt thetfizassetlie. Eafih mo ueaso asmem ers ors 1 mg erac sin e UNITED STATES PATENTS cassette in such manner that all tubes pass through the g z t i operational location in turn while strictly maintaining aruc e a h 3,487,862 1/1970 Soderblom 1 23/259 x Order of Sequence throughout the Open 3,525,591 8/1970 Jungner ct alt, 23/253 R 3,544,272 12/1970 Vaills 23/259 X 9 Claims, 4 Drawing Figures grip/e merf/bn Sheet 1 of 3 US. Patent Dec. 2, 1975 mgg g EQQE SS gg t m Q US. Patent Dec. 2, 1975 Sheet 2 of3 3,923,463
U.S. Patent Dec. 2, 1975 Sheet 3 of 3 3,923,463
APPARATUS FOR PERFORMING CHEMICAL AND BIOLOGICAL ANALYSIS This invention relates to a system of analysis and automated apparatus for the techniques of competitive protein binding including radioimmuno assay and radiometric assay. These techniques are widely used to measure the concentration in liquids of substances hereinafter described as the ligand" which bind to specific antibodies or other macromolecules hereinafter described as specific antibody.
Ligand of the species to be measured, labelled with a radioisotope, is added to a reaction tube containing a quantity of the unknown sample liquid, before or after the addition of specific antibody. The labelled ligand and the ligand in the unknown sample compete for binding to the specific antibody. In similar tubes, in each assay, known concentrations of unlabelled ligand are included to provide reference standards. The amount of ligand is determined by separating antibody bound ligand from free ligand by centrifugation or filtration and by counting the amount of labelled ligand in the precipitate or liquid phase.
To perform such reactions accurately, it is necessary to dispense the sample and reagents accurately, to ensure complete mixing of reagents and diluents, to incubate all samples and reference standards for the same time at the same temperature and to separate antibody bound ligand from free ligand efficiently. Ifis necessary to present reaction tubes in orderly sequence at one or more locations for these operations, to carry these tubes from one location to another, to maintain their initial sequence and to occlude the open end of the tubes to prevent spillage and evaporation. Competitive protein binding assays have been described for several hundred substances and a laboratory may need to use many different assay protocols and to assay batches of very variable size.
Existing apparatus provides arrangements for dispensing samples and reagents into tubes located in carrier racks. Such racks readily get out of sequence and sample identification by manual methods is tedious and inconvenient. Means for diluting samples by constant ratios also exist but existing automatic devices do not allow for variation of ratio from sample to sample, nor do they readily perform serial dilutions as required for reference standards. The rate limiting stage in competitive protein binding assays is generally that of isotope counting which is performed conventionally by nucleonic counters with automatic sample changers. The use of computers has hitherto been limited to performing calculations on data output by nucleonic counters and this entails feeding in sample identification data in addition to the preparation of the initial work sheet.
A hitherto known system provides the means for individually adjusted dilution ratios and for serial dilutions in dilution containers and analysis performed in conveyor belts of incubation pots. This system, however, is an integrated operation which allows no interruption between sample input and data output so that variation of assay protocol, incubation time and counting time is severely restricted and this limits the rate of throughput of samples and applicability of the system.
According to the present invention, there is provided apparatus for analysing a plurality of liquid samples, each sample being in a container or tube, such that a linear series of such tubes forms a rigid or semi-rigid rack, and comprising a cassette within the shell of which a plurality of such racks are to be contained said cassette having apertures in its walls, through which means, operated by apparatus external to the cassette, actuate and advance the racks and tubes sequentially and stepwise past a fixed point and such that a cassette be freely removable from the operating apparatus without disturbing or removing the contained racks and tubes so as to be transportable to other locations, the initial sequence of tubes and racks being maintained throughout.
The present invention facilitates the analysis of samples in small or large batches, permits wide choice of volume and dilution ratio for each sample, choice of volume and sequence of addition of reagents and choice of assay protocol. Further, it minimises the time required for documentation and provides a high rate of sample throughput.
