GB1598129A - Method of and apparatus for automatically determining blood group characteristics and antibodies directed against blood group characteristics - Google Patents

Description

(54) METHOD OF AND APPARATUS FOR AUTOMATICALLY DETERMINING BLOOD GROUP CHARACTERISTICS AND ANTIBODIES DIRECTED AGAINST BLOOD GROUP CHARACTERISTICS (71) We, BLUTSPENDEDIENST DER LAN- DESVERBANDE DES DEUTSCHEN ROTEN KREUZES NIEDERSACHSEN OLDENBURG UND BREMEN GGmbH, a non-profit making corporate body under the laws of the Federal Republic of Germany, of Eldagsener Str. 38, 3257 Springe 1 Hannover, Federal Republic of Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:: This invention relates to a method of and an apparatus for photometrically determining blood group characteristics and specificities of antibodies harmful to blood, using an analyser operating on the principle of continuous flow analysis and capable of being connected to electronic data processing equipment in the on-line and off-line method.

The conventional manual blood group serological investigation methods involve preparations such as the pipetting of test materials. serum separation, cell washing, etc.-and the carrying out of the testsHIif- ferent test preparations on plates and in reaction tubes in a NaC1-, enzyme-, or high protein medium and in the indirect Coombs test in various temperature ranges (4"C, 20"C, 37C) with differing incubation periods ranging from 20 to 45 minutes. These preparations are in the first place time consuming and in the second place expensive due to the use of undiluted, expensive test serums.Also, especially in large series of analyses such as occur in large blood donor centres, they are accompanied by the risk of error, for example due to the possibility of confusion of the preparations when preparing specimens and the possibility of error in transfer in preparing documentation and records. Such errors can result in serious consequences in regard to health in blood transfusions, and indeed in certain circumstances, even in death.

From this it is evident that the conventional manual investigation methods, on account of the stated disadvantages, are suitable only to a limited extent for carrying out blood group serological investigations in large numbers and in a relatively short time.

This is true especially when, for instance, in the case of a catastrophe, blood groups need to be determined rapidly for a very large number of persons or when, as is necessary in regional blood donor centres, a large number of tested blood preserved stocks must be maintained economically for use by hospitals.

It is an object of the present invention to provide an improved method of and apparatus for automatically determining blood group characteristics and specificities of antibodies harmful to blood whereby the disadvantages referred to above shall be materially reduced.

According to the present invention there is provided a method of determining blood group characteristics and specificities of antibodies harmful to blood using an analyser having at least one channel operating on the principle of continuous flow analysis and capable of being connected to an electronic data processor, including the steps of sequentially taking samples from blood quantities to be investigated, by means of a sampler, cycling the sampler from a sampling location to a washing location, washing the sampler at the washing location, and returning it to the sampling location after the taking of each sample. controlling the time of each cycle of the sampler, generating a signal representative of the termination or commencement of a sampler cycle, photometrically analysing each sample taken and generating a record of the sample analysis, the record being in the form of an analogue curve, and comparing at least part of the analogue curve with at least part of a reference curve to selectively permit or not permit an electronic identification of the sample record with said generated signal according to the result of the comparison.

According to another aspect of the invention there is provided an apparatus for determining blood group characteristics and specificities of antibodies harmful to blood comprising means operable to supply blood quantities to a sampling location, a sampler operable to take a sample from the blood quantity at the sampling location, means for cyclically displacing the sampler from the sampling location to a washing location and returning the sampler to the sampling location, means for introducing wash liquid into the sampler at the washing location, photometric means for photometrically analysing each sample taken, timing means for control ling the displacing means and the time of each cycle of the sampler3 means responsive to the end or beginning of each cycle of the displacing means to generate an electric signal operable to identify the sample. means responsive to the photometric means to generate a record of the analysis of each sample, the record being in the form of an analogue curve and means for comparing at least part of the analogue curve with at least part of a reference curve to selectively permit or not permit an electronic identification of the sample record with said generated signal according to the result of the comparison.

