EP1129337A1

EP1129337A1 - Blood analysis device and method for establishing a serodiagnosis

Info

Publication number: EP1129337A1
Application number: EP99950590A
Authority: EP
Inventors: Hermann Leistner; Bertram HÖLLIG
Original assignee: Stratec Biomedical Systems AG
Current assignee: Stratec Biomedical Systems AG
Priority date: 1998-11-13
Filing date: 1999-10-01
Publication date: 2001-09-05
Also published as: DE19852504A1; WO2000029833A1

Abstract

The invention relates to a blood analysis device and method for establishing a serodiagnosis. The method includes performing a test and assessing results of said test in an objective and reliable manner. The inventive device comprises a sample holder (14) having at least one cavity (12) for receiving blood samples (18) and reagents; a light source (24) for illuminating the cavity (12) to be analysed; a colour image acquisition device (10) for 2D-scanning of the cavity (12) to be analysed, which is used for delivering image signals associated to a respective colour; an analog/digital converter (50) for converting image signals into digital image data associated to image points and divided into fundamental colours, in the form of image points; an image processing unit (16) for processing said image data, used for reproduction and/or classification of reaction patterns of a blood sample (18), which are formed in said cavity (12).

Description

Blood analysis arrangement and method for serodiagnostics

description

The invention relates to an arrangement and a method for blood analysis for the. Serodiagnostics, especially for the acquisition, evaluation and reproduction of image data in blood group serology.

In serodiagnostics or blood group serology, specific detection reactions based on antigen-antibody reactions are triggered in the sample substances and their appearance is assessed for blood group determination or also for in vitro diagnosis of infectious diseases. For example, in hemagglutination tests, erythrocytes in blood samples are clumped specifically via antigens that are membrane-bound or fixed in some other way, and the agglutination patterns of the precipitated complexes are assessed semi-quantitatively according to a rating system graded from negative (no agglutination) to fourfold positive (strong agglutination). As an alternative, so-called solid-phase methods for the search and identification of erythrocytic antibodies and for cross-testing have also been proposed, in which positive reactions appear as a layer of bound erythrocytes on specially prepared solid phases.

In this context, it is known to present the test substances in the cavities or wells of microtiter plates and the reaction patterns visually with the naked eye to judge. It is considered disadvantageous that this subjective type of determination can lead to misjudgements, especially in transition phases, which may have serious consequences, for example in the event of intolerance to blood transfusions. In addition to experience and expertise, visual observation requires a lot of time, especially for series analyzes. In addition, the reaction processes only permit visual evaluation in a narrowly limited time window.

Proceeding from this, the object of the invention is to provide an arrangement and a method for test evaluation in serodiagnostics, with which preferably precise test results can be determined reliably, preferably automatically, in an economical and repeatable manner.

To achieve this object, the combinations of features specified in independent patent claims 1 and 13 are proposed. Advantageous refinements and developments of the invention result from the dependent claims.

The analysis arrangement according to the invention accordingly comprises: a sample carrier which has at least one cavity for receiving blood samples and detection reagents and is preferably designed as a microtiter plate or microtiter strip, a light source for illuminating the cavity to be examined, a color image pickup for two-dimensional scanning of the cavity to be examined and color-selective generation of image signals,

- An analog / digital converter for converting the image signals into pixels assigned and pixel by pixel in

Digital image data divided into primary colors, and

an image processing device which processes the image data for reproducing and / or evaluating the image data, in particular for classifying reaction patterns of the blood sample formed in the cavity.

This enables the test evaluation to be carried out and documented automatically at any time according to defined criteria using a true-to-color and unadulterated image of the cavities to be examined. In particular, different color gradations in the reaction patterns, which might not be distinguishable in the case of a mere black-and-white recording, can be identified to avoid incorrect assessments. The arrangement enables blood tests to be carried out in a rational and extremely reliable manner.

According to a particularly advantageous embodiment of the invention, the image processing device has a video controller and a color screen unit for preferably enlarged color image reproduction of the image data. A classic visual assessment can thus be carried out in parallel or in addition by a specialist, the image being able to be displayed in multiples of the original size. The image processing device is advantageously equipped with a storage medium for storing the image data in association with patient data in order to document the test results and to make them comprehensible at any time.

