Photometric reading device

The present invention relates to a photometric reading device.

Such a photometric reading device may be used for automatically performing analytical operations on liquid samples, in particular for performing serological analysis such as is involved in, for example, blood grouping, the determination of the osmotic fragility of red blood cells, antibody titration, prothrombin tests and cross-matching tests.

One of the most important applications of serological testing is the identification of blood groups. Present methods of determining to which group (ABO or Rh(D)) blood belongs involve mixing samples of red blood cells with several different reagent antisera and observing each test to see if the red cells agglutinate, or clump together. For ABO determinations, complementary tests are performed by mixing samples of plasma with red blood cells of known antigenic specificity. The interpretation of both sets of tests must agree before an ABO group can be assigned.

Conventionally, the analysis is carried out in test tubes or even on a tile. However, this test method is tedious. Systems for ABO/Rh testing have therefore been developed which perform all of the necessary tests in the wells of a disposable called a microplate. The microplate is a clear rigid moulding of plastics material such as polystyrene which is impressed a matrix of, for example 8 x 12, reaction wells. After a dilute suspension of red blood cells is mixed with appropriate antisera and plasma is mixed with red cells of known antigenic specificity, the microplate is centrifuged to pellet the red cells and then agitated to resuspend them. Cells which have agglutinated will settle to the bottom of a well within 1 to 5 minutes, while cells which have not agglutinated will remain in suspension for 10 minutes or more. Thus, a strong positive reaction is characterised by a button of agglutinated cells at the bottom of a well surrounded by a relatively clear supernatant, while a negative reaction is characterised by a uniform suspension of red cells. A weakly positive reaction is seen a s small clusters of cells spread across the bottom of a well.

In such systems, the presence of absence of agglutination in each well of the microplate is examined optically by passing light through it and measuring the transmitted light with a photometer. The type of reaction present in a well is determined from the absorbance of the supernatant. After all of the reactions have been categorised as positive or negative, the pattern of reactions for a sample is compared to template patterns to determine the ABO/Rh test results.

US-A-3533744 describes a photometric reading device comprising a plurality of reading elements, and means for continuously moving a plurality of samples relative to said reading elements, wherein said samples are arranged in at least two substantially parallel rows extending transversely to the direction of relative motion.

The system described in this US patent specification is an automated system in which samples are applied to a sample carrier which is then moved through the machine for reagents to be added, and then to a reading station where the sample carrier comes into registration with a light source and a photometer. The carrier is stopped so that the absorbance can be measured, and the carrier is then advanced until the carrier reaches the reading station. However, this moving and stopping has effectively shaken the liquid in the carrier, which, unless time is left to enable the liquid to settle, can cause disturbance of the liquid meniscus, which in turn can cause unwanted optical aberrations. The alternative, is to make the measurement while the carrier is moving. In this case, the whole sample carrier is illuminated through a very narrow transverse slit, so that the sample is scanned by a very thin transverse band of light during the movement of the sample carrier.

According to the invention, a photometric reading device as defined above also has a plurality of wells provided for receiving said samples, said wells being arranged in a matrix having a plurality of rows and columns, the arrangement of the reading elements being such that the relative movement of the reading elements and the matrix of wells brings the wells in each said row into registration with the reading elements sequentially and individually and is characterised in that only a single well is in registration with a reading element at any one time.

The reading elements can be arranged in several ways and yet still ensure that only a single well is read at any one time. The arrangement is subject to only two constraints. Firstly, the reading elements must be located at different positions along the direction of relative movement of the matrix of wells and the reading elements so that the reading elements come into registration with the individual samples in each row of samples at different times. Secondly, the separation of the centres of the reading elements along the direction of relative movement should not be greater than the separation of the centres of wells in adjacent rows so that all the wells in the first row come into registration with the reading elements before the first of the wells in the second row comes into registration with a reading element. The placing of the reading elements is otherwise not critical, but it is preferred to arrange that, assuming constant velocity of the matrix of wells, the wells come into registration with the reading elements after equally spaced intervals of time. Configurations in which the reading elements are evenly distributed impose least restriction on the size of the reading elements. The actual order in which the samples in the wells are measured is immaterial since the results may be re-arranged by subsequent data processing.

The reading elements may, for example, be arranged in a single row at an acute angle to the rows of wells, or in two rows such that alternate reading elements are staggered.

The magnitude of the acute angle depends on the length of the rows of wells and on the separation of the centres of wells in adjacent rows of wells. The angle is typically less than 20°, usually less than 10°, and particularly approximately 5°.

The continuous relative motion of the wells enables the individual measurements to be spread evenly within the time spent by the samples within the device. As a result the speed of operation of the device can be enhanced.

