EP1955042A1

EP1955042A1 - Method and device for determining the relevance of sample array preparations

Info

Publication number: EP1955042A1
Application number: EP06817469A
Authority: EP
Inventors: Wolfgang Schmidt; Christopher Wrighton; Kurt Zatloukal; Harald ZÖBL; Peter Hecht; Gabor Mehes
Original assignee: 3DHistech KFT
Current assignee: 3DHistech KFT
Priority date: 2005-11-30
Filing date: 2006-11-29
Publication date: 2008-08-13
Also published as: WO2007062443A1; US20110034341A1; JP2009517664A; AT502854B1; AT502854A1

Abstract

The invention relates to a method and to a device (10) for determining the relevance of preparations (4) of sample arrays (1), containing a plurality of individual samples (3) on different positions. A device (11) for non-destructive sampling of preparations (4) of the sample array (1) is provided in order to produce a method and/or a device (10) which can be carried out in rapid manner and preferably, without destroying the preparations (4). Said sample array is connected to a data bank (12) and to a calculation unit (16) in order to process the sampled preparations (4) and in order to produce data fields of each individual sample (3) and in order to select at least one parameter from each individual sample data field and in order to compare said parameter or a value derived therefrom or a combination of parameters or values derived therefrom to at least one threshold value, said data bank containing information on the position of the individual sample (3) of the preparation (4). The invention also relates to a device (17) for displaying the value of the comparison to the at least one threshold value as relevant criterion for the individual sample (3), and to a memory (18) in order to store said relevant criterion.

Description

Method and device for determining the relevance of sample larvae preparations

The invention relates to a method for determining the relevance of preparations of sample arrays, in particular tissue sample arrays, containing a large number of individual samples at different positions.

Furthermore, the invention relates to a device for determining the relevance of preparations of sample arrays, in particular tissue sample arrays, containing a plurality of individual samples at different positions.

For diagnostic and research purposes, it is common in medicine to collect different samples, such as tissue samples, and submit to various investigations. In the case of tissue samples taken from human or animal organisms, it is common practice to bed them in paraffin and, at certain selected locations on the tissue sample, puncture and insert cores into corresponding large cylindrical holes of a paraffin block Tissue microarrays (TMAs) are then usually cut using a microtome and the specimens are examined histologically, for example.

Owing to the large number of sections and individual samples described above, tissue sample arrays are increasingly being supplied with automatic analyzes in order to obtain important information as quickly as possible, in particular for diagnostic or therapeutic purposes. For example, US 2003/0215936 A1 describes a method and an apparatus for the fastest possible and efficient examination of such tissue sample arrays.

Although the following description primarily addresses tissue sample arrays, the present invention is not limited to such samples, but is applicable to preparations of a variety of sample arrays containing a plurality of individual samples. In addition to human, animal and plant tissues, combinations of different tissues of different origin are also used. fertil of the present invention in question. Likewise, material extracted from tissue, such as proteins and nucleic acids, which are applied dropwise to a glass slide can be assayed by the present invention. Furthermore, body fluids such as blood, saliva or the like can be analyzed by living organisms. Finally, cultured cells or parts thereof but also organic or inorganic materials, which are arranged in the form of a sample array, may be present as a preparation.

In the case of TMAs (tissue microarrays), the number of mostly cylindrical individual samples, which are introduced into the paraffin block, usually in the range of several hundred individual samples. In order to be able to obtain as many individual sections as possible from a so-called target block, the introduced individual samples should reach as evenly as possible deep into the paraffin block. In practice, it is in the production of such tissue sample arrays due to different length cylindrical individual samples or "Gores", but also problems in introducing the specimens in the paraffin block to different depths of the samples in the paraffin block, which is why it especially in paraffin block sections in deeper regions comes that individual "Gores" only partially or not exist. For a reliable statement of the subsequent examinations on the sections, it is therefore essential to determine the relevance of the sections. In particular, a statement should be made as to whether or not individual samples are present at certain points in the section.

However, it is also of particular importance for other samples to be able to make a statement about the relevance of the individual samples to the preparation. On the one hand, this is of great importance for the reliability of the statements made after analysis of the sample, in particular for diagnoses in the medical field. On the other hand, the preparations represent a tremendous economic value, which can be increased if a statement can be made about the relevance of the individual samples to the preparation.

At present, such a control is In elaborate manual procedures, samples of the specimens are made under the light microscope, whereby a selection of sections of a paraffin block is examined for relevance control purposes. The sections of the sample array used for the examination are usually histologically stained in order to be able to more easily recognize missing individual samples. For subsequent examinations, these sections are no longer available due to the coloration. In addition, such random examinations do not provide information on the actual relevance of the specimens between the samples. Although this information would be improved with an increase in the number of samples, but then fewer preparations for subsequent investigations are available. In addition, the usually manually performed controls are very time consuming and thus costly.

