SE530748C2 - Determination of the number of white blood cells - Google Patents

Abstract

A measurement apparatus for enumeration of particles or white blood cells in a sample comprises: a holder, which is arranged to receive a sample acqu iring device that holds a sample, an imaging system, comprising a magnifying means and at least one digital image acquiring means, said imaging system b eing arranged to acquire at least one digital image of the sample, and an im age analyser, which is arranged to analyse the digital image for identifying particles or white blood cells and determining the number of particles or w hite blood cells and which is arranged to analyse the digital image for iden tifying particles or white blood cells that are imaged in focus, determining types of these particles or white blood cells, the types being distinguishe d by physical features and determining the ratio of different types of parti cles or white blood cells.

Description

25 30 35 53D 748 2 There are a small number of existing automatic analysis methods for determining the number of white blood cells. The number of white blood cells can be determined using the Coulter principle which is based on determining the cell size and thereby the cell type by sensing an impedance. A method for counting white blood cells according to the Coulter principle is described in US 5,262,302. Measuring equipment according to the Coulter principle is expensive and it is therefore a significant investment. Thus, a hospital, or laboratory, will be reluctant to invest in more than one device. This means that the analysis must be performed in a centralized location and that a patient must wait for analysis results.

The Coulter principle is the dominant automated analysis method currently used. However, some other methods have been described. Such a method for determining the number of white blood cells is described in US 5,585,246. Here, a blood sample must be prepared by mixing with a complex between fluorescent dyes and a ligand, which labels the white blood cells. The sample is inserted into a capillary and irradiated by a laser source which scans over the sample in the capillary. Fluorescence is measured to determine the number of white blood cells. A similar method is described in WO 97/02482, using a fluorescent dye and a laser source scanning over a capillary. This method is suitable for determining the number of white blood cell apheresis products containing a small number of white blood cells. Here the capillary is quite thick and it is necessary to wait until the white blood cells have sunk to the bottom of the capillary before the capillary can be scanned. WO 99/45384 discloses a chamber of varying thickness containing a sample. The varying thickness separates different blood compositions. The blood sample is stained with a dye for distinctive highlighting of at least three different blood cell types in the blood sample. The number of white blood cells can be determined using an optical scanning instrument to look at a part of the chamber.

WO 98/50777 describes a method for estimating the number of somatic cells in milk. The method comprises applying a sample volume to a sample chamber and exposing a set of detection elements to electromagnetic signals that have passed from the sample chamber. The intensities of the detected electromagnetic signals are processed and the results are correlated with the number of cells found in the sample.

There is still a need to speed up and simplify existing automated methods for determining the number of white blood cells so that the assay can be performed by any user, without the need for any training, and so that the measuring device can be relatively inexpensive. This would indicate that the assay can be performed at the place where treatment is performed. Furthermore, since the determination of the number of white blood cells is such a commonly performed assay, any improvement in the assay method would have a major impact on patient care. An analytical method that provides an opportunity to obtain results at the site where treatment is performed would be particularly advantageous.

It may also be advantageous to obtain a differential determination of the number of white blood cells, i.e. to examine the distribution of different types of white blood cells in a blood sample. This differential determination of the number of white blood cells can reveal if the cells are present in a normal distribution, or if any cell type has increased or decreased. The information can be useful in diagnosing specific types of diseases. For example, an increase in neutrophils indicates a bacterial infection, while an increase in lymphocytes is common in acute viral infections.

The differential number of white blood cells can also be obtained by microscopically looking at and manually counting stained blood cells in a Bürker chamber. There are also some automated methods. For example, a differential number can be obtained with the Coulter principle by analyzing the shape and magnitude of the electric pulse generated by a cell passing through an electric field. The shape and size of the pulse can be related to the type of white blood cell that is detected. Such a method is described in US 4,528,274. US 5,123,055 describes another method for identifying different types of white blood cells. This method requires that storle your size and color parameters be analyzed sequentially to distinguish the types of white blood cells.

It is still desirable to speed up and simplify existing methods for differential determination of white blood cell count. It would be particularly advantageous to provide a fast, simple and inexpensive assay method so that the assay can be provided on site.

Summary of the Invention An object of the invention is to provide a simple assay for volumetric determination of the number of white blood cells in a blood sample and differential determination of the number of white blood cells. It is a further object of the invention to provide a rapid assay without the need for complicated devices or extensive sample formulations.

These objects are achieved in part or in full by means of a measuring device, a sampling device and a method according to the independent claims.

Preferred embodiments appear from the dependent claims.

Thus, according to one aspect of the invention, there is provided a measuring device for determining the number of white blood cells in a blood sample.

The measuring device comprises a holder, which is arranged to receive a sampling device comprising a measuring cavity which houses a colored and hemolyzed blood sample, and an electromagnetic radiation source, which is arranged to irradiate the blood sample which is accommodated in the measuring cavity of the sampling device. The measuring device also comprises an image system comprising a magnifying means and at least one device for taking digital images.

The imaging system is arranged to take at least one digital image of the blood sample.

The measuring device further comprises an image analyzer which is arranged to analyze said at least one taken digital image for identifying white blood cells distinguished by selective staining with a staining agent and determining the number of white blood cells in the blood sample and which is arranged to analyze said at least one taken digital image for identification. of white blood cells which are depicted in focus, determining the types of these white blood cells, the types being distinguished by geometric properties of the stained cells and determining the ratio of different types of white blood cells in the blood sample.

According to another aspect of the invention, there is provided a method of determining the number of white blood cells in a blood sample. The method comprises taking a blood sample into a measuring cavity of a sampling device.

The blood sample is mixed with a reagent, comprising a hemolyzing agent for lysing the red blood cells in the blood sample and a staining agent for the selective staining of the white blood cells in the blood sample. The method further comprises irradiating the sample comprising the stained white blood cells, taking at least one digital image of a magnification of the irradiated sample in the measuring cavity. The method further comprises digital analysis of said at least one digital image for identifying white blood cells that have been stained with the selective staining and determining the number of white blood cells in the sample, and digital analysis of said at least one digital image for identifying white blood cells depicted in focus. , determining the types of these white blood cells, the types being distinguished by geometric properties of the stained cells and determining the ratio of different types of white blood cells in the blood sample.

The measuring device and the method according to the invention both enable simple analysis of a whole blood sample. For this purpose, the measuring device is arranged to take at least one digital image of a blood sample, which sample has been mixed with a dye for selective staining of the white blood cells. The selective staining of the white blood cells means that the white blood cells can be distinguished in a digital image and that different types of white blood cells can be distinguished by geometric properties of the cells in the same or in another digital image.

The measuring device and the method are thus arranged to make both a volumetric determination of the number of white blood cells inside the blood sample and a differential determination of the number of white blood cells.

While many existing methods can count different blood cells and also subgroups of blood cells, the measuring device according to the invention is specifically arranged for analysis of white blood cells. The reagent comprises a hemolyzing agent which will lyse the red blood cells in the blood sample. This destroys the ability to determine the number of red blood cells in the sample. On the other hand, the lysis of the red blood cells simplifies the distinction and identification of the white blood cells within the blood sample.

Furthermore, the measuring device is specifically arranged for analysis of the at least one digital image so that the cells which are imaged in focus are identified. This allows an image to be taken of a relatively thick sample while only the cells in focus are counted. This can be used to deal with the fact that the determination of the total number of white blood cells is much easier made than the identification of the type of white blood cells because the identification of the type requires that more details of the cell be analyzed. Thus, by ensuring that only cells in focus are counted, the identification of the white blood cell type can be done in a sample that can be used simultaneously for a statistically reliable volumetric determination of all white blood cells in the sample.

The measuring device and the method according to the invention provide a very simple analysis of a whole blood sample. The analysis does not require complicated measuring device or that advanced steps are performed by an operator. Therefore, it can be performed in direct connection with the examination of a patient without the need for a qualified technician. All you need to do is take a blood sample and mix it with a dye. Then the blood sample can be placed in the holder of the measuring device 10 15 20 25 30 35 530 743 6 and as a direct response to this the measuring device can present analysis results.

The blood sample may in fact be allowed to mix with the reagent in the measuring cavity. Thus, no sample preparation will need to be performed manually. Within a few minutes or less, the reaction of the blood sample with the reagent will have hemolyzed the red blood cells and stained the white blood cells so that the sample is ready for transfer to the optical instrument. The blood sample can be mixed with the reagent by, for example, dispersion or diffusion of the reagent into the blood sample or by active vibration or movement of the sampling device so that a stirring is induced in the measuring cavity.