In a method embodying the invention a specimen of each blood quantity to be investigated is taken for analysis by a sampler controlled by freely adjustable time switches, whereby the working speed of the analyser is determined by the time switches of the sample and thus also the automatically operating electronic results evaluation coupled thereto is regulated.

The electronic evaluation of the analysis, which preferably is carried out in multichannel, is connected to a photometer and picks up the analogue curve pattern of the analysis, digitalisation of the values of the peak points of the curve being carried out.

In this manner, the evaluation of the specimens is synchronised with the sampler, so that the correct result is associated with each specimen.

It is advantageous here for the automatic results evaluation to be triggered by a synchronisation adaptable to the measuring problem, which synchronisation permits simultaneous recording of the curve peaks of all the connected-in analysis channels. The recording is carried out here of the curve peaks in the framework of an electronically operating recording window, resulting from the synchronisation and adjustable percentually to the time of the sampling cycle, with tracking of the analogue curve pattern by means of an automatic drift control of the curve peaks from the cycle time.

For the purpose of carrying out the method embodying the invention. a sampling device is used, which comprises a sampling table, which is equipped with an endless chain of specimens of a sampler which is freely adjustable in its working cycle. The sampler is coupled to a motorised valve, which makes possible rinsing of the sampling needles under various pressure conditions. The liquid samples are supplied, together with the liquid reagents. by means of multi-channel proportioning pumps to an analytical unit, which comprises a larger number of analysis channels, from which the samples are then conducted to a multichannel photometer, the results of which are read and recorded by an electronic evaluation component.

One embodiment of the invention will now be described by way of example, reference being made to the accompanying drawings in which: Fig. 1 is a block diagram illustrating the construction of the apparatus for carrying out the method of the invention Fig. 2 illustrates the sampling needle during rinsing.

Fig. 3 illustrates the sampling needle when obtaining a blood sample, Fig. 4 illustrates the preparation of the sample in diluted form, Fig. 5 illustrates the preparation of the sample without dilution.

Fig. 6 is a flow diagram of the analytical investigation of the sample in an analysis channel, Fig. 7 illustrates the path of the media during analysis through a line of an analytical unit, and Fig. 8 is a block schematic diagram illustrating the operation of the apparatus.

The apparatus illustrated in Fig. 1 has the form of a modular-constructed analysis device, operating methodically according to the principles of continuous flow analysis and capable of adaptation to electronic data processing, the blood group serological investigations being photometrically recorded.

The analysis device according to Fig. 1 comprises a sampling table 1, which can be supplied with an endless chain of samples, which are moved into a sampling position for sampling by a sampler 2. The movement of the samples is electrically controlled through the line 10 by the operation of the sampler 2 which has a working cycle or working frequency freely adjustable within ranges.

The movement of the samples is also controlled by a motorised valve 3, which is coupled to the sampler 2 and which initiates the rinsing of a sampling needle 13 (Figs 2 and 3) after the taking of each sample. The sampling needle 13 is carried on an arm 12 (Figs. 2 and 3) which oscillates to and fro between a sampling position and a washing position. Adjacent to the sampling device, there are several multi-channel proportioning pumps 4, through which the various liquids and the sample itself are supplied through different lines to the sampling preparation and to the actual analysis device 5.

This supply is indicated diagrammatically by the lines 11.

From the analysis device 5 or from the analytical unit, since it constitutes a single closed unit, the prepared samples pass to a multi-channel photometer 6, the reading results of which are evaluated by an electronic evaluation unit 7, 8 and written out in the form of a digital reading.

The analysis device 5 operates on the continuous flow principle, i.e., the liquid samples and the liquid reagents are pumped successively by means of the proportioning pumps 4 through a combined plastics hose and glass tubing system and thus mixed with one another in the necessary manner.

The proportioning pumps 4 possess rollerjournalled rods (not shown) with appropriate supports, so that a plurality of such roller rods are disposed around the periphery of an imaginary cylinder, rolling successively over plastics tubes containing a defined through flow volume and compressing these tubes externally, so that their contents are continually propelled forwards.