An automatic scanning method, in particular for biological samples, with which automatic analyzes of a large number of samples can be carried out, is described, for example, in WO 02/101635 A1. For this purpose, a flatbed scanner is used in combination with an automatic image analysis. The evaluation is aimed at sections of samples and not on sample arrays with a large number of individual samples.

US 2004/0136581 A1 describes a method and a device for the automatic analysis of images of a biological sample, wherein a biological sample prepared with a reagent is placed in a microscope slide and scanned optically. Also, this evaluation is not directed to sample arrays with a large number of individual samples. In addition, the samples are affected or even destroyed by the preparation.

Finally, WO 99/39184 A1 shows a device and a method for automatic image processing and analysis of biological samples arranged in microtiter plates. Also, this evaluation is not directed to sample arrays with a large number of individual samples. The object of the present invention is therefore to provide an abovementioned method for determining the relevance of preparations, which can be carried out as quickly as possible and as far as possible without destroying the preparations. The relevance control should be automatable in order to obtain information on the relevance of the preparations of the sample arrays in the shortest possible time with the lowest possible costs. The disadvantages of the prior art should be avoided or at least reduced.

Another object of the present invention is to provide an above-mentioned device for determining the relevance of preparations of sample arrays, which allows a rapid and reliable relevance control and beyond as simple and robust as possible and inexpensive to produce.

The first object according to the invention is achieved in that preferably each preparation is scanned nondestructively, from the data obtained together with the positions of the individual samples on the preparation data fields of each sample of the preparation are generated, selected from each sample data field at least one parameter and this or one derived therefrom Value or a combination of parameters or values derived therefrom is compared with at least one threshold value, and the comparison value is used as a criterion for the relevance of the individual sample, and those individual samples of the preparation whose relevance is below a predetermined relevance threshold are identified as being unusable, and that the reference value together with the position of the individual sample as well as the positions of all unrecognizable individual samples are stored together with a unique identifier of the preparation.

The method according to the invention thus provides for subjecting any number of preparations or each preparation to a relevance check, which is made possible by the fact that the preparations are preferably examined nondestructively and thus are still available for subsequent examinations. For this purpose, nondestructive treatment and scanning of each preparation and collection of the corresponding data is performed. The positions, The given positions at which the individual samples of a sample array are to be arranged or the determined actual positions of the individual samples may be at which individual samples are present in the sample array. Due to the positions at which individual samples should or should be present, the data can be decomposed into data fields per sample and these data fields are fed to further processing. For this further processing, at least one parameter is selected and this or a value derived therefrom or a combination of parameters or values derived therefrom is compared with at least one threshold value. The comparative value obtained per individual sample is finally used as a criterion for the relevance of the individual sample and stored together with the position information of the individual sample of the preparation. As a result of the method according to the invention, there is thus a data record which contains a relevance criterion for each individual sample of the preparation. It is important that not only an overall statement about the quality of the preparation is made, but also a statement about which individual samples are present or not. In the simplest case, this relevance criterion can be binary, ie only make a statement as to whether the individual sample is present or not. Such a relevance control carried out up to each preparation also makes it possible to use those preparations for subsequent investigations in which various individual samples are not present. The data obtained in the relevance control are included for the following, for example, histological examinations of the preparation, so that the data obtained from defective or non-existing individual samples can be excreted in the preparation and thus can not lead to misinterpretation. Only by means of such an additional data record, which makes a reliable statement about the relevance of the preparation, are automated analyzes of the preparations of sample arrays, in particular tissue sample arrays, possible. The procedure for determining the relevance of preparations may be carried out immediately prior to the examination of the preparations or at an earlier point in time, and the data, together with further information about the preparations, may be stored in a database so that they are available for subsequent examinations. Alternatively to storing the data in one Database, these can also be stored in the so-called flat file format. By means of the method according to the invention, it is possible, for example in TMAs (tissue microarrays), to subject a much larger number of sections of sample arrays to subsequent examinations and thus obtain more information from frequently limited tissue resources for diagnostic, therapeutic and research purposes. Thus, there is a data set that clearly identifies the individual samples deemed to be unusable due to relevance control. Thus, preparations with non-existent or unvaluable individual samples are suitable for subsequent investigations, since nonexistent or destroyed individual samples are clearly identified and the data resulting from subsequent investigations of such individual samples can be discarded. Thus, more preparations of a sample array are available to subsequent studies without the risk of misinterpretation. Of course, a combination of the nondestructive method of the present invention with other methods in which individual preparations are impaired or even destroyed is also possible, thereby providing important additional information.