According to one embodiment, the image system is arranged to provide information about the direction of the light in the captured image, whereby focus shifts in the captured image are made possible. This means that a single image can be used both for determining the total number of white blood cells with analysis of the entire depth of the sample at once and for determining the ratio of different types of white blood cells in the blood sample by analyzing cells in the image when the image is displayed with a part of the thickness of the blood sample in focus. An image including information about the direction of the light into the image can be obtained using a group of small lenses that provide the ability to track rays in the captured image so that different parts of the image can be placed in focus.

According to another embodiment, the imaging system is arranged to take a first and a second digital image of the blood sample using different optical settings. The image analyzer is arranged to analyze the first digital image taken to determine the number of white blood cells in the sample and the image analyzer is arranged to analyze the second digital image taken to determine the ratio of different types of white blood cells in the blood sample.

Thus, the measuring device is specifically adapted to take two digital pictures using different optical settings. This means that the optical settings can be optimized and adapted to, firstly, determine the number of white blood cells within a volume and, secondly, determine a ratio between different types of white blood cells.

The magnifying means may comprise an objective lens which is divided for the various optical settings. This means that the digital images can be obtained by imaging along the same optical path so that the images are centered in the same place in the measuring cavity. This makes the measuring device compact. According to one embodiment, the magnifying means comprises two at least partially separated parts which direct light from an irradiated sample to a first and a second device for taking digital images. This means that the light direction from the sample to the device for taking digital images can be defined within a fixed optical arrangement. Thus, the measuring device can be robust and insensitive to shocks.

The imaging system may further include a beam splitter for directing the light from the objective lens toward the first and second digital image capture devices. This means that the first and the second digital image can be obtained simultaneously, whereby the analysis can be performed very quickly.

The first device for taking digital pictures can be arranged to receive light directly from the beam splitter, i.e. no optical element is arranged between the first device for taking digital pictures and the beam splitter.

Alternatively, the light may be arranged to pass directly from the objective lens to the first digital image capture device. Then, to obtain the second digital image, a mirror can be placed in the beam of light to divert light to the second digital image capture device instead.

The magnifying means may further comprise an ocular lens between the beam splitter and the second device for taking digital images.

Thus, the ocular lens can provide a further magnification of the sample to allow differentiation between different types of white blood cells.

Preferably, the lens package is used and the ocular lens package will then move a virtual main plane within the objective lens package to change the relationship between the image plane and the objective lens package to enable further magnification.

The magnifying means may further comprise an optical element between the beam splitter and the second device for taking digital images for influencing cells which are not located in focus of the imaging system, thereby simplifying the identification of white blood cells imaged in focus.

The optical element enables an image to be taken of a sample thickness that is much greater than the depth of field of the image system. The optical element ensures and enables the cells that are out of focus to be taken into account to increase the security of the measurement. Since the optical element affects the image of cells that are out of focus, the cells that are in focus will be easily identified. The optical element can be implemented as a spatial filter which affects the image of a cell so that the cell edge will comprise an overshoot intensity greater than the background intensity, where the cell is imaged by absorbing light.

Alternatively, the identification of the cells imaged in focus can be accomplished by image analysis alone. For example, the image analyzer may be arranged to analyze edges of imaged cells to assess whether the cell is imaged in focus based on the slope of the intent curve at the edge. Cells that are not in focus will show a slow decrease in intensity at the edges, whereby cells that are in focus will be imaged with a sharp edge represented by a large decrease in intensity at the cell edge. Thus, by analyzing how the intensity varies at an edge of an imaged cell, it can be determined whether the cell is imaged in focus or not.

According to an alternative embodiment, the magnifying means may further comprise a wavefront coding part between the beam splitter and the second device for taking digital images. A wavefront coding portion intentionally distorts the light rays by sending them through a wave plate with a saddle-like shape, which is relatively flat in the middle, but with pointed edges.

This causes a specific optical focusing error, the image looks blurry, but the defocus is the same over a large distance. This wavefront coding part thus increases a depth along the optical axis that can be analyzed. The distortions in the image are mainly determined by the shape of the defocusing wavefront coding part which is accurately known.

Therefore, a computer can remove blur point by point. A computer can decode the image using what is essentially a digital filter and thus creates an image that is sharp over a large depth of field. In this way, the magnifying means can increase the depth of field of the imaging system, which enables a greater depth of the sample to be imaged in focus.

According to another embodiment, a device for taking digital pictures is arranged to take both the first and the second picture and at least a part of the magnifying means is adjustable for taking the first and the second picture using different optical settings. This means that the measuring device only needs to contain a device for taking pictures.

Furthermore, it enables your different optical settings to be used by, for example, providing a main lens that is movable between well-defined positions along the optical axis. The main lens can be arranged to be in contact with an edge at each position, whereby the position of the main lens and the optical settings can be carefully controlled. 10 15 20 25 30 35 530 748 9 The magnifying means can be arranged to have a greater degree of magnification in optical settings used for taking the second digital image than in the optical settings used for taking the first digital image.

This means that details can be seen better in the other digital image, whereby different types of white blood cells can be more easily distinguished from each other.

The magnifying means in the optical settings used for taking the first digital image may have a magnification of 1-50x, preferably 1-20x. Within these magnification ranges, the white blood cells are sufficiently enlarged to be detected while the imaging system can be arranged to image the sample thickness within sufficient focus to determine the number of blood cells in the image. Thus, the imaging system may have depths of field that cover the sample thickness. However, the entire sample thickness does not need to be imaged within the depth of field of the imaging system using the usual definition of depth of field. Cells that are imaged slightly out of focus can still be counted correctly using appropriate image analysis. A low magnification means that a large "depth of field" can be obtained. Thus, a large sample thickness can be enabled and a large volume can be analyzed. However, if a low magnification is used, the white blood cells can be difficult to detect because each blood cell is imaged on very few, such as 3-4 pixels. A smaller magnification can be used by increasing the number of pixels in the captured image, i.e. by improving the resolution of the digital image. In this way, it is possible to use an optical magnification of 1-4x while it is still possible to detect the white blood cells. In this context, the "depth of field" means an extension in a direction along the optical axis that is imaged in sufficient focus to enable image analysis to identify cells located within this length. This "depth of field" may be greater than the usual depth of field defined by the optical settings.

The magnifying means in the optical settings used for taking the second digital image may have a magnification of 5-200x, more preferably 5-20x. Within these magnification ranges, the white blood cells are sufficiently enlarged to allow differentiation between different types of white blood cells. A lower magnification can be used using an optical element to amplify cells depicted in focus and to facilitate identification of these cells.

The imaging system may be arranged to obtain the first image with a depth of field of at least the thickness of the measuring cavity of the sampling device. This means that sufficient focus is achieved for the entire sample thickness so that the entire thickness of the measuring cavity can be analyzed simultaneously in the digital image of the sample. Thus, it is not necessary to wait for the white blood cells to settle in the measuring cavity, thereby shortening the analysis time. However, there may be a need to wait for a reaction that causes the red blood cells to hemolyze and to wait for movements caused by the introduction of the sample into the measuring cavity to subside. These waiting times would be very short, of the order of 30 seconds or less.

By choosing not to focus very sharply on a specific part of the sample, sufficient focus is achieved for the entire sample thickness to enable identification of the number of white blood cells in the sample. This means that a white blood cell may be slightly blurred and still be considered the focus of the depth of field. Thus, the analyzed volume of the sample can be well defined by the thickness of the measuring cavity and the size of the digital image that defines the cross-sectional area of the measuring cavity being imaged.

The image system may be arranged to obtain the first image with a depth of field in the range of 50-200 micrometers. This depth of field can be adjusted to correspond to the thickness of the measuring cavity. A thickness of at least 50 micrometers means that the measuring cavity does not force the blood sample to be smeared into a single layer, which enables analysis of a larger blood volume over a small cross-sectional area.

Thus, a sufficiently large volume of blood sample can be analyzed to give reliable values for the number of white blood cells using a relatively small image of the blood sample. Furthermore, it is difficult to achieve a depth of field exceeding 200 micrometers while producing a digital image with sufficient magnification. It is even difficult to achieve a depth of field that exceeds 170 micrometers.

The image system can be arranged to obtain the second image with a depth of field in the range 20-60 micrometers. This can be achieved by imaging a part of the thickness of the measuring cavity. In this case, only this part of the thickness of the measuring cavity is depicted in focus. The second digital image is then analyzed by including only white blood cells that have been imaged in sufficient focus to determine their type. Since the second digital image is used to determine the relationship between different types of white blood cells, it is not important to image a well-defined volume. Thus, it is possible to obtain a suitable first and second image by imaging the same part of the measuring cavity. However, the second image may alternatively be taken so as to image another part of the measuring cavity, this part may have a thickness corresponding to the depth of field of the image system to obtain the second image.