For a high processing speed, it is favorable if the image processing device has an image processor coupled to an image memory.

The required color sensitivity and resolution can be achieved in that the color image sensor is formed by a video camera which has a semiconductor image sensor, in particular a CCD image sensor, and operates in three basic color levels. With regard to an increase in the resolution, it is also advantageous if the color image sensor has an imaging optical system that can be focused on the cavity to be examined. In this context, sample processing is simplified by a positioning device for the sequential positioning of a plurality of cavities of the sample carrier in a measuring position.

In order to avoid image errors, it is advantageous if the light source is formed by a voltage-stabilized electrical white light source and, if appropriate, a diffuser arranged downstream of the white light source. A further improvement is achieved by a gray filter that can be connected upstream of the color image sensor for intermittent calibration of the homogeneity and intensity of the light source. It is also advantageous if the color image sensor is an im ultraviolet and infrared wavelength range blocking and in the visible range transparent measuring light filter is arranged.

To simplify the automatic documentation of the analysis data, it is provided that patient data which can be scanned by means of the color image pickup are preferably applied as a bar code to the sample carrier.

In procedural terms, the above-mentioned task is solved by the following procedural steps:

a cavity of a sample carrier designed to hold blood samples and test reagents is positioned in a measuring position, the cavity in the measuring position is illuminated and scanned two-dimensionally by means of a color image sensor with color-selective generation of analog image signals,

- The image signals are assigned in pixels and digitally divided into basic colors

Image data implemented,

- The image data are reproduced and / or evaluated by means of an image processing device, in particular for classifying reaction patterns formed in the cavity.

As an alternative or in addition to the automatic evaluation, the image data can optionally be displayed on a display unit as a preferably enlarged color image of the detected cavity in connection with evaluation results. It is also provided that the image data can be stored on a storage device in association with patient data.

In order to enable a reaction-specific differentiation, it is proposed that the pixels are assigned a characteristic value for area segmentation of the reaction patterns in accordance with the color components of the assigned image data or a color tone derived therefrom. An objective test evaluation is achieved in that coherent pixel areas with corresponding characteristic values are determined, that area features of the pixel areas such as diameter, center of gravity, area content and compactness are determined and that the reaction pattern is classified by comparing the area features with empirical default values.

A further simplification in image processing and evaluation is achieved by assigning binary characteristic values to the pixels for generating color-specific black and white images.

In an agglutination test, clumped erythrocytes appear as dark red sediment, while in a solid phase test, an erythrocyte sediment is obtained in reverse as an expression of a negative reaction. It is therefore particularly advantageous for the highly sensitive detection and separation of such reaction characteristics to be uniformly assigned a binary characteristic value with a color tone in the red-black area. Further test-relevant statements can be obtained from the fact that with an agglomerate tination or solid phase test, the pixels are uniformly assigned a binary characteristic value with a hue in the yellow-brown area.

In order to obtain a gray value image of the cavity to be measured with gray levels that are as clearly defined as possible, it is advantageous if a gray value is determined for each pixel in accordance with the color components of the assigned image data or a color tone derived therefrom. A preferred evaluation option then provides that the frequency of the individual gray values of the gray value image normalized to the total number of pixels is determined as a gray value distribution curve and that parameters for classifying a reaction pattern of the blood sample located in the cavity are derived from the gray value distribution curve . This can advantageously be done by determining a threshold value from a minimum of the gray value distribution curve and by assigning a first characteristic value to the pixels with gray values above the threshold value and a second characteristic value to the image points with gray values below the threshold value for segmenting the reaction pattern.

A further embodiment of the method provides that a gray-value curve is determined as a function of the pixel position from the gray-scale image for at least one linear pixel sequence, and that a reaction pattern is classified from parameters of the gray-value curve by comparison with predetermined threshold values. The invention is explained in more detail below with reference to an exemplary embodiment shown schematically in the drawing. Show it

1 shows a block diagram of an arrangement for blood analysis in blood group serology;

2 shows a microtiter strip with several cavities and solid phase tests with differently designed reaction patterns in a broken top view;

FIG. 3 shows an enlarged representation of the central cavity according to FIG. 2 with differently shaped and color-distinguishable reaction areas; FIG.