Advantageously, the preparation is optically scanned by sample arrays and the resulting image is stored. By such an optical method, the preparation is not affected and is therefore still available for subsequent investigations. Therefore, theoretically, all preparations may be subjected to an assay to determine relevance without reducing the number of preparations available for subsequent studies. In addition, the optical scanning is possible with very high speed and also automated, whereby many specimens can be examined in a short time.

The preparations can be irradiated with light and a picture of translucent light taken. This simple method includes a light source and a camera or flatbed scanner that picks up the transmitted light passing through the specimen. In this way, for example, missing individual samples can be detected particularly easily. With the transmitted light method, a negative of the captured image can be made, which facilitates the detection of missing or destroyed individual samples due to contrast differences.

As an alternative or in addition to the transmitted-light method, the preparations are preferably excited with light, in particular laser light, and an image of the resulting autofluorescence radiation is recorded and stored. The exploitation of autofluorescence, d.i. the resulting radiation from elements excited with light of a particular wavelength is another suitable method of investigation which does not destroy the sample. Especially in the case of specimens of tissue sample array specimens, in which individual, circular specimens are usually bedded in paraffin, the autofluorescence examination is particularly suitable since, in contrast to conventional tissue specimens, paraffin causes less fluorescence radiation and thus a clear contrast between the specimen and the surrounding paraffin As a result of this high contrast between individual sample and paraffin, the statement as to whether a single sample is present or not can be explained by particularly simple methods

Perform image processing procedure. The simpler the evaluation method, the less computer power is required for the fastest possible automatic passage of relevance control.

Especially with tissue samples from human or animal organisms, light sources of different wavelengths or light sources with a broader wavelength range, for example mercury lamps, and various filters can be used. In the case of a fluorescence microscope, for example, ultraviolet lamps and three different filters having, for example, the following characteristics are employed.

For example, in the case of fluorescence scanners, lasers at two different wavelengths are combined with highly specific fluorescent dyes, e.g. CY3 (indocarbocyanine) or CY5 (indodicarbocyanine). For example, CY3 can be excited at 530 nm and emits light with a wavelength of 595 nm. CY5 is excited at 630 nm and emits fluorescence radiation at 680 nm.

Better results can also be achieved by exciting the preparation of the sample arrays with combined light of different wavelengths. In the case of such "multispectral imaged ^λΛ , different light sources are used, and as a result more information is obtained. For example, lasers such as argon ions or helium / neon lasers are available as light sources. Moreover, instead of lasers, light sources with a wide wavelength range can be used to detect the presence or absence of the individual samples. For example, mercury lamps or fiber optic devices can be used as light sources.

In order to facilitate the subsequent processing of data, the images of the preparations are preferably stored in a standardized format, for example in TIFF or JPG format. This also allows the application of existing image processing programs and does not require conversion of the data prior to the examination.

Advantageously, the image of the preparation is transformed into at least one binary image. A binary image consists of a matrix of logical zeros and logical ones, from which the probability of the existence of a single sample can be determined and thus a statement about the relevance of the single sample can be made. Such binary images are generated so that the parameter used is compared with one or more thresholds. If more than one para- meter, several binary images can be generated, which can be combined later in the algorithm.

The recorded images of the preparations can be filtered according to various aspects. Both mechanical filters, which are placed in front of the camera or the like for taking the pictures, as well as electronic filters, which are traversed by the image data, are used.

As a parameter selected from each sample data field and used to determine the relevance of the preparations, the fluorescence intensity can be used, which is preferably averaged over the cross-section of the single sample and used as a parameter to determine the relevance of the individual samples. For example, the fluorescence intensity in two directions, in particular in the two main axis directions, can be detected via the normally circular cross section of the individual sample and a statement about the relevance of the individual sample can be made from the resulting distribution. The data is compared with a predetermined threshold and then the comparison value used as a criterion for the relevance of the single sample. The respective threshold value can result from empirical values or can also be determined automatically by means of standardized statistical methods, for example so-called box-plot methods. When using the fluorescence intensity as a parameter, the values are preferably set in proportion to the intensity of the surrounding pixels and a distribution of the fluorescence intensity over the pixels of the image is generated. As derived values of a parameter, for example, the variability in fluorescence intensity or the like can be used.

In this case, the unusable individual samples of a sample of the sample array can be summed up. From this sum, a statement can be made about the number of still usable individual samples of a preparation.

If the sum of the unworkable samples of a preparation is compared with a given limit, and if If the sample of the sample array preparation is identified as being unusable, the preparation can be excluded from subsequent investigations. This makes sense in the case of a particularly large number of unworkable individual samples per cut, since this makes it possible to avoid subsequent particularly delicate, expensive and possibly also very costly investigations.