The electromagnetic radiation source may be arranged to radiate a wavelength corresponding to an absorption peak of the colorant.

Consequently, the stained white blood cells containing an accumulation of dye will be detected by an indication of low light transmittance in the digital images.

The electromagnetic radiation source may contain a laser source.

The laser source can produce light of an inverted wavelength that matches the absorbance of the dye. Furthermore, the laser source produces collimated light, which minimizes the interference from scattered light, so that a point with low light transmittance will be clearly distinguished.

The electromagnetic radiation source may alternatively contain an LED. This source of radiation may still provide sufficient radiation conditions to properly distinguish white blood cells from another substance in the sample.

The image analyzer may be arranged to identify areas of high light absorption to determine the number of white blood cells in the blood sample.

The image analyzer may further be arranged to identify black or dark dots in the image. Since the dyes can accumulate in the nuclei of the white blood cells, the light absorbance may have peaks at separate points.

These dots will form black dots in the digital image and can be classified as white blood cells.

The image analyzer may be arranged to electronically magnify said at least one captured image. While the sample is enlarged to take an enlarged digital image of the sample, the taken digital image itself can be electronically enlarged to facilitate distinction between objects imaged very close to each other in the taken digital image.

The image analyzer may be arranged to distinguish different types of white blood cells by analyzing the shape and size of identified areas of high light absorption in said at least one digital image. Because different types of white blood cells have different sizes, the type of white blood cell can be identified by determining the size of the blood cell. Furthermore, the different types of white blood cells can be stained differently, which gives the identified parts of the image different shapes. This can also be used to identify the type of white blood cell. A differentiated number of white blood cells specifying the ratio of three different types of white blood cells can be obtained by analyzing only the size of the white blood cells. A differential determination of the number of white blood cells that distinguishes five different types of white blood cells may require further blood cell characteristics to be examined.

For example, one can examine a number of nuclei per cell, an intensity of radiation transmitted through the blood cell or the shape of the blood cell.

According to a further aspect of the invention, a sampling device is provided for determining the number of white blood cells in a blood sample. The sampling device comprises a measuring cavity for receiving a blood sample. The measuring cavity has a first and a second predetermined solid thickness defined between inner walls of the measuring cavity, the first thickness being adapted for volumetric determination of the total number of white blood cells in the blood sample and the second thickness being adapted for determining a ratio of different white blood cells in the blood sample. .

The sampling device further comprises a reagent, which is arranged in dried form on a surface defining the measuring cavity. The reagent comprises a haemolysing agent for lysing red blood cells in the blood sample and a staining agent for the selective staining of white blood cells in the blood sample.

The sampling device provides an opportunity to directly obtain a whole blood sample into the measuring cavity and provide it for analysis. There is no need for sample preparation. The blood sample can actually be sucked into the measuring cavity directly from a stuck finger of a patient. Providing the sampling device with a reagent enables a reaction in the sampling device which prepares the sample for analysis. The reaction is initiated when the blood sample comes in contact with the reagent. Thus, there is no need for manual sample preparation, which makes the analysis particularly suitable to perform directly in an examination room while the patient is waiting.

Since the reagent is provided in dried form, the sampling device can be transported and stored for a long time without affecting the usefulness of the sampling device. Thus, the sampling device with the reagent can be manufactured and prepared long before performing the blood sample analysis.

While many existing methods can count different blood cells and also subgroups of blood cells, the sampling device according to the invention is specifically arranged to perform determination of the number of white blood cells. The reagent comprises a hemolyzing agent which will lyse the red blood cells in the blood sample. This destroys the ability to determine the number of red blood cells in the sample. On the other hand, the lysis of the red blood cells facilitates the differentiation and identification of the white blood cells in the blood sample. The dye produces a label on the individual white blood cells. This allows individual viewing or individual detection of the white blood cells. The white blood cells can be detected, for example, by scanning the measuring cavity or by obtaining an image of the measuring cavity.

The sampling device further provides a first measuring cavity thickness which is specially arranged to facilitate volumetric determination of the number of white blood cells. The measuring cavity may have a sufficient thickness to enable analysis of a fairly large volume of blood sample and therefore enable a good statistic for volumetric determination of the number of white blood cells. The number of white blood cells can thus be obtained by summing the number of individually detected white blood cells in a defined volume.

The sampling device also provides a second measuring cavity thickness which is specially arranged to facilitate the distinction between different types of white blood cells. In this regard, the second thickness may be thinner than the first thickness, which allows imaging of the entire second thickness within a depth of field for greater magnification. Such a larger magnification may be needed in distinguishing between different types of white blood cells compared to imaging for the sole purpose of determining the total number of white blood cells, regardless of type, within the blood sample.

The sampling device may comprise a body part having two flat surfaces which form inner walls for defining said measuring cavity. The flat surfaces may be arranged at a predetermined distance from each other to determine the sample thickness of an optical instrument. This means that the sampling device provides the optical instrument with a well-defined thickness, which can be used for accurate determination of the number of white blood cells per unit volume of the blood sample. A volume of an analyzed sample will be well defined by the thickness of the measuring cavity and an area of the sample being imaged. Thus, the well-defined volume can be used to associate the number of white blood cells with the volume of the blood sample in the volumetric determination of the number of white blood cells.

The measuring cavity preferably has a first, even thickness of 50-200 micrometers. A thickness of at least 50 micrometers means that the measuring cavity does not force the blood sample to be smeared into a single layer, which enables analysis of a larger blood volume over a smaller cross-sectional area. Thus, a sufficiently large volume of blood sample can be analyzed to obtain reliable values for the number of white blood cells using a relatively small image of the blood sample. The first thickness is more preferably at least 100 micrometers, which enables analysis of an even smaller cross-sectional area or a larger sample volume. Furthermore, the first thickness of at least 50 micrometers and more preferably 100 micrometers also facilitates the manufacture of the measuring cavity which has an inverted thickness between two flat surfaces.

For most samples arranged in a cavity having a thickness of not more than 200 micrometers, the number of white blood cells is so low that only small deviations due to white blood cells arranged overlapping each other occur. However, the effect of such deviations may be related to the number of white blood cells and can thus, at least to some extent, be managed by means of statistical correction of results at least for large values for the number of white blood cells. This statistical correction can be based on calibrations of the measuring device. The deviations can be even smaller for a measuring cavity having a first thickness of not more than 170 micrometers and even smaller for a measuring cavity having a first thickness of not more than 150 micrometers, whereby a simpler calibration can be used. This thickness may also not require any calibration of overlapping blood cells.

Furthermore, the first thickness of the measuring cavity is small enough to enable the measuring device to obtain a digital image such that the entire depth of the entire measuring cavity can be analyzed simultaneously. Since a magnifying means is to be used in the measuring device, it is not easy to achieve a large depth of field. Therefore, the first thickness of the measuring cavity would preferably not exceed 150 micrometers for simultaneous analysis of the entire thickness of the digital image. The depth of field can be arranged to handle a first thickness of the measuring cavity of 170 micrometers or even 200 micrometers.

The measuring cavity preferably has a second uniform thickness of 20-60 micrometers. This second thickness of the measuring cavity would allow imaging of the entire second thickness within a depth of field for a magnification needed to distinguish between different types of white blood cells. Furthermore, the second thickness can still allow imaging of a sufficient volume, which enables analysis of a significant number of white blood cells. This would make it possible to determine the ratio of different types of white blood cells with good statistical certainty. It is typically desirable to analyze the type of 200 white blood cells.

The sampling device may be provided with a reagent which has been applied to the surface dissolved in a volatile liquid which has evaporated to leave the reagent in dried form.

It has been appreciated that the reagent is preferably dissolved in a volatile liquid prior to introduction into the measuring cavity. This means that the liquid can be efficiently made to evaporate from the limited space of the measuring cavity during manufacture and preparation of the sampling device. The reagent may preferably be arranged in dried form in the part of the measuring cavity which is of the first thickness.

The reagent may preferably be dissolved in an organic solvent and more preferably dissolved in methanol. Such solvents are volatile and can be suitably used for drying the reagent on a surface of the measuring cavity. the staining agent may be arranged to selectively stain the nuclei of the white blood cells. This means that the white blood cells can be identified as colored dots and are therefore easily counted in a digital image. Furthermore, the size of the color dots can be used to identify the type of white blood cells, since different types of white blood cells have different sizes. the coloring agent may be from the group of hematoxylin, methylene blue, methylene green, methylenazur, cresol violet acetate, toluidine blue, gentian violet, sudan counterparts, gallocyanin, and fuchsin counterparts, or any combination thereof. However, it will be appreciated that the colorant is not limited to this group, but many other substances may be considered.

