WO1989000698A1

WO1989000698A1 - Continuous flow analysis

Info

Publication number: WO1989000698A1
Application number: PCT/GB1987/000519
Authority: WO
Inventors: Theodore Robert Wills; Donald Norman Rossington
Original assignee: Wessex Instrumentation Ltd.
Priority date: 1985-11-08
Filing date: 1987-07-17
Publication date: 1989-01-26

Abstract

In continuous flow equipment for blood group analysis, air bubbles are introduced into the liquid flow at fittings (8, 33, 35) under control of an air valve (10). Individual samples drawn in via probes 4p, 4rc are separated by periods of bubbled flow passing on lines 231-10 to reaction manifolds 31-10. Periodically the air valve (10) is controlled to produce a recognisable code of air bubbles, thereby identifying adjacent samples in the flow. Downstream of the reaction manifolds 31-10 the code is read at the reaction detection stations (30).

Description

CONTINUOUS FLOW ANALYSIS

The present invention relates to continuous liquid flow analysis in which liquid samples to be analysed are periodi- cally introduced into an upstream region of a continuous liquid flow and are tested in a downstream region of the flow.

As used in, for instance, blood group analysis where blood samples are introduced into a saline flow and reacted with various reagents also introduced into the flow, it is a feature of the analysis that by the time a particular blood sample has passed along the flow path of the analysis equipment to the position in which result of the reactions can be determined, a considerable number - typically 20 - of further samples will have been taken and introduced into the upstream region of the flow path. Thus it is essential to be able to precisely correlate the determined result with the correct sample taken. This correlation can be difficult when the analysis equipment includes a plurality of parallel channels each having slightly different transit times for the samples from introduction to result determination.

Traditionally correlation of results with samples, or phasing as it has been called, has been carried out by periodically introducing a so-called phasing sample amongst the normal samples being analysed. The analysis of a phasing sample is known, thus when the determined result of the reactions corresponds at the results-determination- position with that expected of a phasing sample one can be reasonably confident that it is the phasing sample which has reached this position. Slight discrepancies due to different transit times in the different channels can thus be moni¬ tored and compensated for. However, the use of phasing samples is not entirely satisfactory and it is an object of this invention to enable the avoidance of their use. It is well known to introduce air bubbles or drops of non-misσible liquid into the liquid flow because these bubbles or drops have a scavenging effect on the internal walls of the pipes forming the flow path through the equip¬ ment. The present invention uses such bubbles or drops to codefy the flow whereby the samples can be positively identified without the use of phasing samples.

According to one aspect of the invention there is provided a method of identifying samples in continuous liquid flow analysis consisting in the steps of injecting gas or non-miscible liquid in a recognisable code into an upstream region of the liquid flow in association with at least one sample and reading the bubble code at a downstream region of the liquid flow. According to another aspect of the invention there is provided continuous liquid flow analysis equipment including means for injecting gas or non-miscible liquid in a recog¬ nisable code into an upstream region of a liquid flow path through the equipment and means for reading the bubble code at a downstream region of the liquid flow path.

Preferably gas is injected with the code embodied in the length of some of the bubbles.

The analysis of samples in continuous flow equipment normally involves the processing of information from ten or more channels in the equipment, an operation for which an appropriately programmed micro-computer is particularly apt. Accordingly, the upstream injection of bubbles in a recog¬ nisable code and their downstream reading is preferably under control of the computer. Nevertheless it is envisaged that the generation of the code and its identification may be carried out by dedicated devices.

Preferably the means for reading the code includes a detector able to identify the occurrence (or not) of the reactions in the analysis channels. Preferably means is provided for synchronizing the bubble code - and a normally provided non-coded bubble injection between intermittent codes - with passage of the bubbles through a peristaltic pump of the equipment.

Further, means is preferably provided for ensuring that both a red-cells sample and a plasma sample taken simul¬ taneously into respective channels are synchronously identi¬ fied by the same bubble code.

To help understanding of the invention, a specific embodiment thereof will now be described with reference to the accompanying drawings in whichs-

Figure 1 is a front view of continuous liquid flow analysis equipment;

Figure 2 is a schematic view of a plasma sampling circuit in the apparatus of Figure 1; Figure 3 is a front view on a larger scale than Figure

1 of one reaction manifold of the apparatus;

Figure 4 is a schematic view of a red-cells sampling circuit in the apparatus of Figure 1;

Figure 5 is a diagrammatic view of a normal air bubble pattern passing through a peristaltic pump of the apparatus of Figure 1; . and

Figure 6 is a diagrammatic view of a coded air bubble pattern approaching a detection station in a manifold in one reaction channel of the apparatus of Figure 1. The equipment shown in the drawings is a ten channel blood group analyser 1, in which blood samples are drawn in the form of a plasma sample and a red-cells sample from one

2 of a series of sample test tubes and each sample is split into five for passage to five separate channels, including reaction manifolds 3,...3_ιn.