Likewise, the preparation can be classified on the basis of the determined sum of unworkable individual samples and a classification value can be stored together with a unique identifier of the preparation. This important information can be used for subsequent examinations of the preparation. For example, it may be expedient to use for particularly time-consuming and cost-intensive investigations only those preparations in which no or only a very small number of individual samples are unusable, which thus have particularly high quality. On the other hand, in studies which are quick and cheap to carry out, a preparation with a large number of unrecognizable individual samples, ie a preparation of low quality, can be used and thereby provide important information. The unique identifier of the preparation can be provided, for example, by an identification number, which can also be arranged in the form of a barcode next to the preparation, for example on the glass carrier. By reading the barcode, the information stored in a database about the relevance of the preparation can then be called up and used for subsequent examinations.

In addition to the above methods, a microscopic image of the preparation can also be taken and used for relevance determination. Such microscopic images may contain further interesting information. By superimposing the microscopic image with the image, which results, for example, from fluorescence radiation, the statement about the relevance of the individual samples of the preparation can be improved.

In addition to or as an alternative to the fluorescence intensity, the geometric shape of the individual sample can also be determined from the single sample data be used as a parameter to determine the relevance of the individual samples. For example, from the single sample data field, the contour of the individual sample can be determined by different image recognition methods and compared with the ideal shape of the individual sample, for example the circular shape. If the determined form of the individual sample is too great, it can be rejected as being unusable. For example, the introduction of cylindrical specimens into the paraffin block sometimes results in tissue sample array deformations which may distort subsequent assay results.

To be able to carry out the relevance check as quickly as possible, preferably several preparations are automatically processed sequentially or in parallel and the data obtained on the relevance of the individual samples of the preparations stored together with an identifier of the preparations. Thus, data on the relevance of the preparations can be collected and stored after preparation of the preparations of sample arrays. These data are then available for a selection of preparations for specific subsequent examinations.

The theoretical positions of the individual samples on a preparation, in particular Gewebsprobenarray, are preferably predetermined and stored. These position values can be used to generate the data fields of each individual sample of the preparation.

According to a further feature of the invention, it is provided that the actual positions of the individual samples on a preparation are determined from an image of the preparation. This can be done, for example, by using the so-called "Region Growing." Using this mathematical method, certain pixels of the image, the so-called seed points, are specified by means of a random generator and a certain number of surrounding pixels are included in the calculation The intensity of the seed point is compared with the intensities of the surrounding points and included in the calculation.The "Region Growing" method is particularly suitable for determining the size of surfaces, the number of surfaces, the edge length of Surfaces, to fit a curve to the edge of a surface, to calculate the center of gravity and higher moments, and the circular variance or elliptic variance. The determination of the actual position of the individual samples may be carried out by applying the "region growing" method followed by a method of determining the center of the individual samples.

Furthermore, the actual positions of the individual samples can be compared with the possibly stored, theoretical positions and, if deviated, the predefined position information can be corrected accordingly. The so-called "gridding" analyzes the data and maps them onto a usually rectangular grid, which can be used to counteract the fact that when making, for example, sections from sample arrays, the pattern of the individual samples is often distorted is clearly defined, relevance control can also be performed more reliably.

The second object according to the invention is also achieved by an abovementioned device for determining the relevance of preparations of sample arrays, in which a device for non-destructive scanning of the specimens of the specimen arrays is provided, which scanning device has a database containing the information about the positions of the specimens of the specimens and is connected to a computer unit for processing the sampled preparations and for generating data fields per individual sample and for selecting at least one parameter from each individual sample data field and for comparing this parameter or a value derived therefrom or a combination of parameters or values derived therefrom with at least one threshold value , and in that a device for displaying the value of the comparison of the at least one parameter or a value derived therefrom or a combination of parameters or values derived therefrom with the at least one Threshold as a criterion for the relevance of the single sample, and a memory for storing this relevance criterion is provided together with the position of the single sample of the preparation. A device for determining the relevance of preparations of sample arrays according to the present invention is therefore usually made a computer unit which is connected to a corresponding scanning device and processes the information obtained accordingly.

In this case, the scanning device is preferably formed by a light source and a device for receiving an image of the preparation. In the case of the transmitted-light method, the light source above the preparation and the scanning device are arranged below the preparation, so that the scanning device can detect the light transmitted through the preparation. In the case of autofluorescence, the light source and the scanning device are arranged above the preparation.

The recording device can be formed by a fluorescence scanner, which records the fluorescence radiation of the preparation, which was excited by a corresponding light source.

The light source can be formed by a laser, wherein the wavelength is adapted to the particular circumstances and the use of any fluorochromes.

Likewise, a plurality of light sources may be provided in different wavelength ranges or else a light source which emits light in a very broad wavelength range.

Furthermore, a device for transforming the recorded image of the section into at least one binary image can be provided.