The haemolysing agent may be a quaternary ammonium salt, a saponin, a bile acid such as a deoxycholic acid, a digitoxin, a snake venom, a glucopyranoside or a nonionic detergent of the Triton type. However, it will be appreciated that the hemolysing agent is not limited to this group, but many other substances may be considered.

The sampling device may further comprise an inlet for the sample, which inlet connects the measuring cavity to the exterior of the sampling device, the inlet being arranged to take up a blood sample. The inlet for the sample may be arranged to draw up a blood sample with a capillary force and the measuring cavity may further draw blood from the inlet into the cavity. In addition, the sampling device may be arranged to first draw the sample into the part of the measuring cavity which is of the first thickness. Part of the sample can then be transported further with capillary force into the part of the measuring cavity which is of the second thickness. As a result, the blood sample can be easily inserted into the measuring cavity by simply moving the inlet of the sample so that it is in contact with blood. Then the capillary forces of the inlet of the sample and the measuring cavity will draw a well-defined amount of blood into the measuring cavity. Alternatively, the blood sample can be aspirated or drawn into the measuring cavity by applying an external pumping force to the sampling device. According to another alternative, the blood sample can be taken up into a pipette and then inserted into the measuring cavity by means of the pipette.

The sampling device can be of the disposable type, i.e. it is arranged to be used only once. The sampling device provides a kit for determining the number of white blood cells, since the sampling device is capable of receiving a blood sample and contains all the reagents needed to hand over the sample to the cell count. This is made possible in particular because the sampling device is adapted for use only once and can be designed without considering the possibilities of cleaning the sampling device and reapplying a reagent.

In addition, the sampling device can be formed of plastic material and thereby manufactured at a low price. Thus, the use of a disposable sampling device can still be cost effective.

Brief Description of the Drawings The invention will now be described in more detail by way of example and with reference to the accompanying drawings.

Fig. 1 is a schematic view of a sampling device.

Fig. 2 is a schematic view of a measuring device according to a first embodiment.

Fig. 3 is a schematic view of a measuring device according to a second embodiment.

Fig. 4 is a flow chart of a method according to an embodiment of the invention.

Fig. 5 is a schematic view of a device for a movable lens according to an alternative to the measuring device shown in Fig. 3.

Fig. 6 is a schematic view of a device according to another alternative to the measuring device shown in Fig. 3.

Fig. 7a shows recorded intensities for a cross section of a cell to be analyzed, when the cell is placed in focus for a measuring device according to fi g 3. Fig. 7b shows recorded intensities for a cross section of a cell to be analyzed analyzed, when the cell is placed out of focus of a measuring device according to fi g 3.

Detailed Description of Preferred Embodiments Referring to Fig. 1, a sampling device 10 according to a first embodiment will be described. The sampling device 10 is disposable and should be discarded after use for analysis. This means that the sampling device 10 does not require complicated handling.

The sampling device 10 is preferably formed of a plastic material and can be manufactured by injection molding. This makes the manufacture of the sampling device 10 simple and inexpensive, whereby the costs of the sampling device 10 can be kept down.

The sampling device 10 comprises a body part 12, which has a base 14, which can be touched by an operator without causing any disturbance in the analysis results. The base 14 may also have projections 16 which can fit a holder in an analysis device. The projections 16 can be arranged so that the sampling device 10 is correctly positioned in the analysis device.

The sampling device 10 further comprises a sample inlet (sample inlet) 18. The sample inlet 18 is defined between opposite walls within the sampling device 10, the walls being arranged so close together that a capillary force can be created in the sample inlet 18. The sample inlet 18 communicates with the sampling device 10 to enable of blood being drawn into the sampling device 10. The sampling device 10 further comprises a chamber for counting white blood cells in the form of a measuring cavity 20 arranged between opposite walls inside the sampling device 10. The measuring cavity 20 is arranged to communicate with the sampling inlet 18. The walls defining the measuring cavity 20 are arranged closer to each other than the walls of the sample inlet 18, so that the capillary force can draw blood from the sample inlet 18 into the measuring cavity 20.

The measuring cavity 20 has a first part 20a which is of a first thickness and a second part 20b which is of a second, smaller thickness. The first part 20a communicates with the provin | oppet18, the second part 20b communicating with the first part 20a. Thus, a capillary force can draw blood from the first part 20a of the measuring cavity 20 into the second part 20b.

The walls of the first part 20a of the measuring cavity 20 are arranged at a distance from each other of 50-200 micrometers. The first part 20a is more preferably at least 100 micrometers thick. Furthermore, the first part is more preferably not more than 150 micrometers thick. The distance is equal over the entire first part 20a. The thickness of the first portion 20a defines the volume of blood being examined. Since the test result is to be compared with the volume of the blood sample being examined, the thickness of the first part 20a must be very accurate, i.e. only very small variations of the thickness are allowed within the first part 20a and between first parts 20a of different sampling devices 10. The thickness allows analysis of a relatively large sample volume in a small area of the cavity. The thickness theoretically allows white blood cells to be arranged on each other within the first part 20a. However, the number of white blood cells in the blood is so low that the probability of this happening is very low.

The first part 20a of the measuring cavity 20 is specially adapted for volumetric determination of the total number of white blood cells in a blood sample. The entire thickness of the first part 20a is intended to be imaged within a depth of field of an imaging system. Then an image can be analyzed and the number of existing white blood cells in the image can be counted for volumetric determination of the number of white blood cells.

The sampling device 10 is typically adapted to measure the number of white blood cells above 0.5 x 109 cells / liter of blood. At lower white blood cell counts, the sample volume is too small to allow the counting of statistically significant amounts of white blood cells. Furthermore, when the number of white blood cells exceeds 12 x 109 cells / liter of blood, the effect of white blood cells being arranged overlapping each other begins to become significant in the measured number of white blood cells. At this number of white blood cells, the white blood cells will cover approximately 8% of the cross-section of the sample which is irradiated if the thickness of the first part 20a is 140 micrometers.

Thus, in order to achieve the correct number of white blood cells, this effect must be taken into account. Therefore, a statistical correction of the values of the number of white blood cells above 12 x 109 cells / liter of blood can be used. This statistical correction increases with increasing number of white blood cells because the effect of overlapping blood cells is greater for larger numbers of white blood cells. The statistical correction can be determined by calibrating a measuring device. Alternatively, the statistical correction can be determined at a general level by setting measuring devices for use in connection with the sampling device 10. This statistical correction is of the same order of magnitude as statistical corrections currently performed in analysis devices using the Coulter principle. The sampling device 10 is intended to be used for analyzing the number of white blood cells larger than 50 x 109 cells / liter of blood.

The second part 20b of the measuring cavity 20 is specially arranged for determining a ratio between different types of white blood cells in a blood sample. The entire thickness of the second part 20a is intended to be imaged within a depth of field of an imaging system. Then an image can be analyzed and the number of white blood cells of each type present in the image can be counted to determine the ratio of different types of white blood cells.

The walls of the second part 20b of the measuring cavity 20 are arranged at a distance of 20-60 micrometers from each other. The distance is equal over the entire second part 20b. Since the assay is primarily intended for comparing the number of different types of white blood cells with each other, it is not critical to know the exact volume being analyzed. Therefore, the thickness of the second part 20b must not be as exact as the thickness of the first part 20a. The thickness of the second portion 20b must allow a sufficient number of white blood cells to be analyzed to obtain statistically significant results. Furthermore, as mentioned above, the thickness of the second part 20b should be adapted for imaging in its entirety within a depth of field of an imaging system.

Thus, all white blood cells within the sample are imaged in focus and the analysis of the sample is not hindered by noise in the image from parts of the sample that are imaged out of focus. The second portion 20b is thinner than the first portion 20a to allow the use of a larger magnification while allowing the entire second portion 20b to be imaged within a depth of field of the imaging system. The larger magnification may be needed to enable not only the counting of the total number of blood cells, but also the determination of the type of white blood cells.

A surface of a wall of the measuring cavity 20 is at least partially coated with a reagent 22. The reagent 22 may be freeze-dried, heat-dried or vacuum-dried and applied to the surface of the measuring cavity 20. When a blood sample is taken into the measuring cavity 20, the blood comes into contact with the dried reagent 22 and initiates a reaction between the reagent 22 and the blood.

The reagent 22 is applied by inserting the reagent 22 into the measuring cavity 20 using a pipette or dispenser. Reagent 22 is dissolved in a volatile liquid, such as an organic solvent such as methanol, upon introduction into the measuring cavity 20. The solvent with the reagent 22 can fill the measuring cavity 20. Then drying is performed so that the solvent evaporates and the reagent 22 adheres to the surfaces of the measuring cavity 20. Since the reagent is intended to be dried on the surface of a limited area, the liquid will have a very small surface which is in contact with the surrounding atmosphere, making evaporation of the liquid much more difficult.