The relative motion of the matrix of wells with respect to the reading elements can be effected by moving the samples with respect to stationary reading elements or by moving the reading elements with respect to a stationary matrix of wells.

In a preferred embodiment, the matrix of wells is comprised of a microplate, eg a rectangular microplate containing 8 x 12 sample wells. If the direction of movement of the microplate relative to the reading elements is parallel to the long axis of the microplate, there are 12 rows of 8 samples to be measured. On the other hand, if the direction of movement of the microplate relative to the reading elements is parallel to the short axis of the microplate, there are 8 rows of 12 samples. The sample wells are preferably round-bottomed.

Preferably, the number of reading elements is the same as the number of wells in each row of wells so that corresponding wells in each row are measured by the same reading element.

In practice, where conventional microplates have been used, a relatively powerful polychromatic light source, eg a tungsten filament lamp, has been employed, together with expensive interference filters to remove light of unwanted wavelengths (eg all except the green). It has been known to split the light beam from the lamp and lead it through optical fibres to the individual wells of the microplate, and also through comparison channels to enable fluctuations in the output of the lamp to be compensated. Alternatively, readers in which a light beam is used to read one well at a time, but using a comparison channel to take account of light variations have been used. It has also been found desirable to chop the light beams mechanically to provide a further check on the calibration of the photometers (the so called dark current comparison).

In an embodiment of the invention, the reading elements each comprise a light source and a corresponding light detecting means. The light detecting means may be photodetectors, for example, photodiodes.

Light sources which are particularly suitable for use in a device of the invention are light emitting diodes (LEDs). LEDs have rapid on/off characteristics which enable the samples to be moved continuously and smoothly between a collection of LEDs and corresponding photodetectors, the arrangement of the LEDs and photodetectors being such that each LED and its corresponding photodiode is activated for a very brief time at the time when the appropriate well is in its optimum position for examination. The very rapid on/off characteristics of the LEDs and the provision of an LED for each well (as opposed to one part of a split beam), enables the LEDs to be activated individually so that there is no scattered light interference from one well to another. The effect of moving the wells during the finite time of measurement is to "smear" out the beam, but this is made acceptable by the very short response time of the LEDs.

In an embodiment, a photometric reading device further comprises a reading head comprising said plurality of reading elements, and having first and second plates spaced to define a pathway for said matrix of wells, said light sources being carried by said first plate and said light detecting means being carried by said second plate, and wherein said moving means is arranged to move said matrix of wells along said pathway.

If any agglutinate is allowed to obstruct any part of the light beam a high absorbance will result, which will be interrupted as negative. To ensure that this does not happen the photometric reading head preferably further comprises means for supporting said matrix of wells at an angle to the horizontal during the relative movement of the reading elements and the matrix of wells. For example, said matrix of wells may be supported at 17° to the horizontal. This encourages the agglutinate to collect in one part of the well. Alternatively, a horizontal microplate containing wells provided with V-shaped, U-shaped or inclined bases may be used.

It is preferred to position the light sources such that during measurement the measurement beam does not pass through the part of the sample where agglutinate has collected. Also, the effective diameter of the beam is preferably small relative to the sample diameter. This helps to ensure that there is good discrimination in the measurement between positive and negative reactions.

When the light passing through a sample is detected and measured it is necessary to subtract from the result the "dark current" for that particular detector. The latter will be affected by any stray light that can penetrate the measurement area, so that usually it is necessary to take special care to exclude all ambient light. The use of LEDs with their rapid switching allows a "dark current" measurement to be made just before each light transmission measurement so that the final result is essentially insensitive to variation in ambient light.

The LEDs preferably produce substantially monochromatic (eg. ± 20nm bandwidth) light of a wavelength susceptible to absorption by the liquid to be tested, eg the wavelength of the light should be in the green region of the spectrum (ca 570nm) when the liquid comprises blood cells.

LEDs consume very much less electrical power than filament bulbs. Thus the heat dissipated within the equipment is considerably reduced and power supplies may be simple, small and inexpensive. Furthermore LEDs have a long life and are unlikely to need replacement within the life of the instrument. In certain embodiments of the invention the LEDs are used in pulsed mode and are each switched on for only about five milliseconds for each sample measured. Thus the total switch-on time for each LED during a year's typical use might be only ten minutes. By way of contrast filament bulbs must be run continuously whilst the instrument is switched on. LEDs also have emission characteristics which are extremely stable and their use can enable comparison channels to be dispensed with. LEDs are also physically small and may thus be contained in the space immediately adjacent to each vessel, thereby obviating the need for fibre optics in multi-channel designs.