In order to increase the relevance of the data obtained, a filter device for filtering the recorded images of the preparations may be provided. As already mentioned above, these may be filters which are arranged in front of the recording device in terms of hardware, but also filters which make a correction of the data obtained by software.

In addition, a microscope for receiving the preparations may be provided to provide additional information for determining relevance. In order to allow the quickest possible analysis, a device for automatic supply and removal of the preparations can be provided.

Likewise, a magazine for receiving a plurality of preparations may be provided, from which the preparations for relevance determination are automatically removed and returned. Thus, a semi-automated relevance determination of the preparations can be achieved.

Preferably, a device for determining the actual positions of the individual samples is provided on the preparation. Thus, the real position of the individual samples, which often does not agree with the desired position, can be reliably determined.

Finally, a device for correcting the positions of the individual samples on the preparation can be provided, which is connected to a device for receiving the preparation for determining the actual positions of the individual samples. As a result, the distortions of the grid of the arranged individual samples which are usually produced when a sample array is being cut can be corrected.

Hereinafter, the present invention will be described with reference to the accompanying drawings.

Show:

Fig. 1 is a schematic diagram illustrating the preparation of sections from sample arrays;

FIG. 2 is a plan view of an example of a section of a sample array; FIG.

3 is a block diagram illustrating the method for determining the relevance of preparations of sample arrays according to the present invention;

FIGS. 4a and 4b show examples of an existing and a nonexistent or heavily damaged individual sample of a preparation;

FIGS. 5a and 5b show the measurement results in the individual samples according to FIG Figures 4a and 4b using the fluorescence intensity as a parameter;

Fig. Βa and βb show two possible data sets of the individual samples according to Figs. 4a and 4b;

FIG. 7 shows the measurement result of a further embodiment of the method according to the invention utilizing autofluorescence; FIG. and

8 shows a block diagram of an embodiment of the device for determining the relevance of preparations of sample arrays.

1 shows a schematic diagram for illustrating the production of preparations 4 or sections from sample arrays 1, in particular tissue sample arrays (TMAs tissue microarrays). The sample array 1 consists of a cuboid paraffin block 2 into which individual cylindrical samples 3, in particular tissue samples, are introduced. Due to the different origin of the individual samples 3, but also due to mechanical problems when introducing the individual samples 3 into the paraffin block 2, the depth of the individual samples 3 in the paraffin block 2 is different in practice. In the preparation of preparations 4 or sections from the sample array 1, which is preferably carried out with a microtome, especially in deeper regions of the sample array 1 often missing individual samples 3 at different positions. These missing individual samples 3 on the preparations 4 are not available in subsequent histological or histopathological examinations and thus reduce the information obtained from the examinations. It is therefore very important to be able to make a statement about the relevance of the preparations 4.

As shown schematically in the left image of FIG. 1, a selection of preparations 5 or sections of the sample array 1 for quality control (QC) is used according to the prior art. The preparations 5 are usually histologically stained and made a statement about the presence or absence of a single sample 3 on the preparation 5 due to color differences in the microscope image. The products 5 are no longer available for other examinations after relevance control. This reduces the total number of used In addition, a relevance check carried out according to the state of the art offers relatively insecure information since it is not possible to make any reliable statements about their relevance for the preparations between the randomly selected preparations 5.

There is therefore a need for a method and a device for determining the relevance of the preparations 4 of a sample array 1, which are not destroyed by the relevance control and thus are still available for subsequent investigations.

FIG. 2 shows a plan view of a preparation 4 of a sample array 1, in which the individual samples 3 are arranged in a specific pattern, which permits an unambiguous assignment of the individual samples 3. In the example shown, the columns are binary-coded with a part of the holes for the individual samples 3. Thus, it is not possible after preparation of the preparations 4 to confuse the individual samples 3, for example, by turning or twisting the glass carrier. The position of the individual samples 3 in the sample array 1 or on the preparation 4 is clearly assigned.

3 shows a block diagram for illustrating the method according to the invention for determining the relevance of preparations 4 of sample arrays 1. Starting with the production of a sample array 1 according to block 101, the preparations 4 are produced from the sample arrays 1 according to block 102. Thereafter, according to block 103, the relevance control is initiated without destroying the preparations 4. According to block 104, the preparations 4 of the sample arrays 1 containing a plurality of individual samples 3 are scanned non-destructively, resulting in a data field of the preparation 4. The non-destructive scanning of the preparations 4 can be carried out, for example, by transmitted light and / or by the use of autofluorescence. According to block 105, the information of the positions of the individual samples 3 on the preparation 4 is used to generate data fields of each individual sample 3 of the section 4. In this case, the theoretical position of the individual sample 3 on the preparation 4 or after the comparison of the determined actual positions of the individual samples 3 on the preparation 4 with the theoretical positions (according to the "grid These individual sample data fields are now processed in accordance with block 106 such that at least one parameter is selected from the single sample data field and this or a value derived therefrom or a combination of parameters or values derived therefrom is compared with at least one threshold according to block 107 and the comparison value is used as the relevance criterion for the individual sample 3 and stored together with the position of the individual sample 3 of the preparation 4. The result of the relevance control obtained in block 107 can be combined with results of additional examinations of the preparation 4 according to block 108 and in block 109 These results of additional examinations of preparation 4, created in block 108, may be different, also manually performed, methods which provide additional information in block 110, and the data is stored. Relevance determination creates important information which improves the interpretation of the data from the preparations 4 of the sample arrays 1 and permits a better utilization of the preparations 4.