The equipment may include two teleprinters and six modules, five of which are, for convenience, arranged together to form the sample processing unit whilst the sixth module is described as the control unit.
As already specified, multiple tubes are located in a multiplicity of racks which in turn are located within a cassette. The cassettes can be transferred manually between different modules. These features permit all the tubes for one assay to be kept together, the initial sequence of tubes to be rigidly maintained and the simultaneous processing of different assays employing wide variation in analytical protocol.
Another novel feature in the preferred embodiment of the invention is the control unit which is pro grammed to control the operations of the modules. This unit also counts simultaneously the radioactivity from a multiplicity of reactions and is programmed to compute the results of assays from these counts and to present the data in any desired form at the appropriate terminal. This feature greatly increases the throughput of the assay system whilst reducing the number of controls on the processing units. It further avoids the use of a plurality of nucleonic radioactivity counters such as ratemeters or sealers, reduces the opportunity for human error and minimises documentation.
An apparatus and system according to the invention will now be described in some detail by way of example and with reference to the accompanying drawings, in which:
FIG. 1 is a block diagram of the overall system,
FIG. 2 is a pictorial diagram showing detail of a sample processing unit of the system of FIG. I,
FIG. 3 is a detail of filter tape employed in the system, and
FIG. 4 is a diagrammatic pictorial view of a cassette holding reaction tubes.
Referring firstly to FIG. 1, the control unit 1 incorporates a small on-line computer 2 with electronic data registers 3 and interface unit 4, radioactivity counting locations 9 defined between two rows of photomultiplier units 5 arranged in pairs and connected to corresponding discriminator units 6 that feed signals to the interface, and means for advancing tape 7 bearing radioactive locations from a supply spool 8 to the counting locations 9 and thence to a take up spool 10.
The control unit is programmed by three classes of data. Class I data pertain to the instruction and operational language of the computer unit and identification of storage locations in its data register and these are normally stored permanently within the data register.
3 Class II data pertain to the analytical procedure and protocol for a specific assay and are normally input at the beginning of any assay operation. Class III data pertain to individual samples within an assay and are input by the operator during the first stage of each assay.
Data are input via any suitable terminal but a teleprinter 51 with paper tape punch and reader is the preferred form. Data are stored in two ways. Data controlling functions, such as dispensing, transferring and dilution by the sample processing unit 52, are retained in the electronic data register 3 of the control unit 1. These data, together with all other data relating to an assay, are stored as a punched tape which is prepared during the course of the sample input. The paper tape is retained for use at a later stage in the analysis. In addition, all Class II and III data are typed out by the input teleprinter 51 using a simple conversational language to provide the work record. No further records or data input are required of the operator. The remaining functions of the control unit will be described after the cassette and sample processing modules.
Referring now to FIG. 4, showing a cassette 14 containing reaction tubes for samples to be processed, part of the technique to be described is based on a principle of moving racks 11, holding tubes 12 at uniform pitch, stepwise past a fixed operation point 34. The racks are of uniform length and are arrayed within the shell or cassette 14 in two bands 11A, 1118. Each bank consists of a plurality of racks 1 I placed side by side along their long sides. The racks in the two banks are more or less end to end but are so staggered that the front rack of one bank and the rear rack of the other bank are free to move endwise and thus be moved from one bank to the other. Such a movement is carried out stepwise by levers and pushrods in the base module 15 (FIG. 2) which receives the cassette 14 and these, operating through apertures 16 in the walls of the cassette, move each rack in turn past the operation location 34. On completion of the endwise displacement of two racks, each bank of racks 11A, 11B is displaced as a whole at right angles to the stepwise movement by an amount equal to the thickness of a rack so that the original staggered disposition of the banks is regained and the next racks in the sequence are aligned for endwise movement.