The samples are drawn from the test tube 2 via a pair of probes 4_p and 4_rc. The plasma probe 4_p extends into the test tube 2 only so far as to reach the plasma layer 5 whilst the red-cells probe 4 extends through the plasma layer into the red-cells layer 5 . The samples are drawn up into the probes by tubing leading to a peristaltic pump 6. The pump incorporates a considerable number of lines 7. These lines are of tubing having varying internal cross- sectional areas whereby differing flow rates can be achieved in differing lines, as is conventional.

The plasma samples and the red-cells samples pass along similar tubing circuits, although there are differences in the circuits as will be described. Figure 2 shows plasma sampling,, Saline solution is pumped in a 3«4 ml/min line 7, to an air injection fitting 8. Air to be injected in accordance with the invention is supplied in line 9 at 22" water gauge under control of a Reedex switch 10 to the fitting 8. The resulting bubble containing saline solution is passed on line 10 to a sample valve 11. This valve comprises a movable bar 12 and a pair of abutments 13 ,

13 _β The line 10 divides into two, one line 10 passing between the bar 12 and the abutment 10 . whilst the other pr^F line 10ws p ^easses between the bar 12 and the abutment 13ws

Usually the bar 12 will be spring biassed towards the abutment 13 WS to close the line 10W5, whereby the bubbled saline solution passes through line 10 . When a sample is taken an air cylinder, not shown, is actuated to move the bar 12 towards line 10 and close it, thereby diverting the saline flow via line 10ws to waste. usually with the plasma probe 4 up it receives the bubbled saline solution at its T fitting 14. Part of the saline is pumped towards the analysis manifolds and part is^" passed down the probe to. scavenge it. At the bottom of the probe 4 - in its withdrawn position - is a wash fitting 15. This comprises a tube 16 which is open at both ends and surrounds the probe⁸s tip to the top of which tube is pumped saline solution on line 17. The bottom of the tube 16 is positioned just below .the bottom end of the probe in its withdrawn position. A line 18 withdraws laterally from the bottom of the tube 16 the wash saline line 17 and the bubbled saline passing down the probe and out at its bottom end. By pumping on the line 18 at a faster rate than the sum of the wash flow and the flow down the probe, no leakage from the bottom end of the tube 16 occurs. When a sampr-le is to be taken, ^ι the pvrobes 4p,'4re are moved smartly down into the test tube 2 by an air cylinder 19 at the same time as the bar 12 in the sample valve 11 is moved. As a result the supply of bubbled saline to the plasma probe 4 on line 10 is stopped; and a plasma sample is drawn up into the probe, typically for five seconds, and pumped towards the analysis manifolds. The sample, and the following bubbled saline when the probe 4 is withdrawn and the bar 12 returned, passes through a delay coil 20, corresponding in length to the bromelin coil for red-cells as described below. From the delay coil, the sample/saline is passed on line 21 to a five way splitter 22 to five 0.32 ml/min lines 23 1, ...23D_ and to a 1.6 ml/min line

24 passing excess to waste. The lines 23l, ...23o_ are some of the peristaltic pump lines 7 and they pass their sample/ saline flow to respective reaction manifolds 3 ...3_.

1 5

At the inlets to these manifolds respective reagents, known blood group red-cells with rouleaux enhancing agents in the case of the plasma channels, are added on others of the lines 7. These reagents are pumped from cooled and agitated storage bottles 25. In the manifolds, the sample/ saline flow passes through reaction coils 26 to saline dilution inlets 27. Here diluting saline solution is pumped in to break up the rouleaux. After passing through further mixing and settling coils 28,29, the sample/saline flows are passed to detection stations 30 where the flow is between respective infra red emitters 30 and receivers 30 , shown by way of example in Figure 6_β The occurrence of agglutination reactions is determined in individual channels by measurement of radiation received by the channel"s detector from its emitter. . Agglutination, in clumping the red cells together, permits more radiation to be received due to the reduction in the concentration of the freely suspended red cells. Air bubbles totally occlude the radiation and can thus be detected. From the detection stations the flows pass to waste.