FIGS. 4a and 4b show examples of autofluorescence images of two individual samples 3 of a preparation 4 of a sample array 1. These are schematic images of actual measurement results. The individual sample 3 from FIG. 4 a is almost ideally circular and stands out clearly from the surrounding paraffin block 2. Fig. 4a shows the image of a single sample 3 with very high quality.

In contrast, Fig. 4b shows a severely deformed hole 5 in the paraffin block 2, in which a residue 6 of the single sample 3 or paraffin is present. This single sample 3 is greatly deformed or not present and is therefore not available for subsequent investigations.

FIGS. 5a and 5b show a variant of a method for determining the relevance of sample array sections using the individual samples 3 according to FIGS. 4a and 4b. In this case, for example, the fluorescence intensity I is chosen as the parameter, whose distribution is evaluated as a function of the x and y axes becomes. FIG. 5a shows in diagram 7 the sum of the fluorescence intensity ΣI as a function of the x-axis and diagram 8 the sum of the fluorescence intensity ΣI in the y-direction. The curves of the diagrams 7 and 8 have a point of inflection which shows a local maximum of the fluorescence intensity ΣI substantially in the middle of the single sample 3. The intensity profiles corresponding to the diagrams 7 and 8 can now be further processed, for example an average value can be formed and compared with one or more threshold values. This comparison value is used as relevance criterion for the individual sample 3 and stored together with the position information of the individual sample 3 of the preparation 4, for example in a database. The intensity curves according to FIG. 5a show a particularly high quality of the individual sample 3 according to FIG. 4a.

On the other hand, the curves 7 and 8 of the fluorescence intensities I in the x and y directions in the individual sample according to FIG. 5 b are significantly different and have on the one hand a substantially lower intensity and on the other hand a course deviating significantly from the ideal case, which serves as a criterion for the relevance of the Single sample 3 can be used. In the example shown, the course of the fluorescence intensity I has a local minimum in comparison to the individual sample 3 according to FIG. 5a. From the parameter of the fluorescence intensity I, therefore, a statement about the relevance of the individual sample 3 on the preparation 4 of the sample array 1 can be made particularly quickly and reliably.

FIGS. 6a and 6b show two further possible data sets of the individual samples 3 according to FIGS. 4a and 4b. In this case, the autofluorescence intensity I is represented as a function of the pixel of the recording of the individual sample 3, that is to say of the individual sample data field, and different values are calculated therefrom. These derived parameters, such as extreme values, mean values or the like, as shown by the horizontal lines, can be used as a relevance criterion for the single sample 3. As can be seen from the diagram according to FIG. 6 b, the intensities I differ significantly from those according to FIG. 6 a as a function of the localization of the individual pixels of the data field of the individual sample 3. Figures 6a and 6b show the results of so-called boxplot measurements. The box is the rectangle determined by the quartiles, which comprises 50% of the data. The length of the box shows the interquartile range. This is a measure of the variance determined by the difference of the upper and lower quarti. Another quartile is the median (solid line). The other horizontal lines are called whiskers whose maximum length is 1.5 times the interquartile range and can be determined from the data. The top and bottom dashed lines show the extremes of the upper and lower whiskers. Values that are outside these limits are called outliers and used to determine the relevancy of the sample.

FIG. 7 shows a further example of a simple relevance determination according to the present method, the fluorescence intensity I being represented on a logarithmic scale as a function of the position L of the individual sample 3. The dark dots indicate positions of the individual samples 3 or pixels of the image of the individual sample 3, on which material is present, and the white dots positions of the individual samples 3 without material. Black dots below the manually determined threshold S indicate that material is not available for technical reasons. This is used as a criterion for the relevance of the single sample 3. The determination of the threshold value S can also be determined automatically from the data of the preparation, wherein a change of the threshold value from one passage to another is possible in so-called learning systems.