In order that, in this piping system, no blending shall take place between the specimens flowing continuously at intervals after one another through the lines, and also that better thorough mixing shall be maintained of the reagents and specimen as they flow together, volumes of air are introduced at regular intervals into the liquid stream by the proportioning pumps 4, these volumes of air dividing the liquid stream into segments. that is. into sections, commencing with the sampling by the sampler and continuing to the photometric analysis.

Sampling by the sampler 2 is carried out through the washable sampling needle 13 (Figs. 2 and 3). which is mounted in a suitable manner on the sampler arm 12 and is swung to and fro by motor power through an angle of about 90 between a sampling position (Fig. 3) and a washing position (Fig.

2). The duration of this cycle, sampling, pivotal movement and washing phase, is freely adjustable by timers, so that the speed or frequency, with which the specimens are investigated. can be chosen within ranges.

In the position "sampling", the sampling needle 13 penetrates into a sampling vessel 14 containing the sample 15 and removes a defined volume of the sample, which is determined by the flow volume of the pump hose, the immersion time and the flow speed of the sample. During the taking of the sample, the movement of the wash liquid together with the air bubbles contained therein is stopped, this being done automatically by the aforementioned motorised valve 3 in this position, while the sample is sucked up by a proportioning pump 4 and is supplied through the line 16 to the sampling preparation. The sample substance is shown shaded at 17 in the sampling needle.

After each sampling action, the arm 12 swings together with the sampling needle 13 into the washing position shown in Fig. 2, and washing liquid is forced from above into the sampling needle 13, air bubbles 18 being already contained in the liquid. A portion of this washing liquid, segmented by air bubbles, also flows through the line 16 for the purpose of washing the sampling line leading to the sample preparation.

In Fig. 2, reference 19 indicates the wash vessel together with the wash medium contained therein, which emerges at the bottom from the sampling needle in the direction of the arrow 20.

In Fig. 3, the wash liquid can be seen together with the air bubbles 18 in the upper end 90 of the sampling needle. It is retained there by means of the switched-off motor valve 3 and forms a suspended liquid column.

When the sampling needle 13 is raised and swung back into the washing vessel, due to valve intermediate positions in front of the closed bores of the valve, a light pressure is built up by continuous compression of the liquid column, segmented by air bubbles, in the supply hose (not shown) for the washing medium, which pressure is released when the needle is immersed in the washing vessel.

This lightly pressurised liquid meets, at an angle of 90 , the liquid stream sucked up during sampling and interrupts this stream by its own subdivisions, in that on the one hand it is pumped as a mixture of washing medium and air bubbles into the system and, on the other hand, is pressed outwards into the washing vessel as an excess component of the liquid stream for the purpose of cleaning the sampling needle 13.

The preparation of the sample is shown diagrammatically in Figs. 4 and 5. The sample, taken through the sampling needle 13, flows via the line 16 at 21 into a line, which contains a mixing coil 22 and which divides at 23, so that one portion of the prepared sample passes into line 24 and is discarded, while another portion flows into line 25 and then to a distributor block 26 in which the sample is supplied to a channel in device 5. Reference 4 indicates a proportioning pump. Air is drawn in by the proportioning pump 4 through the line 27 and delivered at 28 into the line into which tlie sample has also been introduced. In this manner, all samples are segmented or separated one from another, so that mixing of samples is prevented.

Together with the air, a diluting liquid, for example a human plasma protein solution, or an 0.9% buffered NaCI solution comprising glucose and albumen additives, is introduced via line 29.

From Fig. 4 of sample preparation with sample dilution, it can be seen thatthediluting liquid flows in the line 29 continuously, while air is introduced via the line 27 at 28, so that air bubbles form in the diluting liquid, and the sample, which is supplied between volumes of washing liquid segmented by air bubbles is introduced through line 16.