Thus, it is advantageous to use a volatile liquid, such as methanol, which enables the liquid to evaporate efficiently from the limited area of the measuring cavity.

According to an alternative manufacturing method, the sampling device 10 may be formed by connecting two parts to each other, one part forming the bottom wall of the measuring cavity 20 and the other part forming the top wall of the measuring cavity 20. This allows the reagent 22 to dry on an open surface before the two parts are connected to each other. Thus, the reagent 22 can be dissolved in water because the solvent need not be volatile.

Reagent 22 comprises a hemolyzing agent and a coloring agent. The haemolysing agent may be a quaternary ammonium salt, a saponin, a bile acid such as a deoxycholic acid, a digitoxin, an urinary toxin, a glucopyranoside or a nonionic detergent of the Triton type. The coloring agent may be hematoxylin, methylene blue, methylene green, methylenazur, cresol violet acetate, toluidine blue, gentian violet, a sudan equivalent, gallocyanin, or a fuchsin equivalent, or any combination thereof. When a blood sample comes in contact with reagent 22, the hemolyzing agent will act to lyse the red blood cells so that the lysed red blood cells are mixed with the blood plasma. Furthermore, the dye will accumulate in the nuclei of the white blood cells.

Reagent 22 should contain sufficient amounts of dye to clearly stain all the nuclei of the white blood cells. Thus, there will often be an excess of colorant which is mixed into the blood plasma. The excess of dye gives a homogeneous, low background level of dye in the blood plasma. The accumulated dye in the white blood cells will be distinguishable from the background level with dye.

Reagent 22 may also contain other ingredients which may be active, i.e. take part in the chemical reaction with the blood sample, or non-active, i.e. not take part in the chemical reaction with the blood sample. The active ingredients may, for example, be arranged to catalyze the haemolysing or coloring action. The non-active ingredients may, for example, be arranged to improve the attachment of the reagent 22 to the wall surface of the measuring cavity 20. In 15 minutes or less than a minute, the blood sample will have reacted with reagent 22 so that the red blood cells have been lysed and the staining agent has accumulated in the nuclei of the white blood cells.

Referring to Fig. 2, a first embodiment of a measuring device 30 for analyzing white blood cells in the blood sample will be described.

The device 30 includes a sample holder 32 for receiving a sampling device 10 with a blood sample. The sample holder 32 is arranged to receive the sampling device 10 so that the measuring cavity 20 of the sampling device 10 is correctly positioned inside the device 30. The device 30 comprises a light source 34 for illuminating the blood sample inside the sampling device 10. The light source 34 may be a light bulb. light. The dye accumulated in the nucleus of the white blood cell nuclei will absorb light of specific wavelengths so that the nuclei of the white blood cells appear in a digital image of the sample. If a color image is taken, the white blood cells appear as specifically colored dots. If a black and white image is taken, the white blood cells appear as dark dots against a lighter background.

The light source 34 may alternatively be a laser or an LED. This can be used to increase the contrast in the image so that the white blood cells can be more easily detected. In this case, the light source 34 is arranged to radiate electromagnetic radiation of a wavelength corresponding to an absorption peak of the dye. the wavelength should further be chosen so that the absorption of the blood masses is relatively low. Furthermore, the walls of the sampling device 10 should be substantially transparent to the wavelength. For example, where methylene blue is used as the colorant, the light source 34 may be arranged to emit light of the wavelength 667 nm.

The device 30 further comprises an imaging system 36 which is arranged on an opposite side of the sample holder 32 relative to the light source 34. Thus, the imaging system 36 is arranged to receive radiation which has been transmitted through the blood sample. The imaging system 36 in this embodiment comprises a magnifying means 38 which is divided into two separate parts. A first part 38a of the magnifying means 38 is arranged to receive radiation which has been transmitted through the blood sample in the first part 20a of the measuring cavity 20. The imaging system further comprises a first imaging device 40, which is arranged to image the first part 20a of the measuring cavity 20, as it is enlarged by the first part 38a of the magnifying means 38. The first portion 38a of the magnifying means 38 38a is arranged to provide a magnification of 1-50x, more preferably 1-20x and most preferably 1-4x. Within these magnification ranges, it is possible to distinguish the white blood cells. The image can be taken with an improved resolution to enable the use of a lower magnification. Furthermore, the depth of field of the first part 38a of the magnifying member 38 can still be arranged to at least correspond to the thickness of the measuring cavity 20.

The first portion 38a of the magnifying means 38 includes an objective lens, or lens system, 42 disposed near the sample holder 32 and an ocular lens, or lens system, 44 disposed at a distance from the objective lens 42.

The objective lens 42 provides a first magnification of the sample which is further enlarged by the ocular lens 44. The magnifying means 38 may include additional lenses to provide a suitable magnification and image of the sample.

The first part 38a of the magnifying means 38 is arranged so that the sample in the first part 20a of the measuring cavity 20, when placed in the sample holder 32, is focused on an image plane of the first imaging device 40.

The first imaging device 40 is arranged to take a first digital image of the sample. The first imaging device 40 may be any type of digital camera such as a CCD or CMOS camera. The pixel size of the digital camera places a limitation on the imaging system 36 in such a way that the circle of uncertainty in the image plane must not exceed the pixel size within the depth of field. However, the white blood cells can still be detected even if they are slightly blurred, and therefore the circle of uncertainty may be allowed to exceed the pixel size while being considered within the depth of field, as they fi niered in this context. As used herein, "depth of field" thus means a length in the direction along the optical axis that is imaged in sufficient focus to enable image analysis to identify cells located within that length. This "depth of field" may be greater than the usual depth of field defined by the optical settings.

The digital camera 40 will take a first digital image of the sample in the first part 20a of the measuring cavity 20, the entire sample thickness being sufficiently focused in the first digital image for counting the white blood cells.

The imaging system 36 will define an area of the first portion 20a of the measuring cavity 20, which will be imaged in the first digital image. The area to be imaged together with the thickness of the first part 20a of the measuring cavity 20 defines the volume of the sample to be imaged.

A second part 38b of the magnifying means 38 is arranged to receive radiation which has been transmitted through the blood sample in the second part 20b of the measuring cavity 20. The imaging system further comprises a second imaging device 41, which is arranged to image the second part 20b of the measuring cavity 20 as it is enlarged by the second part 38b of the magnifying means 38. The second portion 38b of the magnifying means 38 is arranged to provide a magnification of 5-200x, more preferably 5-100x and most preferably 5-20x. Within these magnification ranges, it is possible to distinguish the white blood cells. The image can be taken with an improved resolution to enable the use of a lower magnification. Furthermore, the depth of field of the second part 38b of the magnifying member 38 can still be arranged to at least correspond to the thickness of the measuring cavity 20.

Like the first part 38a, the second part 38b of the magnifying means 38 also comprises an objective lens, or lens system, 43 arranged near the sample holder 32 and an ocular lens, or lens system, 45 arranged at a distance from the objective lens 43. The objective lens 43 provides a first magnification. of the sample, which is further enlarged by the ocular lens 45.

The magnifying means 38 may include additional lenses or other optical components to achieve a suitable magnification and image of the sample. The second part 38b of the magnifying means 38 is arranged so that the sample in the second part 20b of the measuring cavity 20, when placed in the sample holder 32, will be focused up on an image plane of the second imaging device 41.

The second imaging device is arranged to receive a second digital image of the sample. The second imaging device 41 may be of some type of digital camera, such as a CCD or CMOS camera. Since the second image is to be used for the determination of different types of white blood cells, the blur circle in the image plane must not exceed the pixel size within the depth of field. The digital camera 41 will take a second digital image of the sample in the second portion 20a of the measuring cavity 20, the entire sample thickness being sufficiently focused in the second digital image to determine the type of white blood cell.

The imaging system 36 is set to be suitable for imaging blood samples in sampling devices 10. There is no need to change the setting of the imaging system 36. Preferably, the imaging system 36 is arranged in a housing so that the settings are not inadvertently changed.

The device 30 further comprises an image analyzer 46.

The image analyzer 46 is connected to the first and second digital cameras 40, 41 for receiving first and second digital images, the digital images being taken by the digital cameras 40, 41. The image analyzer 46 is 10 15 20 25 30 35 530 748 24 arranged to identify patterns in the first digital image, corresponding to white blood cells, for counting the number of white blood cells present in the digital image. Thus, the image analyzer 46 can be arranged to identify dark spots in a lighter background. The image analyzer 46 may be arranged to first electronically enlarge the digital image before analyzing the digital image.