Finally, FIG. 8 shows a block diagram of a possible device 10 for determining the relevance of preparations 4 of sample arrays 1, comprising a plurality of individual samples 3 at different positions. The device 10 has a device 11 for non-destructive scanning of the preparations 4 of the sample arrays 1. The scanning device 11 may be connected to a database 12, which contains information about the position of the individual samples 3 of the preparations 4. The scanning device 11 is formed in particular by a light source 13, preferably a laser, and a device 14 for receiving a Image of Specimen 4. For further information, a microscope 15 for taking an image of the specimens 4 may be arranged. The scanning device 11 is connected to a computer unit 16, which processes the data of the scanned preparations 4 accordingly and generates data fields per individual sample 3. In the computer unit 16, a parameter from each individual sample data field is selected and this parameter or a value derived therefrom or a combination of parameters or values derived therefrom is compared with at least one threshold value and the comparison value is displayed on a display device 17, for example a screen, and in a memory 18 stored as relevance criterion together with the position of the single sample 3 of the preparation 4. For more efficient processing of the method for determining the relevance of the preparations 4, a device 19 for the automatic supply and removal of the preparations 4 may be provided, which preferably with a magazine 20 for receiving a plurality of preparations 4, which were removed from a corresponding bearing 21 , connected is.

Although the examples mainly deal with tissue sample arrays (TMAs), the present invention, as already mentioned, is applicable to a wide variety of sample arrays containing a plurality of individual samples.

Example of application of the method to the sections of a tissue sample array

The objective method for determining the relevance of sample array preparations is suitable for identifying the available individual samples per preparation. In the present example, a Tissue Microarray (TMA) array of 450 individual "cores." Each tissue sample array contains 30 individual samples of a particular organ or tissue, which are listed in the rows of the following table In order to determine the relevance of preparations of sample arrays, the number of individual samples per tissue can now be determined, which is essential for subsequent examinations of these preparations is. If, on the other hand, only the number of non-existent individual samples or destroyed individual samples without their position were determined, this would provide insufficient information in the present case of a tissue microarray, since, for example, no statement can be made about which tissue type more or fewer individual samples are present. The table below shows the application of the method according to the invention to two sections of a tissue sample array consisting of 450 individual samples from 15 different tissues. Each missing or destroyed individual sample is uniquely identified, so that in the end the number of usable individual samples can be determined. Column 2 of the table shows the number of usable individual samples per tissue type at section no. 4 of the tissue sample array. A total of 14 of the 450 individual samples are defective and thus not usable or missing and are therefore also not available for subsequent investigations. Column 3 of the table shows the result for section # 137 of the same tissue sample array. In this case, 223 out of 450 individual samples have already been destroyed or missing. This clearly shows the problem described above that many individual samples do not protrude so far into the paraffin block and thus are absent in deeper sections of the tissue sample array. For certain examinations or for examinations on certain types of tissue, however, with knowledge of this additional information, it is now also possible to use those sections in which more individual specimens are unusable and the limited number of tissue specimens can be optimally utilized. For example, examination of tissue samples from the cervix, mamma, or ovary at section No. 137 will be useful, since in this case a majority of the individual samples will be present, whereas for example only four out of 30 liver samples will be present. The knowledge of the missing individual samples of a preparation can therefore be used to filter the data for subsequent automated analyzes.

Each individual sample of the tissue sample array usually also contains information about the patient. For example, the 30 individual samples per tissue type in the present example come from 10 different sources, for example 10 different patients. Thus, from the same source Ie or the same patient ideally 3 individual samples. The method according to the invention determines the relevance of each individual sample of the preparation, so that, for example, a statement can be made as to which type of tissue all 3 of the ideally present individual samples can be utilized or from which only 2 or only 1 or no single sample can be utilized , Of course, this information is of considerable importance for certain studies on the preparation. How the information obtained by the method according to the invention are processed, however, depends strongly on the particular application and the subsequent investigations on the preparations.

TABLE

Claims

Claims:

1. A method for determining the relevance of preparations (4) of sample arrays (1), in particular tissue sample arrays containing a plurality of individual samples (3) at different positions, characterized in that preferably each preparation (4) is scanned without destruction, from the data obtained from each individual sample (3) of the preparation (4) are generated together with the positions of the individual samples (3) on the preparation (4), from each individual sample data field at least one parameter selected and this or a value derived therefrom or a combination of parameters or derived values are compared with at least one threshold, and the comparison value as a criterion for the relevance of the single sample

(3) and those individual samples (3) of preparation (4) whose relevance is below a given relevance threshold are identified as being of no value, and that the comparison value together with the position of the single sample (3) and the positions of all unworkable samples (3) together with a unique identifier of the preparation (4).

2. The method according to claim 1, characterized in that the preparation (4) optically scanned and the image is stored.

3. The method according to claim 2, characterized in that the preparation (4) illuminated with light and an image of the translucent light is recorded.