The LED is preferably such that it reaches full intensity from "off" (and vice versa) very rapidly, eg within 50 nanoseconds. This allows measurements to be made in rapid succession for both states of the LED, ie both on and off. High speed measurement also allows reading while the sample is moving without significantly broadening the effective measuring beam width.

The light detecting means may be a conventional photodiode and preferably a photodiode having a high sensitivity at the emission wavelength of the LED, a small physical size, a sensitive area large enough that all light emerging from the sample is detected (the light beam may be focused onto the photodiode by a lens), and a rapid response matching the "on/off" characteristics of the LED.

The means of activating the LEDs, of bringing about relative motion of the samples and the reading elements and of measuring the transmitted light may be coupled with conventional control and data processing apparatus, eg an analog to digital converter and a microcomputer.

A preferred embodiment of the device according to the invention will now be described by way of illustration only, with reference to the accompanying drawings, in which:- <ul id="ul0001" list-style="none"><li>Figure 1 is a detailed view in section of one element of a photometric reading head, and</li><li>Figure 2 shows the arrangement of a set of such photometric reading head elements in relation to the direction of motion of a sample microplate.</li></ul>

A photometric reading device for use in the determination of blood groups comprises a photometric reading head and a sample drive adapted to transport a sample microplate through the reading head.

The reading head comprises an upper plate 1 and a base plate 2, into which are set respectively light-emitting and light-receiving parts of twelve photometric reading elements. As shown in Figure 1, each reading element comprises, in the upper plate 1, a light-emitting diode or LED 20 and a parallel hole collimator 21, and, in the base plate 2, a condenser lens 22 and a photodiode 23. The base plate 2 is covered by a glass screen 3. The LED 20 emits a beam of green, substantially monochromatic light of wavelength 570nm. The beam has a diameter of approximately 2mm within the wells.

Although not shown in Figure 1, the reading head is tilted at 17° to the horizontal to similarly tilt the microplate 10 and thereby encourage any agglutinate contained within the wells 10-1, 10-2 etc of the sample microplate 10 to be offset relative to the central axis of the microplate well 10-1, 10-2 etc, and therefore out of the measurement beam.

A microplate drive (not shown) is arranged to convey a rectangular, 96-well sample microplate 10 through the photometric reading head. The microplate drive comprises a carriage driven by a stepper motor via a reduction gearbox, pulleys and a drive cord.

The wells 10-1, 10-2 etc, of the microplate 10 are arranged in eight rows of twelve. The elements of the reading head are arranged such that, as the microplate 10 passes through the head, corresponding wells in each row of wells, 10-1, 10-2 etc are scanned by one of the reading elements.

In Figure 2, the arrangement of the reading elements 30 with respect to the direction of movement of the sample microplate 10 is shown. The microplate 10 is moved in the direction of the arrow past a row of reading elements 30 arranged in a line at an angle of approximately 5° to the direction of the rows of wells A, B, C, D, of the microplate 10. In an alternative arrangement (not shown) the reading elements 30 are staggered in two parallel rows at a similar angle. This alternative arrangement provides maximum space (and hence minimum interference between the elements and maximum flexibility in choice of devices for the units).

The distance d, the separation along the axis of movement of the microplate 10 of the first and last of the reading elements 30 to come into registration with a well in the microplate 10, is slightly less than the distance p, the separation of the centres of the wells in adjacent rows A, B, C, D, .... of the microplate 10. In preferred embodiments, the distance d is approximately equal to [(n-1)/n].p where n is the number of reading elements. In the device illustrated, for example, d is approximately (11 x p)/12.

In operation the microplate 10 is inserted into the carriage and a reading sequence initiated. As each row of wells A, B, C, D, in the microplate 10 passes through the reading head, the reading elements are fired in the order 30-1, 30-2, 30-3, 30-4, 30-5, 30-6, 30-7, 30-8, 30-9, 30-10, 30-11, 30-12. The absorbance of the supernatant in each sample well is measured and transmitted to a host computer (not shown).

Prior to measurement, the microplate 10 may be identified by an attached bar code label. In this case, a separate hand wanded bar code reader (not shown) is used to read the label and transmit the decoded number back to the host computer. A photoelectric trigger at the label position ensures that the only codes accepted are from microplates placed in the instrument's carriage. A successful label read initiates the reading sequence and prevents the microplate from being exchanged.

In an alternative embodiment to that described above, eight reading elements are used and the microplate is moved along a line parallel to its long axis. This arrangement has the advantage that the number of components, and hence the cost, is lower. The total measurement time, however, is somewhat longer if the speed of relative movement of the microplate and the reading elements remains the same.