At the end of a sequence of operations, the racks can be advanced by the levers and pushrods to the original starting position, the first reaction tube of the first rack in any cassette being identified by a suitable marker; in the preferred form, a magnet location in the rack beneath the first tube provides a signal to a fixed sensor located in the base module. Outward displacement of the banks of racks is prevented by side walls 17 and the two banks are separated by a shallow central partition 18. Lips 19 on the racks engage under corresponding lips 19A on the side walls and central partition to prevent upward displacement of the racks. The levers and pushrods which move the racks are linked mechanically to switches in the module base which thus sense the position of the racks. In this way, the precise sequence and location of tubes and racks is maintained and made known to the control unit during operational procedures.
In order to transfer the reaction tubes 12 to another base module, it is simply necessary to lift the cassette 14 from one module and engage it in the corresponding location of another. Various sizes of reaction tube can be accommodated within a rack. Disposable moulded 4 plastics racks with integral containers may be used or racks may consist of a permanent shoe and a disposable" multitube component. In one typical cassette arrangement, a total of 420 reaction tubes of 3 ml volume are accommodated with 15 tubes in each of 28 racks.
When it is necessary to seal the tubes, the cassette 14 is placed in a closed box or a lid 21 is fitted to occlude the open ends of all the tubes 12. The lid may consist of a flexible sheet which is pulled taut over the tubes but in the preferred form, a layer of foam rubber 22 is sandwiched between a rigid top sheet 23 and a smooth plastic under lining 24. In this way, effective occlusion of all the tubes can be obtained at a single action and the manual sealing and unsealing of many tubes by individual stoppers is obviated.
The sample processing unit (FIG. 2), consists of (1) sample insertion module 25, (2) dilution module 26, (3) reagent addition module 27, (4) transfer module 28 and (5) filtration module 29.
The insertion module 25 has the following features. A location in the base module 15 for the reaction tube cassette 14, which location provides the means to move the levers and pushrods and switches which advance the tubes in the cassette to and from the operational location. It incorporates a probe unit 30, pump 31 and wash facilities 32 for the transfer of liquid samples 33 to the reaction tube 12 at the operational location 34 without carry-over of solution from one sample to another. The probe carried on a suitable arm and connected by flexible plastic tubing to the pump unit 31 descends into a sample tube 33 at the sample location. The pump withdraws a quantity of the sample in excess of that required for the reaction. The probe is then elevated and rotated through an arc to a wash and wipe location 32 where the probe descends. In this position, two claws faced by a tape 35 of absorbent paper close on the probe. Drops of liquid left on the outside of the probe are thus removed by the absorbent paper when the probe is raised. After elevation of the probe, the claws open and the paper is advanced. The probe then swings through a further arc to the operational location 34 where it descends into the reaction tube 12, the pump discharges the required volume and the probe is lifted and taken back to the wash location where the probe is washed internally and externally by the action of another pump supplying wash fluid and again the probe is wiped on the outside before returning to the sample input location.
The volume of sample dispensed into each reaction tube 12 is determined by the analytical protocol. The same volume may be dispensed into all tubes or varied according to the requirements for dilution. In the preferred form, the pump takes up or discharges a unit volume at each stroke and the control unit controls the number of strokes in each take up and dispensing operation according to assay protocol.
Operation of the sample insertion module 25 is effected by the operator ensuring that Class I and Class II data have been input to the control unit and Class III data pertaining to each sample are typed on the input teleprinter with the sample tube 23 in the sample location. Typing in the command signal initiates the insertion module-sequence.