4. The method according to claim 2 or 3, characterized in that the preparation (4) with light, in particular laser light, is excited and an image of the resulting fluorescence radiation of the preparation (4) is recorded and stored.

5. The method according to claim 4, characterized in that the recorded image is filtered.

6. The method according to claim 4 or 5, characterized in that the preparation (4) is excited with combined light of different wavelengths.

7. The method according to any one of claims 3 to 6, characterized in that the image of the preparation (4) is stored in a standardized format, for example in TIFF or JPG format.

8. The method according to any one of claims 3 to 7, characterized in that the image of the preparation (4) is transformed into at least one binary image.

9. The method according to any one of claims 4 to 8, characterized in that the recorded images of the preparations (4) are filtered.

10. The method according to any one of claims 1 to 9, characterized in that as a parameter, the fluorescence intensity, preferably averaged over the cross section of the single sample (3) and used as a parameter for determining the relevance of the individual samples (3).

11. The method according to any one of claims 1 to 10, characterized in that the unrecognizable individual samples (3) of a preparation (4) are summed.

12. The method according to claim 11, characterized in that the sum of the unrecognizable individual samples (3) of a preparation (4) is compared with a predetermined limit and when this limit is exceeded, the preparation (4) of the sample array (1) is identified as unusable.

13. The method according to claim 11 or 12, characterized in that the preparation (4) is classified on the basis of the determined sum of unverwertbarer individual samples (3) and a classification value is stored together with a unique identifier of the preparation (4).

14. The method according to any one of claims 1 to 13, characterized in that a microscopic image of the preparation (4) is taken and used for relevance determination.

15. The method according to any one of claims 1 to 14, characterized draws that the geometric shape of the single sample is determined from the single sample data field and used as a parameter for determining the relevance of the individual samples (3).

16. The method according to any one of claims 1 to 15, characterized in that several preparations (4) automatically processed sequentially or in parallel and the data obtained on the relevance of the individual samples (3) of the preparations (4) together with an identifier of the preparations (4 ) get saved.

17. The method according to any one of claims 1 to 16, characterized in that the positions of the individual samples (3) on a preparation (4) are predetermined and stored.

18. The method according to any one of claims 1 to 17, characterized in that the actual positions of the individual samples (3) on a preparation (4) from an image of the preparation (4) are determined.

19. Method according to claim 18, characterized in that the actual positions of the individual samples (3) are compared with the stored positions, and in case of deviation the stored position information is corrected accordingly.

20. Device (10) for determining the relevance of preparations (4) of sample arrays (1), in particular tissue sample arrays, containing a plurality of individual samples (3) at different positions, characterized in that a device (11) for non-destructive scanning of Preparations (4) of the sample arrays (1) is provided, which scanning device (11) with a the information about the positions of the individual samples (3) of the preparations (4) containing database (12) and with a computer unit (16) for processing the scanned Preparations (4) and for generating data fields per individual sample (3) and for selecting at least one parameter from each sample data field and for comparing this parameter or a value derived therefrom or a combination of parameters or values derived therefrom with at least one threshold value, and in that a device (17) for displaying the value of the Comparison with the at least one threshold value as relevance criterion for the single sample (3), and a memory (18) for storing this relevance criterion together with the position of the single sample (3) of the preparation (4) is provided.

21. The device according to claim 20, characterized in that the scanning device (11) by a light source (13) and means (14) for receiving an image of the preparation (4) is formed.

22. The apparatus according to claim 21, characterized in that the receiving device (14) is formed by a fluorescence scanner.

23. Device according to claim 21 or 22, characterized in that the light source (13) is formed by a laser.

24. The apparatus of claim 21 to 23, characterized in that a plurality of light sources (13) are provided in different wavelength ranges.

25. Device according to one of claims 21 to 24, characterized in that means for transforming the recorded image of the preparation (4) is provided in at least one binary image.

26. Device according to one of claims 21 to 25, characterized in that a filter device for filtering the recorded images of the preparations (4) is provided.

27. Device according to one of claims 20 to 26, characterized in that a microscope (15) for receiving the preparations (4) is provided to provide additional information for determining relevance.

28. Device according to one of claims 20 to 27, characterized in that a device (19) for automatic supply and discharge of the preparations (4) is provided.

29. Device according to one of claims 20 to 28, characterized indicates that a magazine (20) is provided for receiving a plurality of preparations (4), from which the preparations (4) for relevance determination are automatically removed and returned again.

30. Device according to one of claims 20 to 29, characterized in that a device for determining the actual position of the individual samples (3) on the preparation (4) is provided.

31. Device according to one of claims 20 to 30, characterized in that a device for correcting the positions of the individual samples (3) on the preparation (4) is provided, which correction device with a device for receiving the preparation (4) for determining the actual positions of the individual samples (3) is connected.