4 shows a simplified representation of a gray-scale image of an agglutination test in a cavity;

FIG. 5 shows a histogram of the gray scale image according to FIG. 4;

6 shows a gray value image of a cavity corresponding to FIG. 4 to illustrate a linear image evaluation; and

7 shows the gray value curve of the image data along the dashed scan line in FIG. 6.

The arrangement for serodiagnostic blood analysis shown symbolically in FIG. 1 enables detection, Storage, playback and evaluation of image data from blood tests on blood samples. Samples which have at least one blood ingredient such as blood plasma, blood serum or cellular elements are regarded as blood samples. The arrangement essentially consists of a color image pickup 10 for the two-dimensional scanning of the sample vessels or cavities 12 of a sample carrier 14 designed as a microtiter strip or microtiter plate and a digital image processing device 16 for determining reaction patterns of the blood samples 18 presented in the cavities 12 together with test reagents.

The sample carrier 14 can be positioned in a measuring chamber 20 by means of a positioning device 22, so that one of the cavities 12 is arranged in a measuring position. To illuminate the cavity 12 to be measured, a light source 24 is used, which comprises a high-frequency white light source 28 voltage-stabilized by means of a stabilizer circuit 26 and a scatter plate 30 arranged between the white light source and the cavity.

The color image pickup 10 is formed by a video camera 32 which has a CCD image sensor 34 operating in the three basic color levels red, green, blue (R, G, B). Imaging optics 36 are arranged in front of the CCD image sensor 34 and enable the sensor surface to be focused on the facing inner wall of the cavity 12 in the measuring position. An interchangeable lens 40, which can be actuated by means of an actuator 38, serves to interpose various optical elements in the beam path Bring cavity 12 and color image pickup 10. A measuring light filter 42 of the exchangeable optics 40 transmits the measuring light emanating from the cavity 12 in the visible wavelength range and blocks in the UV and IR ranges. A gray filter 44 enables calibration of the light source 24 by means of blank image recordings, on the basis of which the homogeneity and intensity of the illumination can be checked. A concave lens 46 is provided to focus the color image pickup 10 on the surface of the sample carrier 14 in order to scan a bar code marking applied there for sample and subject identification.

The analog RGB image signals supplied by the color image sensor 10 can be converted in an analog / digital converter 50 into image data or pixels assigned to digital image data. The captured cavity 12 is mapped onto a two-dimensional pixel matrix, three image data being assigned to each pixel as RGB values. Alternatively, these RGB values can be converted into a so-called HSI color model, in which pixel-by-pixel values for the hue (H), the saturation (S) and the brightness (I) are available. This digital image data can be further processed in an image processor 52 coupled to a RAM image memory and, if necessary, displayed on a monitor 56 as a true-to-original, preferably enlarged color image via a video controller 54 or on a permanent memory 58 in association with patient data, preferably in a database format lay down. FIG. 2 shows a microtiter strip 14 which is coated on the walls 60 of its cavities 12 with a reactive solid phase layer made of immobilized erythrocytes with selected antigenic patterns. Antibodies from sera or plasmas can be bound to these erythrocytes. An erythrocyte double layer or a cell lawn 62 is formed when sufficient amounts of erythrocytic antibodies are present in the sample to be examined. Accordingly, a 4-fold positive reaction is shown in the left cavity in FIG. 2, while a weaker positive reaction pattern (++) is shown in the middle cavity. Negative results (-) can be seen on the buttons of unbound cells which collect by centrifugation as erythrocyte sediment 64 in the center of a cavity 12 (FIG. 2, right).

The reaction patterns can be mathematically analyzed using the image data obtained using different methods. An overall result is derived from the results of the individual methods, whereby their different restrictions and inaccuracies can be taken into account by appropriate mathematical weighting.

A first evaluation method makes direct use of the color information contained in the image data in order to be able to extract evaluation features from test-specific areas of the reaction pattern. First of all, to segment the reaction patterns into areas that belong together, the pixels according to the color components of the assigned a characteristic value to assigned image data or a color tone derived therefrom. For the pixel areas with matching characteristic values, surface features such as diameter, center of gravity, surface area and compactness can be determined, on the basis of which the reaction pattern in the cavity is classified by comparison with empirically determined default values.