When a complete batch of reference standards and samples have been dispensed, the racks 11 are returned to their initial sequence and the cassette 14 is transferred manually to the dilution module 26, as indicated ent to the sample and other pump or pumps remove similar amounts through a multi-channel probe. Thus a series of dilution steps may be performed. Between each step, the sampleand diluent are mixed by the operation of an additional pump with a reciprocating action operating a plunger in one channel of the probe. The volume dispensed or taken up by each pump action may be adjustable over a wide range but, in the preferred form, one or more fixed volumes are dispensed or taken up at each stroke and repetitive the peripheries of which seal on the under surfaces of the margins of the filter locations 36 and a cavity within the pressure plate communicates with a vacuum source. Elevation of the pressure plate and activation of the vacuumsource causes a negative pressure to be transmitted across the filter membrane and for the contents of the reaction tubes 12 to be drawn through the flexible tubing to the filters where the precipitates are strokes are used to give any multiple of these fixed volumes. The operation of the dispensing and takeup pumps is controlled by the control unit 1 according to Class II and III data. Once the dilution sequence has been initiated, no further intervention is required by the operator.
On completion of dilution and return of the reaction tubes 12 to the start position, the cassette 14 is transferred to a corresponding location on the reagent dispensing module 27. As the reaction tubes step to the operational location, one or more reagents are dispensed in the precisely required volume by a probe and pump unit of this module. Pump operations may be controlled by settings on the module itself or by programming the control unit. On completion of dispensing and mixing, the occlusive lid 21 is applied to the batch of tubes and the cassette incubated at the desired temperature for the period required. The reagent dispensing module 27 also incorporates a peristaltic pump so that when required a suspension of charcoal or precipitating agent can be dispensed from an agitated solution into the reaction tubes.
On completion of incubation, the cassette 14 is placed on the transfer module 28 from whence the contents of the reaction tubes 12 are transferred to filter locations 36 on the filtration module 29, the action of the two modules being closely integrated. This transfer is effected in the preferred form from five reaction tubes at a time but any convenient number may be used. Five probes descend into five adjacent tubes. The probes are connected by flexible tubes to five corresponding hemispherical domes rigidly mounted on the filtration unit 37. Each probe contains a second channel through which wash solution is pumped from a supply bottle.
Filtration is performed at the locations 36 through a cellulose acetate, or glass fibre membranes, or filters of similar porosity, mounted at intervals over perforated segments of a flexible plastic carrier tape 7, and sealed to the tape around the margins of the filters. The carrier tape is further marked at fixed intervals by holes 41 (FIG. 3) or indentations so that its position may be precisely controlled by sensors fixed to the filtration module signalling to the control unit. The plastic tape 7 is supplied from a spool 38 and fed through a series of locations to a take up spool, the tape advancing stepwise by five locations at a time. At the first five locations 39 on the filtration module 29, the membranes are wetted with a protein solution. At the second five locations, the five hemispherical domes are sealed against the upper margin of each filter disc 36 by the elevation of a pressure plate 40 which bears on the lower surface of the filter tape 7. This plate also has five suction areas retained on the filters. Wash solution is pumped into the reaction tubes and this is also drawn through the filters. Wash solution is also pumped to aseries of outlets peripherally disposed at each filter location so as to provide uniform washing over the whole area of the filter membrane. In the preferred form, the filtrate proceeds to waste but counting of filtrate radioactivity is an alternative to counting precipitate radioactivity. On completion of filtration and washing, the pressure plate 40 is lowered and the probe assembly on the transfer module 28 elevated/Both tape 7 and reaction tubes 12 then advance five locations: At the next station 42 the filter tape 7 is dried by a fan heater and on emerging from this station, transparent'adhesive tape'is applied tothe'filter bearing surface of the carrier tape. After completion of one or more batches of samples, the tape is rewound to its initial sequence and is then transferred to the supply spool position 8 in the radioactivity detector station. I 3
Thus, in the manner just described, radioactive com-. ponents from a multiplicity of reactionsoccurring in said tubes are simultaneously transferred to a corresponding multiplicity of filters on the continuous tape. FIG. 1 shows a radioactivity detector station at which photo multiplier devices 5 convert the radioactivityat the multiplicity of filter sites into-signals which are accumulated and counted directly in electronic data registers 1. The accumulated totals associated with prior instructions are stored at other locations in the register assembly 1, and computer means 2 performs calculations and transfers the accumulated totals and results of the calculations to appropriate output terminal means denoted at 54.