In the coil 22 these constituents are mixed together and finally supplied to the distributor block 26 for passing to the actual analysing device.

The distributor block 26 possesses connections corresponding to the number of analysis channels used.

If diluting of the sample is not necessary, then the sample is supplied between air bubble-segmented washing liquid volumes directly to the distributor block for passing further to the analysis device in the manner illustrated in Fig. 5.

In Fig. 6, the analysis device is now shown diagrammatically. The sample passes, for example as in Fig. 5, into the line referenced 30 and flows, propelled by the proportioning pump 4, into the analysis channel 31 with its mixing coils Ml to M4.

Before the sample enters the analysis channel 31, however, there is already flowing through this channel an acidified glucose solution, uniformly subdivided by air bubbles, which is introduced via the line 31 and into which the air 32 is introduced at slight excess pressure to uniformly segment this liquid stream of the previously introduced reagent. Therefore, before the sample enters via the line 30, an air-segmented liquid stream is already present, flowing continuously, in the analysis channel 31.

After the sample has been added via the line 30, further reagents are now pumped in via the further lines 34, 35, 36 and 38, also in continuous flow, these reagents being necessary for the investigation. Thus, a serological reagent flows in via line 34, a hexadimethrin solution via line 35 and a sodium citrate solution via line 36 and the haemolysis agent via the line 38. These individual solutions are now successively mixed with the sample in the mixing coils Ml to M4, namely first the sample with the acid glucose solution, then in coil M2 mixing with the hexadimethrin solution, etc. Finally, the mixing coil M3 is followed by a sedimentation length S, which consists of a glass coil lying horizontally.

In the region of mixing coil M2, a nonspecific agglomeration of red blood corpuscles takes place. In the case of a negative serological reaction, this non-specific agglomeration is again broken up after the addition of the sodium citrate solution, acting as a re-suspension agent, via the line 36 and subsequent mixing together. A uniform distribution of the red blood corpuscles in the individual sample segments is thereby simultaneously produced, so that after the sedimentation range in the horizontally lying glass coil S, only a small number of red blood corpuscles is removed from the system via the branch 37.

If, however, a positive reaction takes place after the mixing and incubation range, between the serological reaction partners (blood liquid, test serum, red blood corpuscles, test blood corpuscles), then the nonspecific blood corpuscle agglomerations (agglutinates) change into true cell agglomerations (so-called haemo-agglutination reaction), which cannot again be broken down even after the adding of the resuspension agent, that is the sodium citrate solution, so that the agglutination remains permanently.

In the region of the sedimentation coil, the blood corpuscle clumps collect due to gravity at the bottom of the sedimentation glass coil, and these sedimented blood corpuscles are then removed out of the system at position 37 by the liquid stream flowing past them. This is carried out by suction or pumping out, since the line 39 passes through the proportioning pump and so to discharge.

By contrast to a negative serological reaction, therefore, a much higher proportion of red blood corpuscles are here removed from the continuous liquid stream. In the negative reaction, therefore, a large quantity of red blood pigment is therefore supplied to the photometer for photometric evaluation, but in a positive reaction less red blood pigment is supplied.

A haemolysis agent solution is supplied via the line 38 from the proportioning pump into the analysis channel 31 in the direction of flow downstream of the discharge point 37.

The red blood corpuscles in the remaining portion of the liquid are thereby destroyed.

In this way, the red blood pigment (haemoglobin) contained therein is released which, after venting of the analysis channel via the duct 40, flows into a through-flow cuvette, in which the photometric determination of the blood pigment is carried out at a wavelength of 420 nm. The analysis liquid, now free of air bubbles, is pumped back by the proportioning pump 4 via the line 41 and to discharge.

The value measured by the photometer is supplied to the electronic evaluator unit 7, 8.

A different serological reaction behaviour of the blood samples leads to a different analogous measuring curve, which is detected by the electronic evaluator component and further processed.