The red-cells channels are similar to the plasma channels as regards their manifolds 3_g...3 _ and receive rouleaux enhancing anti-rhesus and ABO anti-sera reagents from storage bottles 31. However the sampling arrangements are different as shown in Figure 3. A pumped bromelin solution on line 32 at 1.6 ml/min is added to the red cells to activate them. This solution has air bubbles added at fitting 33. To control flow of red-cells samples taken by the probe 4Tc in the same manner as the plasma samples, the red-cells/saline flow from the probe 4 must be pumped at 2.9 ml/min within the length of line 34. This interferes with the pattern of air bubbles added at fitting 35 due to the large size of the line 34 so that the flow in line 34 must be de-bubbled at fitting 36, from which part of the red-eelIs/saline flow is passed to waste. The required flow passes to a mixing fitting 37 which is preferably incorporated in the fitting 36. Thence the flow passes to a bromelin reaction coil 38. The delay coil 20 has a length to match this coil 38 whereby the plasma and the red- cells samples taken at the same time are passed through the reaction manifolds more or less in step with each other.

From the bromelin coil the flow is split at a splitter 39 similar to the five way splitter 22 and thence to the pump and the reaction manifolds 3 6_...31_4n0. The analysis equipment is under control of a micro computer 40 which controls the Reedex air switch for injecting the air bubbles at fittings 8, 33 and 35 and which receives information from the detection station detectors.

In the preferred arrangement the computer is programmed to provide that the Reedex switch is operated to provide a regular injection of air for, say, 0.4 seconds every 2 seconds. In order to minimise the possibility of the bubbles in the lines 7 passing through the peristaltic pump being broken by the rollers 41 of the pump, bubbles are preferably arranged to pass through the pump in such position in the lines that they are equally spaced either side of the rollers, preferably one bubble passing through the pump between each successive pair of pump rollers. The bubbles in the individual lines can.be brought into phase with each other by lengthening or shortening individual ones of the pump lines 7. In order to synchronize the bubbles in all the channels to the pump rollers, once they are mutually in synchronism, the time of injection of the air bubbles by the Reedex switch can be advanced or retarded by the computer. An optical detector 42 is provided on the pump to detect the passage of rollers past a specific point. Conveniently this is in the form of a slotted disc 43 having a slot 44 for each roller. An infra red emitter and detector pair detects when light can pass from the emitter to the detector in the presence of a slot. This triggers the Reedex switch via the computer. A suitable variable delay from the time of slot detection to triggering of the switch tunes the positioning of the bubbles between the rollers.

One or more sample(s) drawn from their test tube(s) is/are labelled in their passage through the analysis equipment by coding the air bubble pattern immediately prior to or immediately following the samples. Conveniently this is effected by modifying the air bubble pattern into a readily recognisable code. In the preferred embodiment the code is achieved by providing an air bubble injected for 0.2 sec. in place of a normal 0.4 sec. bubble and then a 0.8 sec. bubble instead of the next 0.4 sec. bubble, with this pattern repeated for- three cycles, i.e. for twelve seconds. This code when placed equally between two five second samples taken thirty seconds apart remains between the samples even although they can spread slightly by dilution. The code is readily detected at the detection stations 30 by the computer recognising the code. The presence of an air bubble in one of the channels occludes transmission from the channel's infra red emitter 30 to the receiver 30 , whereby the receiver output is modulated by the code. Thus the satis¬ factory operation of each channel can be monitored by the arrival of the code at the detector stations.

On start-up of the equipment, once the flow pattern has settled down, an initial bubble code is generated and then the first sample is passed through the equipment. Because of individual differences between the channels, the code will arrive at the channels' detection stations slightly spread out in time. Thereafter the samples should arrive at the stations at regular 30 second intervals provided that the samples are taken at such intervals. After say thirty minutes - 60 samples - the code is regenerated and should arrive at the individual stations thirty minutes after the code arrived at each for the first time. Small tolerances are permitted but, should the code not arrive when expected _r the channel is assumed to be at fault and the operator of the equipment is alerted by the software and the results obtained are identified as being unreliable.

It is important that a plasma sample and a red-cells sample taken together should both be identified by the code if either is. For this it is important that the air bubble code reinserted with the bromelin on line 32 at fitting 33 in the red-cells line should have arrived at the mixing fitting 37 at the same time as the code reaches the point downstream of the plasma probe 4 by the same amount. To provide this, the line 32 will be of a length to give a delay of say one minute, namely the time taken on the plasma line for the code to pass from the air bubble fitting 8 to the point equivalent to mixing fitting 37. The invention is not restricted to the details of the above described embodiment. For instance each sample may be identified by being preceded or succeeded by a unique air bubble code. This would require a code having more bits than the three cycles of dot-dash code for an appreciable number of samples and hence the delay between samples may be increased.