At the radioactivity detector station, the tape 7 is transported by pinch rollers 53 with drive mechanisms and is thus fed through five counting locations 9. Movement of the carrier type is controlled as on the filtration unit and the counting time is determined by the assay protocol. At each of the five counting locations, there is an opposed pair of photomultiplier tubes 5, the output from which passes via a discriminator 6 and distribution unit to the electronic data register 3 for counting.
Since the counting efficiency of photomultiplier tubes is variable, a tape bearing radioactive filter locations is first advanced one step at a time through the five count locations 9. The relative efficiency of each pair of tubes 5 is thus determined and a correction factor is then applied automatically to the counts received from each location.
When the carrier tape 7 for an assay batch is placed in the detector station of the control unit, the corresponding paper tape is fed into the tape reader on an output teleprinter 54. As radioactivity counting proceeds, the totals for each counting location are associated with the corresponding data on the punch tape. The reference standard line is then computed according to the programme and the concentrations of ligand in the samples are determined and statistical analyses are performed according to standard analytical proce- 7 dure. The assay data are output to the teleprinter or al ternative terminal.
1. Apparatus for analysing a plurality of liquid samples, each sample being in a tube, and comprising a cassette having a shell in which a plurality of such tubes are to be contained in sequence. said cassette having apertures in its walls through which means, operated by apparatus external to the cassette, actuate and advance the tubes sequentially and stepwise past a fixed point, said cassette being freely removable from the operating apparatus, without disturbing the contained tubes, to other locations, the initial sequence of tubes being maintained throughout.
2. Apparatus according to claim 1, wherein the eassette is provided with a lid which occludes the open ends of all the tubes contained in the cassette.
3. Apparatus according to claim 1, wherein the cassette carries racks of moulded plastics in which the tubes are an integral part of the rack.
4. Apparatus according to claim 1 wherein said tubes are arrayed in linear series in substantially rigid racks to be received within the cassette shell, and said actuating means advances the racks with the tubes therein sequentially past said fixed point.
5. In combination with the apparatus of claim 1, liquid analysis apparatus comprising at least one module with means to carry out a physical operation on the liquid samples and with a location to receive a selected one of said cassettes at a time and means to actuate perative elements which advance the tubes inside said cassette sequentially past a fixed point.
6. Liquid analysis apparatus according to claim 5 wherein operations of dispensing, transferring and diluting liquid samples are performed by means of pumps actuated by signals received from a control unit which incorporates an electronic data register, according to a sequence of instructions supplied to the control unit by a human operator in at least one prior operation and which instructions may be varied according to the requirements of analytical protocol.
7. Apparatus according to claim 5, wherein liquid transfer and dispensing operations are performed by pumps with fixed stroke volume, multiples of this fixed volume being transferred by repetitive action of such pumps depending on signals from a control unit with a data register operating on prior instructions.
8. Liquid analysis apparatus according to claim 5, comprising means to transfer radioactive components from a multiplicity of reactions occurring in said tubes simultaneously to a corresponding multiplicity of filters on a continuous tape, photomultiplier devices to convert the radioactivity at a multiplicity of filter sites into signals which are accumulated and counted directly in electronic data registers, the accumulated totals associated with prior instructions being stored at other locations in the register, and means to perform calculations and to transfer the accumulated totals and results of the calculations to appropriate output terminals.
9. In combination with apparatus according to claim 8, a tape of strong flexible plastics material, bearing at appropriate intervals a series of locations where the tape is perforated and where the perforations are surmounted by filter discs adherent to the tape at the margins of the filter discs, said tape additionally having holes or indentations to aid in the correction registration of the tape at operational locations.
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|U.S. Classification||422/66, 250/328|
|International Classification||G01N33/483, G01N35/02|