This means that the image analyzer 46 can much more easily distinguish white blood cells that are imaged close to each other, although the electronic magnification of the digital image will make the digital image slightly blurred.

The image analyzer 46 can calculate the number of white blood cells per volume of blood by dividing the number of white blood cells identified in the first digital image by the blood sample volume, which is well as described above. The volumetric determination of the number of white blood cells can be shown on a display of the device 30.

The image analyzer 46 is further arranged to identify patterns in the second digital image, which correspond to a white blood cell, for counting the number of white blood cells present in the digital image. The image analyzer 46 will further analyze the shape and size of each detected white blood cell to determine the type of white blood cell. Thus, the image analyzer 46 may be arranged to identify dark dots in a lighter background as white blood cells. The image analyzer may be arranged to first electronically enlarge the digital image before analyzing the digital image. This means that the image analyzer 46 can much more easily distinguish white blood cells that are imaged close together, although the electronic magnification of the digital image will make the digital image somewhat blurred. The image analyzer 46 will then determine the type of white blood cell primarily by determining the size of the imaged white blood cell. White blood cells that have been determined to be 7-10 micrometers in diameter are classified as lymphocytes. White blood cells that have been determined to be 10-15 micrometers in diameter are classified as granulocytes. White blood cells that have been determined to be 17-25 micrometers in diameter are classified as monocytes. Furthermore, granulocytes can be identified by the presence of two or more dots within a cell corresponding to two or more nuclei. This can be used to improve the determination made in the size classification.

As described above, a determination of the number of white blood cells can be achieved differentiated into three parts by only analysis of the size of the detected blood cells. The determination of the number of white blood cells differentiated into five parts, with granulocytes further differentiated as eosinophils, neutrophils and basophils, can be obtained using additional assay parameters. The determination of the triple number of white blood cells can also be improved by using these parameters. Thus, the assay can further examine the shape of the detected blood cells. The assay can also examine the intensity of the radiation emitted by the detected blood cells.

The image analyzer 46 can calculate the number of white blood cells of each type.

Typically, the image analyzer 46 can count and classify a certain number of white blood cells, say 1000. The percentage or ratio of each type of white blood cell can then be determined as the number of white blood cells classified as belonging to the type divided by the total number of white blood cells analyzed. A statistically significant measure can be determined by analysis of approximately 200 white blood cells. However, it is desirable that a larger number of white blood cells be analyzed to improve the statistics. Furthermore, the image analyzer 46 can be arranged to analyze only white blood cells which are imaged in sufficient focus for the correct classification. In addition, where two or more white blood cells are very close together, they may be difficult to separate properly and thus such white blood cells may be completely rejected by the image analyzer 46. On the other hand, since the image system 36 is arranged to image the entire thickness of the measuring cavity 20, part 20b in focus, the image analyzer 46 can perform a volumetric determination of the number for each type of white blood cell based solely on the second digital image.

The image analyzer 46 can be realized as a processor unit which includes codes for performing the image analysis.

With reference to fi g 3, a second embodiment of a measuring device 130 for analysis of white blood cells in a blood sample will be described.

The device 130 includes a sample holder 132 for receiving a sampling device 110 with a blood sample. The device 130 is arranged to receive sampling devices 110, the measuring cavity 120 being evenly thick over the entire surface to be imaged. Thus, the measuring cavity 120 has a thickness corresponding to the first part 20a of the measuring cavity 20 of the sampling device 10 according to the embodiments described with reference to Fig. 1 above. The sample holder 132 is arranged to receive the sampling device 110 so that the measuring cavity 120 of the sampling device 110 is correctly positioned inside the device 130.

The device 130 includes a light source 134 for illuminating the blood sample within the sampling device 110 in a manner similar to the light source 34 in the first embodiment.

The device 130 further comprises an imaging system 136 which is arranged on an opposite side of the sample holder 132 relative to the light source 134. Thus, the imaging system 136 is arranged to receive radiation which has been transmitted through the blood sample. The imaging system 136 in this embodiment is arranged to take a first and a second digital image along the same optical path so that the images are centered at the same point in the measuring cavity 120. The first and second digital images are still taken using different optical settings.

This can be achieved in a number of different ways, which will be described below.

As shown in Fig. 3, the imaging system includes a magnifying means 138 which includes a common part and two separate parts. The magnifying means may thus comprise an objective lens, or lens system, 142, which is arranged near the sample holder 132 and which is divided for the two optical settings for taking both the first and the second digital image. The objective lens 142 provides a first magnification of the sample. The imaging system 136 may further include a beam splitter 139 for directing light in two different directions toward a first and a second imaging device 140, 141, which imaging devices may be of any type of digital camera, such as a CCD camera.

The magnifying means 138 includes a first ocular lens, or lens system, 144, which is disposed between the beam splitter 139 and the first digital camera 140. The objective lens 142 provides a first magnification of the sample, which is further enlarged by the ocular lens 144. The magnifying means 138 may include additional lenses for providing an appropriate magnification and imaging of the sample in the first digital image.

The magnifying means 138 further comprises a second ocular lens or lens system 145, which is disposed between the beam splitter 139 and the second digital camera 141. The objective lens 142 provides a first magnification of the sample, which is further destroyed by the ocular lens 145. The magnifying means 138 may include additional lenses for providing appropriate magnification and imaging of the sample in the second digital image. The objective lens 142 and the ocular lens 145 are implemented as lens packages and the ocular lens package 145 will then move a virtual principal plane within the objective lens package 142 to change the relationship between the image plane and the objective lens package 142 to allow further magnification, while the sampling device 110 is not moved. 35 530 748 27 lens lens package 142. In this way, different magnifications can be obtained in the first and second digital images.

In particular, the magnifying means 138 shown in this embodiment includes an optical element 147 which amplifies imaging of white blood cells located in focus. This improves the possibilities for identifying which blood cells are imaged in focus and must thereby be taken into account when distinguishing different types of white blood cells.

The optical element 147 enables the taking of an image with a sample thickness which is much greater than the depth of field of the part of the imaging system 136 which takes the second digital image. The optical element 147 ensures that the cells that are out of focus can be discarded to increase the security of the measurement. Since the optical element 147 affects the image of cells that are out of focus, the cells in focus will be easily identified. The optical element 147 may be implemented as a spatial filter which affects the image of a cell so that the cell edge will include an excess of intensity greater than the background intensity, where the cell is imaged by absorbing light. This can be easily detected in image analysis and therefore, these cells can be quickly discarded.

According to an alternative embodiment, the magnifying means may comprise a wavefront coding part between the beam splitter 139 and the second digital camera 141. The wavefront coding part may thus replace the optical element 147.

A wavefront coding portion intentionally distorts the light rays by passing them through a wave plate of saddle-like shape, which is relatively flat in the middle, but with pointed edges. This causes a specific optical focusing error, the image looks blurry, but the defocus is the same over a large distance. This wavefront coding part thus increases a depth along the optical axis that can be analyzed. The distortions in the image are mainly determined by the shape of the defocusing wavefront coding part, which is accurately known. Therefore, a computer can remove blur point by point. A computer can decode the image using what is essentially a digital filter and thus creates an image that is sharp over a large depth of field. In this way, the magnifying means can increase the depth of field of the imaging system, which enables a greater depth of the sample to be imaged in focus.

When replacing the beam splitter, the imaging system 136 may include a mirror or other element (not shown) for directing substantially all of the light from the sample toward a selected one of the two digital cameras 140, 141. The mirror may then be rotated 530 748 28 or moved to shift the camera 140, 141 viewing the sample. This allows more light to pass to the digital cameras 140, 141 and thus provides better lighting conditions for shooting. However, the two images cannot be recorded simultaneously and the image system 136 will need moving parts.

Alternatively, one of the cameras may be arranged to see the sample when the mirror is completely removed from the optical path.

According to another alternative, the objective lens 142 may provide all the magnification needed to obtain the first image. Thus, light can be sent directly from the beam splitter or mirror to the first digital camera 140.

According to yet another alternative, no objective lens is shared by the first and second optical settings. Thus, a beam splitter or mirror may be provided near the sample holder 132 and the magnifying means 138 may include both an objective lens and an ocular lens in both optical paths between the beam splitter and the first digital camera 140 and between the beam splitter and the second digital camera 141.

