Patent Description:
Sample analyses of the above kind are frequently carried out using microtiter plates, the wells of which contain a piece of sample-containing substrates. An example of such analysis is blood screening of neonates. Such analysis comprises collecting of blood samples from neonates by impregnating blood samples to certain areas of fibrous cards (substrates) so as to form sample spots on the cards. The samples are let to dry onto the cards. Typically the cards are sent by mail into the central laboratory where the cards are fed to a manual or automatic card handling apparatus, which punches one ore more small-diameter sample disks, from each of the sample spots. The sample disks may be of a diameter of a few millimeters depending on the amount of the sample needed for the analyte test and also the size of elution container for example, if microtiter plate well, diameter of <NUM>. The disks are conveyed to wells of the microtiter plate so that one sample well includes typically exactly one disk. The wells are then subjected to different (bio)chemical preparation steps depending on the chemistry applied. Thereafter the amount of analyte(s) in the wells is measured, for example fluorometrically. In a typical assay, the sample disks are first submerged in a suitable incubation buffer within the wells in order to elute (e.g. extract or dissolve) analyte from the disk to the buffer. The amount of the analyte can further be detected for example optically, i.e. using fluorescence, time-resolved fluorescence, absorbance or luminescence measurements or using radioactive labeling or mass spectroscopy
It is crucial to the analysis that each well contains a sample disk, which is correctly punched from fibrous substrate card and the impregnated sample from the disk is totally extracted into the buffer Otherwise the analysis of the sample is not reliable.

However, it sometimes may happen that not all wells are containing a sample disk for various reasons. Possibly the disk is not successfully punched in a puncher, the disk is lost in the puncher or during transport of the plate from the puncher to the analysis apparatus, or the disk is lost within the analysis apparatus. In particular, fibrous disks are prone to static electricity (see e.g. <CIT>) accumulated during punching or later, which increases the risk of losing a disk. On the other hand, a sample disk may not contain enough blood due to erroneous punching, or stick to the upper portion of well walls, in which cases elution of the sample also cannot take place.

Traditionally, an operator of the analysis apparatus checks that there is a disk in each well by visual inspection before placing the plate into the apparatus or after the measurement again depending on the type of chemistry used. This is however time-consuming and susceptible to human errors. Afterward detection is not always possible if a disk-remover unit is used. In addition, losing a disk within the apparatus or improper elution of the sample, even if a well contains a disk can not be detected by visual inspection.

<CIT> and <CIT> disclose methods for analysis of blood samples from dried blood spot samples eluted from blood disks. Before analysis, a sample of the eluant is transferred from the elution container to another container to avoid blocking of light path by the sample disk present in the elution container. However, for the simplicity and reliability of the process, analysis from a container still having the disk therein would be preferable.

AutoDelfia® system from PerkinElmer is has means for controlling the success of elution. This instrument is equipped only with time-resolved fluorescence detection and to detect missing spots it uses a modified protocol of time-resolved measurement immediately after elution and before removing the disk. From these measurements one can evaluate probability of unsuccessful elution but the procedure used is complicated and sets up some expectations for plate maps and possible number of missing disks. Although the method finds all missing spots, it also produces false alerts, which means extra costs and time delays for screening.

It is an aim of the invention to find a simple and reliable apparatus for automatically verifying successful elution while the sample disk or the like substrate is within the elution well, thus ensuring safe analysis of blood samples and also avoiding extra testing due to false alerts. Furthermore, it is an aim of the invention to achieve a solution to controlling elution irrespective of whether the substrate is floating on or submerged within the incubation buffer contained in the well.

The aims of the invention are achieved by an apparatus as defined in the independent claim. Thus, it has been found that automatic detection of elution can be carried out by directing light to a sample well at a wavelength or wavelength range which is absorbed by at least one elutable component of the sample but at least partly transmitted by the sample substrate (i.e. the sample disk). The calculated absorbance value of the amount of light transmitted through the well and further detected by a detector, is indicative of the degree of real elution irrespective of the position or presence of the sample disk in the well.

Advantageous embodiments of the invention are the subject of dependent claims.

In particular, it has been found that fibrous sample substrates, such as sorbent papers or the like, have a sufficient optical transmission around the absorption peak of haemoglobin, that is, approximately <NUM>. On the other hand, haemoglobin is easily eluted from blood samples and thus serves as a good indicator of the degree of their elution.

According to embodiments in accordance with the invention, the sample disk is present in the sample well during analyte detection. This is crucial if the screening assay is such that the disk is not removed from the sample well in a separate disk removal step before the measurement of the analyte. An example of an assay which can be carried out without removing the disk is the GALT assay widely used in screening newborn babies.

According to one embodiment the eluted sample is transferred into another plate without transferring the disks. Here the present invention is exploited to verify that the transferred sample is valid by absorption measurement.

According to one embodiment, the elution detection according to the invention is performed automatically in parallel or successive manner for a plurality of sample wells in an optical sample measurement apparatus. In particular, the detection provides significant advantages in connection with high-throughput screening using a plurality of stored (e.g. stacked) microtiter plates each containing an array of samples, as the risk of obtaining false screening results due to human errors or losing of disks within the screening apparatus or incomplete elution of samples is practically avoided.

In addition, the apparatus preferably includes.

According to the present invention, the light source and detector used in determining the degree of elution of the sample are used also by said optical measurement unit for the measurement of the amount of the analyte. Accordingly, no separate units are required reducing manufacturing costs and processing times. The degree of elution of the sample is adapted to be determined using the optical measurement unit before or after the optical measurement of the analyte in each sample well. If the actual analyte measurement is performed e.g. fluorometrically the analyzator could be easily equipped with a simple filter photometer for absorbance measurement.