Claims

CLAI S

1. A method of identifying samples in continuous liquid flow analysis consisting in the steps of injecting gas or non-miscible liquid in a recognisable code into an upstream region of the liquid flow in association with at least one sample, reading the bubble code at a downstream region of the liquid flow and identifying the sample in accordance with its sequential relationship to the bubble code.

2. A method of identifying samples as claimed in claim 1, wherein the bubble code labels a particular sample immediately preceding or succeeding it, whereby this sample is identifiable, and other samples are taken into the liquid flow at regular intervals after the labelled sample, whereby they can be identified by reference to their time of arrival at the downstream region after the labelled sample.

3. A method of identifying samples as claimed in claim 2, wherein samples are labelled successively with a predetermined number of non-labelled samples being taken between the labelled samples.

4₀ A method of identifying samples as claimed in claim 3, wherein the gas or non-miscible liquid is injected into the upstream region of the liquid flow between the non- labelled samples in non-coded form.

5. A method of identifying samples as claimed in claim 1, wherein each sample in the liquid flow is identified by the bubble code preceding or succeeding it in the liquid flow. 6. A method of identifying samples as claimed in any preceding claim, wherein the bubble code is effected by varying the size of the bubbles whilst keeping their injection frequency constant.

7. A method of identifying samples as claimed in any one of claims 1 to 5, wherein the bubble code is effected by varying the injection frequency of the bubbles whilst keeping their size constant.

8. Continuous flow analysis equipment, including means for injecting gas or non-miscible liquid in a recognisable . code into an upstream region of a liquid flow path through the equipment and means for reading the bubble code at a downstream region of the liquid flow path.

9. Continuous flow analysis equipment as claimed in claim 8, wherein gas is injected into the liquid flow path and the injection means comprises an electro-mechanical gas switch and a computer for controlling operation of the switch whereby the injected gas is codified as required.

10. Continuous flow analysis equipment as claimed in claim 8 or claim 9, wherein the bubble-code reading means is provided by analysis detection means.

11. Continuous flow analysis equipment as claimed in claim 10, wherein the analysis detection means comprises an infra-red emitter and an infra-red receiver arranged on opposite sides of the flow path at the downstream region of the liquid flow path, the presence of a bubble between the emitter and the receiver being detectable by the virtually total occlusion of emitted radiation reaching the receiver.

12. Continuous flow analysis equipment as claimed in any one of claims 8 to 11, wherein the equipment includes a plurality of channels between which the sample is divided and a peristaltic pump for pumping the channels, each channel being of adjustable length upstream of the pump whereby bubbles of the code in each channel pass in synchronism through the pump between its rollers. 13. Continuous flow analysis equipment as claimed in claim 12, including means for synchronising bubble injection with the rollers of the pump whereby the bubbles in all channels can be synchronised together to pass through the pump between its rollers. 14. Continuous flow analysis equipment as claimed in claim 13, wherein the means for synchronising bubble injection comprises a detector for detecting passage of the pump rollers past a specific point and a variable delay for timing bubble injection. 15. Continuous flow analysis equipment as claimed in any one of claims 8 to 14, wherein the analysis equipment is adapted to analyse blood samples and has both plasma and red-cells sampling circuits, the red-cells sampling circuit including means for bromelin injection to the samples and a bromelin reaction coil and the plasma sampling circuit including a delay coil of a length corresponding to the bromelin coil whereby the red-cells samples and the plasma samples are synchronised.

1 _β Continuous flow analysis equipment as claimed in claim 15, wherein the bubbles are gas bubbles and the equipment includes sampling probes downstream of bubble injection points in saline flow to the probes, means for lowering the probes together into a blood sample and means for interrupting bubbled saline flow to the probes when the probes are lowered.

17. Continuous flow analysis equipment as claimed in claim 16, wherein a wash fitting is provided for the bottom of each probe when raised, the wash fitting comprising a tube surrounding the bottom of the probe and having a radial wash saline inlet at its upper end and a radial saline withdrawal outlet at its lower end, the arrangement being such that pumping from the outlet at at least the rate of flow of wash saline to the inlet and saline down the probe avoids free flow from the probe. 18. Continuous flow analysis equipment as claimed in claim 16 or claim 17, including means in the red-cells sampling circuit for removing bubbles from the saline flow from the red-cells probe, a fitting for injecting the gas bubbles - suitably coded - to the bromelin flow upstream of its mixture point with the saline/sample flow from the red- cells probe and a delay between the air injection fitting and this mixture point for synchronising the reintroduced bubble code with the bubble code in the plasma sampling circuit.