According to a further alternative, the first and the second digital image are obtained by means of a digital camera. In this case, the magnifier 138 must be replaced or changed to change the optical settings to obtain the two digital images. Thus, an objective lens 242 may be movable between two well-defined positions, as shown in Fig. 5. The objective lens 242 may thus be arranged to move along the optical axis and come into contact with a stop, for example an edge of the optical axis comes into contact with a projection 250, 252. The distance between the objective lens and the sampling device can thus be accurately controlled to control the magnification of an image to be taken. In any of the above-described alternatives, the first digital camera 140 is arranged to image the measuring cavity 120 with a first optical setting provided by the magnifying means 138. The magnifying means 138 is thus arranged to provide a magnification of 1-50x, more preferably 1-20x and most preferably 1-4x. within these magnification ranges, it is possible to distinguish the white blood cells.

The image can be taken with an improved resolution to enable the use of a lower magnification. Furthermore, the depth of field of the magnifying member 138 can still be arranged to at least correspond to the thickness of the measuring cavity 120.

As described with reference to Fig. 2, the first digital camera 140 is arranged to take a first digital image of the sample. The first digital camera 140 looks at the sample so that the thickness of the entire measuring cavity 120 is within the depth of field as defined for the first embodiment. The imaging system 136 will define an area of the measuring cavity 120 that will be imaged in the first digital image. The area imaged together with the thickness of the measuring cavity 120 de fi nines the volume of the sample being imaged.

Further, in any of the above-described embodiments, the second digital camera 141 is arranged to image the measuring cavity 120 with a second optical setting provided by the magnifying means 138.

The magnifying means 138 is arranged to provide a magnification of 5-200x, more preferably 5 ~ 100x and most preferably 5-20x. within these magnification ranges, it is possible to distinguish the white blood cells.

The image can be taken with an improved resolution to enable the use of a lower magnification. However, since the second digital image looks at the same part of the measuring cavity 120 as the first digital image, the larger magnification used in the second optical setting can prevent the entire thickness of the measuring cavity 120 from being imaged within a depth of field. The second digital image will thus depict white blood cells in focus, but it will also depict white blood cells and other parts of the blood sample that are out of focus, causing a blurred image in the image. Under these conditions, the optical element 147, as described above, will improve the possibilities for identifying cells that are imaged in focus and are easier to classify.

The magnifying means 138 can advantageously be arranged to place a top part of the thickness of the measuring cavity 120 in focus in the image plane of the second digital camera 141. This means that the disturbances in the parts of the blood sample that are out of focus are relatively small. However, it is conceivable that any part of the measuring cavity 120 is imaged in focus in the second digital image. Furthermore, the magnifying means 138 may be arranged to typically image a measuring cavity thickness of 20-60 micrometers in focus.

According to yet another alternative, shown in fi g 6, only one digital image is taken. However, this digital image must provide information about the direction of detected light. This means that the digital image contains information about not only the detected radiation but also a place in the room from which the detected radiation was emitted. This digital image can then be displayed in a way so that the focus of the digital image can be shifted as desired. The digital image can thus firstly be used for counting the total number of white blood cells within the depth of the entire measuring cavity and secondly, by focusing shifts to a part of the thickness, for determining the ratio between different types of white blood cells in the sample. This option can be implemented to include a light source 334, an objective lens 342 and a digital camera 340. These parts can be implemented in a manner similar to that described above. The device further includes a set of small lenses 360 provided in the optical path between the sampling device 110 and the digital camera 340. The set of small lenses 360 enables tracking of light rays in the captured image so that different parts of the image can be placed in focus.

To return to fi g 3, the device 130 further includes an image analyzer 146. The image analyzer 146 is coupled to the first and second digital cameras 140, 141 for receiving the first and second digital images taken by the first and second digital cameras. 140, 141.

Alternatively, the image analyzer 146 only receives a digital image including information about the direction of the light, as described in the paragraph above. The image analyzer 146 is arranged to analyze the first and second digital images in a manner similar to that described for the image analyzer 46 according to the first embodiment above. However, since the second digital image can be obtained by imaging only part of the sample thickness within a depth of field, the image analyzer 146 may have to handle the second image more carefully.

First of all, the image analyzer 146 will only analyze white blood cells that have been identified as depicted in focus. This is possible because the image analyzer 146 only determines the ratio of different types of white blood cells and therefore does not need to know the exact volume of the sample being analyzed. Cells imaged out of focus can become blurred in such a way that the image analyzer 146 could determine incorrect cell sizes and therefore classify the cells incorrectly. Thus, by ensuring that only cells imaged in focus are analyzed, the security of the analysis can be improved.

In identifying out-of-focus cells, the image analyzer 146 may use the excess intensities at the edges, produced by the optical element 147, of the out-of-focus white blood cells. Fig. 7a illustrates recorded intensities for a cross-section of a blood cell depicted in focus, Fig. 7b illustrating recorded intensities for a cross-section of a blood cell depicted from focus. It is clear that the intensities at the edges of the cell can be used by the image analyzer 146 to remove cells that are out of focus. According to an alternative, the image analyzer 146 may analyze the edges of the imaged cells to determine whether the cell is imaged in focus, based on the slope of an intensity curve at the edge. Cells that are not in focus will show a slow decrease in intensity at the edges while cells in focus will be imaged with a sharp edge represented by a large decrease in intensity at the cell edge. Thus, by analyzing how the intensity varies at an edge of an imaged cell, it can be determined whether the cell is imaged in focus or not.

The image analyzer 146 is further arranged to determine the size of the white blood cells depicted in focus. The determined size can then be used to classify the white blood cells in a manner similar to the method described above with reference to the first embodiment. Since the second digital image may be somewhat blurred and difficult to analyze, the image analyzer 146 may be arranged to count and classify only a relatively small number of white blood cells, say 200. This may still be sufficient to form a statistically significant result of the relationship between different types of white blood cell sample. Alternatively, the image analyzer 146 may be arranged to measure size and verify whether a cell is imaged in focus within the same image processing step. Consequently, the size of each imaged cell is determined, but only the cells depicted in focus are considered when determining the ratio of different types of white blood cells in the sample.

The image analyzer 146 can be realized as a processor unit which includes code for performing the image analysis.

Referring to Fig. 4, a method for volumetric determination of the number of white blood cells will be described. The method involves taking a blood sample in a sampling device, step 102. An undiluted sample of whole blood is taken up in the sampling device. The sample can be taken from capillary blood or venous blood. A sample of capillary blood can be dropped into the measuring cavity directly from a stuck finger of a patient. The blood sample comes into contact with a reagent in the sampling device which initiates a reaction. The red blood cells will be lysed and a stain accumulates in the nuclei of the white blood cells. Within a few minutes after taking the blood sample, the sample is ready for analysis. Alternatively, a blood sample is taken and mixed with a haemolysing agent and a staining agent before introduction into the sampling device. The sampling device is then placed in an assay device, step 104. An assay can be initiated by pressing a button on the assay device. Alternatively, the assay is initiated automatically when the apparatus detects the presence of the sampling device.

The sample is irradiated, step 106, and a first and a second digital image of the sample are taken, step 108, using various optical settings. The sample is irradiated with electromagnetic radiation of a wavelength corresponding to an absorption peak of the dye. This means that the digital image will contain black or darker dots at the sites of the nuclei of the white blood cells.

The captured digital images are transferred to an image analyzer which performs image analysis of the first and second digital images, step 110.

The image analyzer counts the number of black dots in the first digital image for a volumetric determination of all white blood cells in the blood sample. The image analyzer also analyzes the size and shape of a certain number of black dots in the second digital image to classify the white blood cells and to obtain a ratio of different types of white blood cells in the blood sample.

It should be emphasized that the preferred embodiments described herein are in no way limited and that many alternative embodiments are possible within the scope of the scope of the appended claims.