To simplify the evaluation, color-specific black and white images can be generated by assigning binary parameters. 3, a first black-and-white image is obtained by assigning a characteristic value "black" to the pixels of the cell lawn 62 on account of their color tone in the yellow-brown area, and a characteristic value "white" to the remaining pixels. Correspondingly, the sediment area 64, the pixels of which have a hue lying in the red-black area, is shown separately as a second black and white image data set. Then, based on the mean size of the black area in the first image and at the same time a pronounced black area in the second image, a 2-fold positive reaction (++) can be concluded.

Further evaluation methods are based on an indirect gray value analysis of the reaction patterns. For this purpose, a gray value is determined from the color components of the image data or their color tone to produce a gray value image. A two-dimensional evaluation then provides that the frequency of the individual gray values normalized to the total number of pixels is used as a gray value distribution curve is determined, and that extreme values and integral values of curve sections for the classification of a reaction pattern are determined.

As an example of this method, a simplified gray value image of an agglutination test is shown in FIG. 4, in which erythrocytes adhered to one another have formed a compact agglutinate 66. The dashed boundary line 68 encloses an evaluation area whose gray value distribution is shown as a gray value distribution curve or histogram 70 in FIG. 5. Curve 70 has two clearly distinguishable peak areas 72, 74, which are separated by a local minimum at gray value 45, area 72 being assigned to the light image background and area 74 to dark cell clumps. The calculation of the cumulative frequency or the curve integral below the single minimum found enables an empirical classification of the reaction picture into a 4-fold positive class in the given case.

In general, various characteristics of curve 70 can be used for classification, in particular the number of local minima, the number of iterative search steps until a minimum is first found, the cumulative frequencies below a first and possibly a second minimum and the cumulative frequency in a range by the absolute maximum, the latter as a measure of the uniformity of the background. Alternatively, an area segmentation analogous to the color evaluation described above is also conceivable, A minimum value is determined for the gray value distribution curve and a first characteristic value is assigned to the pixels with gray values above the threshold value and a second characteristic value is assigned to the pixels with gray values below the threshold value.

A further evaluation or classification option results from a line-related analysis of the gray value image. In this case, for at least one linear pixel sequence or scan line 76 (FIG. 6), a gray value curve 78 is generated from the gray value image, as is shown in FIG. 7 depending on the pixel position. Characteristics of curve 78 are determined as evaluation criteria. For this purpose, the maximum and minimum gray values are determined in a first step and provided with a percentage discount or surcharge. The intersection points 80, 80 ', 82, 82' with the curve 78 are searched for the resulting gray value levels. The gradients GRAD at points 80, 82 and 80 ', 82' and the intersection of the straight lines defined by these pairs of points are then calculated as parameters. Further parameters result from the difference in contrast DIFF between points 80, 82 and 80 ', 82' and the diameter DIAM between points 82, 82 '. In addition, the continuity and the variance of the gray values as a measure of the homogeneity of the image content are checked in sections. The results are assigned by comparing the parameters obtained with empirical threshold values. A strongly positive reaction requires, for example, a diameter DIAM larger than 100 pixels, a contrast difference reference _D IFF of more than 25 and a gradient GRAD of more than 45 °.

Claims

claims

1. Arrangement for blood analysis for serodiagnostics with

a) at least one cavity (12) for receiving blood samples (18) and detection reagents, preferably designed as a microtiter plate or microtiter strip, (14),

b) a light source (24) for illuminating the cavity (12) to be examined,

c) a color image sensor (10) for two-dimensional scanning of the cavity to be examined

(12) and color-selective generation of image signals,

d) an analog / digital converter (50) for converting the image signals into image points assigned and image-wise divided into primary colors,

e) an image processing device (16) processing the image data for reproducing and / or evaluating the image data, in particular for classifying reaction patterns of the blood sample (18) formed in the cavity (12).

2. Arrangement according to claim 1, characterized in that the image processing device (16) has a video controller (54) and a color screen unit (56) for preferably enlarged color image reproduction of the image data.

3. Arrangement according to claim 1 or 2, characterized in that the image processing device (16) has a storage medium (58) for storing the image data in association with patient data.

4. Arrangement according to one of claims 1 to 3, characterized in that the image processing device (16) has an image processor (52) coupled to an image memory.