The invention offers advantages over the art, especially by eliminating/controlling the effect of the presence or position of the disk on the signal measured, and thus being able to determine the degree of real elution. Along the invention, defined in appended claim <NUM>, the use of absorption measurement or the like in the instrument gives a clear and absolute signal from every sample alone without any complicated data analysis. Furthermore, it does not need any special plate map or any minimum number of wells or history data. The measured signal is a direct criterion for the quality of the test. In other words, a threshold absorbance value below which the proper elution can be assumed not to have taken place can be reliably determined in all possible situations that may occur in the well (e.g. disk floating, disk submerged, disk stuck to the a wall, disk tilted, disk removed from the well).

As defined herein, "incubation buffer" is a solution typically comprising analyte specific reagents such as substrates, cofactors, label molecules, antibodies, enzymes, and buffer components.

Further embodiments and advantages of the invention are explained in the detailed description with reference to the attached drawings.

The present invention can be used for automatically detecting whether a blood disk currently is or has been in a well of the microtiter plate. This is achieved by measuring the absorbance of light in the well at a suitable wavelength absorbed by haemoglobin eluted from the blood disk to buffer within the well.

The general principle of the elution detection is illustrated in <FIG>. Light <NUM> is emitted by a light source <NUM> through an optical filter <NUM> which is adapted to limit the wavelength band to the desired range, for example, around <NUM>. The small portion of the light <NUM> is directed to the reference detector <NUM> via a partial reflector 13A. The remaining light is further directed via mirror 13B to the sample well <NUM> from above. Overall there are used lenses to collect light from the light source and to focus it in to the sample. The lenses are not shown on the figure. The well <NUM> is typically part of a well array of a microtiter plate. The well <NUM> contains incubation buffer <NUM> and a sample disk <NUM>. In the figure, the disk <NUM> is shown submerged position. Transmitted light <NUM> is collected and focused to the detector <NUM> using the lens system.

Absorption measurement could be performed by many different ways. In addition to the embodiment disclosed in <FIG>, the light source and the detector can be located below and above the sample, respectively. In the case of single containers, in turn, the absorbance can be measured through side walls of the container.

If a well based on the measurement is detected as not containing a well-eluted sample, the actual measurement of the analyte may be skipped or the measurement results may be disregarded in respect of such well.

The threshold signal strength may be individually determined for each assay or series of assays based on the real patient data or it may be pre-programmed to the apparatus based on prior experiments or calculations, for example. Examples <NUM> - <NUM> later in this document clarify the efficiency of the invention.

The following measurement examples illustrate the efficiency of the invention in practice. In the measurements, different analytes of blood samples impregnated to sorbent sample disks were measured by absorbance of the wavelength of <NUM>. Haematocrite values of <NUM>, <NUM>, <NUM> and <NUM> were used, corresponding to haemoglobin values of <NUM>/dl, <NUM>/dl, <NUM>/dl and <NUM>/dl. (The lower limit for the haemoglobin values of <NUM>-day old neonatals is <NUM> - <NUM>/dl). The two lowest haematocrite values were measured both when the disk is in the well and when the disk was removed from the well. In addition, wells with white disks (i.e. disks not containing an impregnated sample), wells containing only buffer and empty wells were measured. In the case of GALT (example <NUM>), additional samples (calibrators A-F and controls N and AbN prepared from sheep blood) were measured.

The results of the measurements are shown in Tables <NUM> - <NUM> and <FIG>. The Figures shows graphs of absorbance values and their relative frequencies of incidence and for each sample type (the integral area of each curve amount to <NUM>). Samples with no blood, that is, the three undesired well conditions (white disks, no disks and empty wells) are drawn with solid lines and blood samples are drawn with dotted lines. As can be seen, in each case the three undesired conditions could be reliably distinguished from the cases where the wells contained a real blood sample (by selecting a threshold absorbance signal at the gap between the two groups of measurements.

Claim 1:
An apparatus for optical analysis of a sample eluted from a sample substrate (<NUM>) to an incubation buffer (<NUM>) contained in a sample well (<NUM>) of a sample holder loadable into the apparatus, the apparatus comprising;
- a plurality of microtiter plates, each microtiter plate comprising an array of sample wells (<NUM>), the microtiter plates serving as said sample holder, and
- an optical measurement unit,
characterized in that;
- the apparatus comprises a sample substrate (<NUM>) contained in one sample well (<NUM>) of said array of sample wells, which sample well (<NUM>) contains an incubation buffer (<NUM>),
- the optical measurement unit comprises:
∘ a light source (<NUM>) which is configured to direct light (<NUM>) through the sample well (<NUM>) containing the sample substrate (<NUM>) and the incubation buffer (<NUM>) at a predefined wavelength or wavelength range absorbed by the sample eluted from the sample substrate (<NUM>) but transmitted by the sample substrate (<NUM>), and
∘ a detector (<NUM>) which is configured to detect light (<NUM>) transmitted through the sample well (<NUM>),
- the optical measurement unit is adapted to measure an amount of predefined analyte in the sample eluted from the sample substrate (<NUM>) contained in the sample well (<NUM>) by means of the light source (<NUM>) and the detector (<NUM>), and in that
- the apparatus comprises a computing unit adapted to calculate, based on an amount of light (<NUM>) transmitted through the sample well (<NUM>) and detected by the detector (<NUM>), an absorbance value indicative of a degree of elution of the sample in the sample well (<NUM>) containing the sample substrate (<NUM>) and the incubation buffer (<NUM>), said amount of light transmitted through the sample well by said optical measurement unit before or after the optical measurement of the amount of the predefined analyte in the sample well (<NUM>).