Fig. 7 of the drawing shows a portion of the flow channel of the sample preparation component. Reference 42 designates the hose used with a defined cross-section 43. The sample contained in the tube is blocked at both sides by air 44, between which flows dilution solution 45 with air bubbles 46 contained therein.

The electronic evaluator unit 7, 8 is capable of switching from one to six channels and picks up the analogous photometric signals directly from the photometer and digitalises the values of the curve peaks.

These values are transmitted, with the association of a multi-digit sample number and an analysis identification block, which consists for example of data about the measuring position number, the date, method number, dilution, cycle time, recording window time and the photometric basis value output, to a data carrier or directly to an electronic data processing unit.

The transmitting of the analysis identification block is carried out with two working cycles of the sampler. In the first working cycle, fixed data for example, such as measuring place number, date, method number and dilution are transmitted, whereas in the second working cycle, for example, variable values, such as sample cycle time, electronic recording window time calculated thereon, and the photometric basis values of the switched-in channels are transmitted.

After transmitting of this basic information. the evaluator device commences automatically to search for exactly defined-in regard to form and time--curve patterns for the automatic bringing into operation of the system (so-called synchronisation).

The curve conditions to be fulfilled can be varied in the device and adapted to the measuring problem. Prerequisites for an automatic bringing into operation of results evaluation are firstly the satisfying of the required curve patterns, for example, curve peak point height at least 50% ofthe photometric reading range and fall of curve pattern relative to the peak point by at least 50% within the sample cycle time, and secondly guaranteed entry of all analysis samples during the sample cycle time for purposes of evaluation in the photometer cuvettes.

If these requirements for the analysis synchronisation are not satisfied, a fault announcement is made by the evaluator device.

If the required conditions are satisfied, the evaluator unit takes on the work, by associating the photometric values with a multi-digit sample number. The analogue curve patterns are now monitored for a phase displacement in both directions out of the recording window constructed electronically within the sample cycle time and referred percentually thereto. If such displacements occur, fault announcements are likewise given out by the evaluator device.

The blood quantities to be analysed are supplied by the sampling table 1 to the sampling location at which they are sampled for analysis by the sampler 2 which continuously passes through a cycle of taking a sample and being washed at a washing location. The results of the sample analysis and other data are monitored and correlated with the sampler cycle. The sampler cycle is illustrated in Fig. 8 and is represented by the letters a to h. Unit I represents the control for rinsing or washing the sampler 2, unit II represents the control for taking the blood sample.Unit III represents the lifting and pivoting mechanism for the sampler 2 and unit V which is shown within the evaluator unit 7 operates to detect the cycle time and count the number of cycles as a result of pulses (which can be generated electrically, hydraulically, or pneumatically) transmitted along line 9 by control switch S. When the control switch S is operated to start-up the apparatus, the sampler 2 is washed and the washing step is denoted by the reference a.

After this washing step, the time period of which can be controlled by unit I, the lift and pivot mechanism III takes over as represented by reference b and the sampler 2 is raised, pivotted through 90 and lowered as represented by reference c. Time control is now transferred to unit II as represented by reference d and after a selected time for taking the sample as represented by reference e, the lift and pivot mechanism III takes over as represented by reference f and the sampler 2 at the sampling location is raised, turned back through 90 from the sampling to the washing location and lowered. The reference g represents its return to the washing location and the reference h its lowering to the initial or starting position.On the return of the sampler I to its initial or starting position, the switch S or an associated switch sends a pulse along line 9 to the unit V. The sampler cycle continues repeatedly until the switch S is again operated to stop the operation. Each pulse transmitted to the unit V therefore represents one sampler cycle and although described as occurring at the end of a cycle can equally be generated at the commencement of a cycle. The unit V counts the pulses so that each sample and the analysis thereof can be identified and correlated with the count or its position in the sequence of pulses and the interval between pulses represents the sampler cycle time which can be controlled as required by adjustable time switches in the units I and II.