5. Arrangement according to one of claims 1 to 4, characterized in that the color image sensor (10) is formed by a semiconductor image sensor (34), in particular a CCD image sensor having video camera (32) working in three basic color levels.

6. Arrangement according to one of claims 1 to 5, characterized in that the color image sensor (10) has a focusable on the cavity to be examined (12) imaging optics (36).

7. Arrangement according to one of claims 1 to 6, characterized by a positioning device (22) for sequential positioning of a plurality of Ka vities (12) of the sample carrier (14) in a measuring position.

8. Arrangement according to one of claims 1 to 7, characterized in that the light source (24) by a voltage-stabilized electrical white light source

(28) and optionally a diffuser (30) arranged downstream of the white light source.

9. Arrangement according to one of claims 1 to 8, characterized by a gray filter (44) which can be connected upstream of the color image sensor (10) for intermittent calibration of the homogeneity and intensity of the light source (24).

10. Arrangement according to one of claims 1 to 9, characterized by a upstream of the color image sensor (10), blocking in the ultraviolet and infrared wavelength range and permeable in the visible range, measuring light filter (42).

11. Arrangement according to one of claims 1 to 10, characterized in that on the sample carrier (14) by means of the color image sensor (10) scannable patient data (48) are preferably applied as a bar code.

12. Arrangement according to one of claims 1 to 11, characterized by an interchangeable lens (38, 40) controllable by means of the image processing device (16) optional positioning of filters or imaging lenses in front of the color image sensor (10).

13. Method for blood analysis for serodiagnostics in which

a) a cavity (12) of a sample carrier (14) designed to hold blood samples (18) and test reagents is positioned in a measuring position,

b) the cavity (12) in the measuring position is illuminated and scanned two-dimensionally by means of a color image sensor (10) with color-selective generation of analog image signals,

c) the image signals are converted into digital image data assigned to pixels and divided into primary colors pixel by pixel,

d) the image data, in particular for the classification of reaction patterns formed in the cavity (12), are reproduced and / or evaluated by means of an image processing device (16).

14. The method according to claim 13, characterized in that the image data on a color screen unit (56) as a preferably enlarged color image of the cavity (12) may be displayed in conjunction with evaluation results.

15. The method according to claim 13 or 14, characterized in that the image data are stored on a non-volatile storage means (58) in association with patient data.

16. The method according to any one of claims 13 to 15, characterized in that a characteristic value for area segmentation of the reaction patterns is assigned to the pixels in accordance with the color components of the assigned image data or a color tone derived therefrom.

17. The method according to claim 16, characterized in that coherent pixel areas (62,64) are determined with matching parameters, that area features of the pixel areas (62,64) such as diameter, center of gravity, area and compactness are determined, and that the reaction pattern by comparison the area features are classified with empirical default values.

18. The method according to claim 16 or 17, characterized in that the pixels for the generation of color-specific black and white images are assigned binary characteristic values.

19. The method according to any one of claims 16 to 18, characterized in that in an agglutination or solid phase test, a uniformly assigned binary value is assigned to the pixels with a color tone in the red-black area.

20. The method according to any one of claims 16 to 19, characterized in that in an agglutination or solid phase test, the pixels with a color tone in the yellow-brown area are assigned a binary characteristic value.

21. The method according to any one of claims 13 to 15, characterized in that a gray value for generating a gray value image (66) of the cavity to be measured (12) is determined for each pixel in accordance with the color components of the associated image data or a color tone derived therefrom .

22. The method according to claim 21, characterized in that the frequency of the individual gray values of the gray value image normalized to the total number of pixels is determined as a gray value distribution curve (70), and that from the gray value distribution curve (70) parameters for classifying a reaction pattern of the in the cavity ( 12) blood sample can be derived.

23. The method according to claim 22, characterized in that a threshold value is determined from a minimum of the gray value distribution curve (70), and that the pixels with gray values above the threshold value have a first characteristic value and the pixels with A second characteristic value for area segmentation of the reaction pattern is assigned to gray values below the threshold value.

24. The method according to claim 21, characterized in that a gray value curve (78) is determined as a function of the pixel position from the gray value image (66) for at least one linear pixel sequence (76), and that from parameters of the gray value curve (78 ) a reaction pattern is classified by comparison with predetermined threshold values.