Claims (54)

10 15 20 25 30 35 530 748 33 E A measuring device for determining the number of white blood cells in a blood sample, said measuring device comprising: a holder, which is arranged to receive a sampling device comprising a measuring cavity containing a colored and hemolyzed blood sample, an electromagnetic radiation source, which is arranged to irradiate the blood sample. accommodated in the measuring cavity of the sampling device, an image system, comprising a magnifying means and at least one device for taking digital images, said image system being arranged to take at least one digital image of the blood sample, and an image analyzer, which is arranged to analyze said at least one taken digital image for identifying white blood cells distinguished by selective staining with a staining agent and determining the number of white blood cells in the blood sample and which is arranged to analyze said at least one taken digital image for identifying white blood cells imaged in focus, determining these white blood cell types, wherein t the types are distinguished by geometric properties of the stained cells and determination of the ratio of different types of white blood cells in the blood sample. Measuring device according to claim 1, wherein the image system is arranged to provide information about the direction of the light in the captured image, whereby shift of focus in the captured image is made possible. The measuring device according to claim 1, wherein the imaging system is arranged to take a first and a second digital image of the blood sample using different optical settings and wherein the image analyzer is arranged to analyze the first taken digital image for determining the number of white blood cells in the sample and the image analyzer is arranged to analyze the second digital image taken to determine the ratio of different types of white blood cells in the blood sample. Measuring device according to claim 3, wherein the magnifying means comprises two at least partially separate parts, which direct light from an irradiated sample to a first and a second device for taking digital images. Measuring device according to claim 4, wherein the magnifying means comprises an objective lens which is divided for the different optical settings. The measuring device according to claim 5, wherein the image system further comprises a beam splitter for directing light from the objective lens towards the first or the second device for capturing digital images. 10 15 20 25 30 35 530 748 34 Measuring device according to claim 6, wherein the first device for taking digital images is arranged to receive light directly from the beam splitter. Measuring device according to claim 6 or 7, wherein the magnifying means further comprises an ocular lens between the beam splitter and the second device for taking digital images. Measuring device according to any one of claims 6-8, wherein the magnifying means further comprises an optical element between the beam splitter and the second device for taking digital images for influencing cells not located in the focus of the imaging system, thereby identifying white blood cells imaged in focus is facilitated. The measuring device according to claim 3, wherein the image analyzer is arranged to analyze edges of imaged cells to determine whether the cell is imaged in focus based on the slope of an intensity curve at the edge. Measuring device according to claim 3, wherein a device for taking digital images is arranged to take both the first and the second image and wherein at least a part of the magnifying means is adjustable for taking the first and the second digital image using different optical settings. Measuring device according to any one of claims 3-11, wherein the magnifying means is arranged to have a greater degree of magnification in optical settings used for taking the second digital image than in the optical settings used for taking the first digital image. Measuring device according to claim 12, wherein the magnifying means used in the optical settings for taking the first digital image has a magnification of 1-50x, more preferably, 1-20x. Measuring device according to claim 12 or 13, wherein the magnifying means used in the optical settings for taking the second digital image has a magnification of 5-200x, more preferably, 5-20x. Measuring device according to any one of claims 3-14, wherein the imaging system is arranged to take the first image with a depth of field of at least the thickness of the measuring cavity of the sampling device. Measuring device according to claim 15, wherein the imaging system is arranged to take the first image with a depth of field in the range 50-200 micrometers. Measuring device according to claim 15 or 16, wherein the image system is arranged to take the second image with a depth of field in the range 20-60 micrometers. 10 15 20 25 30 35 530 748 35 A measuring device according to any one of claims 15-17, wherein a volume of an analyzed sample in the first digital image is dominated by the thickness of the measuring cavity and an area of the sample being imaged. Measuring device according to any one of the preceding claims, wherein the electromagnetic radiation source is arranged to radiate a wavelength corresponding to an absorption peak of the colorant. Measuring device according to any one of the preceding claims, wherein said electromagnetic radiation source comprises a laser source. A measuring device according to any one of claims 1-19, wherein said electromagnetic radiation source comprises an LED. Measuring device according to any one of the preceding claims, wherein the image analyzer is arranged to define areas with high light absorption for determining the number of white blood cells in the blood sample. The measuring device according to claim 22, wherein the image analyzer is arranged to identify black dots for determining the number of white blood cells in the blood sample. Measuring device according to any one of the preceding claims, wherein the image analyzer is arranged to electronically enlarge said at least one taken image. Measuring device according to any one of the preceding claims, wherein the image analyzer is arranged to distinguish different types of white blood cells by analyzing the shape and size of identified areas with high light absorption in said at least one digital image. A sampling device for determining the number of white blood cells in a blood sample, said sampling device comprising: a measuring cavity for receiving a blood sample, said measuring cavity having a first and a second predetermined thickness defined between inner walls of the measuring cavity, said first thickness being adapted. for volumetric determination of the total number of white blood cells in the blood sample and said second thickness is adapted to determine a ratio of different types of white blood cells within the blood sample, and a reagent arranged in dried form on a surface defining the measuring cavity, said reagents include a haemolysing agent for lysing red blood cells in the blood sample and a staining agent for selectively staining white blood cells in the blood sample. 10 15 20 25 30 35 530 748 36 The sampling device according to claim 26, wherein the sampling device comprises a body part having two flat surfaces forming inner walls for defining said measuring cavity. The sampling device of claim 27, wherein the planar surfaces are arranged at a predetermined distance from each other to determine a sample thickness of an optical instrument. A sampling device according to any one of claims 26-28, wherein the measuring cavity has a first uniform thickness of 50-200 micrometers. The sampling device according to claim 29, wherein the measuring cavity has a first uniform thickness of at least 100 micrometers. A sampling device according to claim 29 or 30, wherein the measuring cavity has a first uniform thickness of not more than 150 micrometers. A sampling device according to any one of claims 26-31, wherein the measuring cavity has a second uniform thickness of 20-60 micrometers. A sampling device according to any one of claims 26-32, wherein the reagent has been applied to the surface dissolved in a moist liquid which has evaporated to leave the reagent in a dried form. A sampling device according to any one of claims 26-33, wherein the staining agent is arranged to selectively stain the nuclei of the white blood cells. A sampling device according to any one of claims 26-34, wherein the colorant is one of the group of hematoxylin, methylene blue, methylene green, methylenazur, cresol violet acetate, toluidine blue, gentian violet, sudan counterparts, gallocyanin, and fuchsia equivalent equivalents. A sampling device according to any one of claims 26-35, wherein the haemolysing agent is a quaternary ammonium salt, a saponin, a bile acid, a digitoxin, a snake venom, a glucopyranoside or a non-active detergent of the Triton type. A sampling device according to any one of claims 26-36, further comprising a sample inlet connecting the measuring cavity to the exterior of the sampling device, said inlet being arranged to take a blood sample. A sampling device according to any one of claims 26-37, wherein the sampling device is of a disposable type. A method of determining the number of white blood cells in a blood sample, said method comprising: taking a blood sample into a measuring cavity of a sampling device, wherein the blood sample is mixed with a reagent comprising a hemolyzing agent for lysis of the red blood cells in the blood sample and a stain for selectively staining the white blood cells in the blood sample, irradiation of the sample comprising the stained white blood cells, taking at least one digital image of an enlargement of the irradiated sample in the measuring cavity, digital analysis of said at least a digital image for identifying white blood cells stained by the selective staining and determining the number of white blood cells in the sample, and digital analysis of said at least one digital image for identifying white blood cells depicted in focus, determining the types of these white blood cells, the types being distinguished by geometric properties of the colored c or determination of the ratio of different types of white blood cells in the blood sample. The method of claim 39, wherein the blood sample is mixed with the reagent in the measuring cavity. ' A method according to claim 39 or 40, wherein said at least one digital image is taken so that the information about the direction of the light is recorded in the captured image, whereby focus shift in the captured image is enabled and wherein the analysis is performed by sequential focus shift. The method of claim 39 or 40, wherein taking at least one digital image comprises taking a first digital image of a first magnification of the irradiated sample in the measuring cavity and taking a second digital image of a second magnification of the irradiated sample in the measuring cavity, the second magnification being larger than the first and the first taken digital image being analyzed to determine the number of white blood cells in the sample and the second taken digital image being analyzed to determine the ratio of different types of white blood cells in the blood sample. The method of claim 42, wherein the measuring cavity has a thickness of 50-170 micrometers and wherein said first digital image is taken with a depth of field that at least corresponds to the thickness of the measuring cavity. The method of claim 43, wherein a volume of an analyzed sample in the first digital image is governed by the thickness of the measuring cavity and an area of the imaged sample. 10 15 20 25 530 748 38 The method of claim 43 or 44, wherein the second digital image is taken with a depth of field in the range of 20-60 micrometers. A method according to any one of claims 42-45, wherein the first digital image is taken using a magnification of 1-200x, more preferably 1-20x. A method according to any one of claims 42-46, wherein the second digital image is taken using a magnification of 5-200x, more preferably 5-20x. A method according to any one of claims 39-47, wherein the sample is irradiated with light of a wavelength corresponding to an absorption peak of the dye. A method according to any one of claims 39-48, wherein said irradiation is performed by means of a laser source. A method according to any one of claims 39-48, wherein said irradiation is performed by means of an LED. A method according to any one of claims 39-50, wherein said assay comprises identifying areas of high light absorption to determine the number of white blood cells in the blood sample. The method of claim 51, wherein said assay comprises identifying black dots to determine the number of white blood cells in the blood sample. The method of any of claims 39-52, wherein said analyzing comprises electronically enlarging said at least one taken digital image. A method according to any one of claims 39-53, wherein said assay comprises distinguishing different types of white blood cells by analyzing the shape and size of identified areas of high light absorption in said at least one digital image.