Also, the unit V can compare the cycle time with a predetermined value and control the time switches in units I and II to maintain the cycle time at such predetermined value. Also, the evaluator unit 7 may include or be associated with a data bank in which data is stored relating to, for example, the method of analysis employed, reagents and strengths of reagents employed. cycle time, which can be released to a memory store or print-out device to provide such information as may be required with each identified sample analysis.

The dotted lines shown in Fig. 8 with references a', b', d', e', f, h', S', I', II' and 9' represent the arrangement where the units I and II are included in the electronic evaluator unit 7 (reference c and g being common to both versions).

As can be seen from the foregoing illustrative embodiment, the present invention provides a simple and economical method and apparatus for carrying out blood group serological investigations with improved reliability and in a rapid, simple and economical manner. A large number of variations, modifications and equivalents of the embodiment described and illustrated will be apparent to those skilled in the art and are intended to be included within the scope of the appended

Claims (9)

claims. WHAT WE CLAIM IS:

1. A method of determining blood group characteristics and specificities of antibodies harmful to blood using an analyser having at least one channel operating on the principle of continuous flow analysis and capable of being connected to an electronic data processor, including the steps of sequentially taking samples from blood quantities to be investigated, by means of a sampler, cycling the sampler from a sampling location to a washing location, washing the sampler at the washing location, and returning it to the sampling location after the taking of each sample, controlling the time of each cycle of the sampler, generating a signal representative of the termination or commencement of a sampler cycle, photometrically analysising each sample taken and generating a record of the sample analysis, the record being in the form of an analogue curve, and comparing at least part of the analogue curve with at least part of a reference curve to selectively permit or not permit an electronic identification of the sample record with said generated signal according to the result of the comparison.

2. A method according to Claim 1, in which the time of each sampler cycle is regulated by freely adjustable time switches, whereby the working speed of the analyser and the time sequence of the identified samples is regulated.

3. A method according to Claim 1 or Claim 2 wherein an electronic evaluation of the analysis is performed by evaluation means which is automatically supplied with the analogue curve readings of the analysis results, and which checks them for a required curve pattern in regard to form and in regard to time sequence and, after the required criteria have been satisfied, transmits the values of the relevant curve peak points in digital form in a data block, with association of the sample number, to data carriers or directly to an electronic data processing installation.

4. A method according to Claim 3 wherein after automatic bringing into operation of the results evaluation, the curve patterns of the analyses are subjected to an electronic window which provides an electronic phase displacement monitoring within the sample cycle time, i.e., an electronic monitoring within a predetermined portion of the cycle sample time.

5. A method according to Claim 4 wherein if the required photometric reading signal conditions are not satisfied and if a phase displacement of the analysis samples occurs out of the electronicrecording window, related percentually to the sample cycle time, fault signals are produced by the evaluator means.

6. A method accordingto Claim 3, Claim 4 or Claim 5, wherein the electronic evaluation of the analysis is performed in a multichannel manner, i.e., simultaneously for a plurality of samples.

7. Apparatus for determining blood group characteristics and specificities of antibodies harmful to blood comprising means operable to supply blood quantities to a sampling location, a sampler operable to take a sample from the blood quantity at the sampling location, means for cyclically displacing the sampler from the sampling location to a washing location and returning the sampler to the sampling location, means for introducing wash liquid into the sampler at the washing location, photometric means for photometrically analysing each sample taken, timing means for controlling the displacing means and the time of each cycle of the sampler, means responsive to the end or beginning of each cycle of the displacing means to generate an electric signal operable to identify the sample, means responsive to the photometric means to generate a record of the analysis of each sample, the record being in the form of an analogue curve and means for comparing at least part of the analogue curve with at least part of a reference curve to selectively permit or not permit an electronic identification of the sample record with said generated signal according to the result of the comparison.

8. A method of determining blood group characteristics and specificities of antibodies harmful to blood substantially as herein described with reference to the accompanying drawings.

9. Apparatus for determining blood group characteristics and specificities of antibodies harmful to blood constructed, arranged and adapted to operate substantially as herein described with reference to the accompanying drawings.