Counting of white blood cells

Technical field [0001] The invention relates to a sampling device, method and system for volumetric calculation of white blood cells in a blood sample.

Background of the invention [0002] Determining the number of white blood cells is often important in connection with treating a patient. This analysis may be needed to diagnose e.g. leukemia, infectious or inflammatory diseases or to monitor treatment. It is desirable to allow the results of the analysis to be obtained as quickly as possible to minimize patient waiting time and allow the physician to make decisions about treatment and diagnosis directly during the first patient examination. It would therefore be beneficial to provide a method of analysis that can be quickly carried out by a doctor or nurse without having to send the test to the laboratory.

[0003] At present, the number of white blood cells is usually obtained by manual procedure, staining the blood sample and microscopic observation of the sample in a special counting chamber, e.g. a B ^ ker chamber. The counting chamber is provided with a grid dividing the chamber into well-defined small volumes. White blood cells can settle at the bottom of the counting chamber to allow microscopic focusing on all cells in the chamber and thus facilitate counting.

Thus, the sample must remain deposited for several minutes before counting. The number of white blood cells can then be determined by counting the number of blood cells per field in the grid. The number of white blood cells is obtained manually by an analyst who must have experience in performing the analysis to enable reliable analysis.

[0004] This analysis is time consuming. In addition, because it is done manually, the results of the analysis may vary depending on the person performing the analysis.

[0005] There are several existing automated methods of analysis for determining the number of white blood cells. The number of white blood cells can be determined for

EP 1 701 150 by means of the Coulter principle, which is based on determining the cell size and thus the cell type by detecting impedance. A method of counting white blood cells according to the Coulter principle is described in US 5,262,302.

[0006] The Coulter principle is the dominant automated method of analysis. However, there are several other ways that have been described. One such method for determining the number of white blood cells is disclosed in US 5,585,246. In this case, the blood sample must be prepared by mixing with a fluorescent dye and ligand complex that tags white blood cells. The sample is introduced into the capillary and is irradiated by a laser source that scans the area above the sample in the capillary. Fluorescence is measured to determine the number of white blood cells. A similar method is disclosed in WO 97/02482, using a fluorescent dye and scanning using a laser source over the capillary. This method is adapted to calculate white blood cells in coating products containing low white blood cell counts. Here the capillary is quite thick and you should wait for white blood cells to settle on the bottom of the capillary before scanning the capillaries.

[0007] WO 99/45384 shows a sample containing a chamber of varying thickness. Different thickness separates different blood compounds. The blood sample is dyed to distinguish at least three different types of white blood cells in the blood sample. White blood cells can be calculated using an optical scanning device to view part of the chamber.

EP 1 161 994 A2 discloses a cuvette for introducing blood and reagent containing products. The cuvette has a hollow main body holding the blood product and a reagent for hemolysis-fluorescence staining. The cuvette is made of colorless and transparent plastic and has a lid made of rubber. Hemolysis-fluorescence staining reagent is added to the cuvette during leukocyte counting. The blood product is then added to the cuvette, and the reagent and blood are mixed and the reagent reacts with the blood product. The cuvette is centrifuged, and after exposure to centrifugal force, it is set on a micro-leukocytometer to count the number of leukocytes in the blood product.

US 5,948,686 discloses a sample chamber comprising a first wall and a second transparent wall. The walls are separated from each other by the thickness of the transverse plane. The thickness of the transverse plane may vary in

EP 1 701 150 depending on hematocrit and target components. The whole blood sample is mixed with the amount of sensitive coloring agent, which may be in liquid or dry form. After mixing the sample, it is introduced into the sample chamber, where the sample stops briefly to allow rollers and gaps to form. The gaps are open areas remaining between the formed rollers, which are clusters of red blood cells. White blood cells are in the gap along with the platelets. White blood cells are detected by the microscope in clear gaps.

[0008] There is still a need to accelerate and simplify existing automated methods for determining the amount of blood cells, so that analysis can be provided at the point of patient care. In addition, since white blood cell counting is such a commonly used analysis, any improvement in the method of analysis would have a major impact on patient care. A method of analysis that would provide results at a patient care point would be particularly beneficial.

Summary of the invention [0009] The object of the invention is to provide a simple analysis for the volumetric calculation of white blood cells. A further object of the invention is to provide rapid analysis without the need for advanced devices.

[0010] These objectives are defined by the sampling device, method and system according to the independent claims. The preferred embodiments are obvious from the dependent claims.

[0011] In this way a sampling device is provided for volumetric calculation of white blood cells in a blood sample. The sampling device includes a measuring cavity for collecting a blood sample, the blood sample being undiluted whole blood that has been directly taken into the measuring cavity. The measuring cavity has a predetermined constant thickness measured such that said thickness, when the device is in use, in conjunction with the area of ​​the sample being imaged determines the volume of the sample being tested. The sampling device further comprises a reagent which is placed in a dried form on the surface defining the measuring cavity, said reagent comprising a hemolytic agent for lysis of red blood cells in a blood sample and a coloring agent for selective staining of white blood cells in a blood sample. The sampling device further includes a sample inlet which is

EP 1 701 150 defined between opposite walls inside the sampling device, said walls being so close together that capillary force can be generated in the sample inlet. The measuring cavity in the sampling device is placed in communication with the sample inlet, and the measuring cavity is defined by two flat surfaces, flat surfaces are arranged at a predetermined distance from each other to determine the thickness of the sample for optical measurement, wherein the flat surfaces of said measuring cavity are arranged closer to each other than said sample inlet walls, so that capillary force can collect blood from the sample inlet into the measuring cavity.

[0012] The sampling device allows direct collection of a whole blood sample into the measuring cavity and providing it for analysis. There is no need for sample preparation. In fact, the blood sample can be drawn into the measuring cavity directly from the patient's punctured finger.

Providing a reagent sample collection instrument allows the reaction in a sample collection instrument to be made ready for analysis. The reaction begins when a blood sample contacts the reagent. So there is no need for manual sample preparation, making the analysis particularly suitable for performing directly in the doctor's room while the patient is waiting.

[0013] Because the reagent is provided in a dried form, the sampling device can be transported and stored for a long time without affecting the usability of the sampling device. Thus, the reagent sample collection instrument can be made and prepared long before the blood sample is analyzed.

[0014] Many existing methods are capable of counting various blood cells and even subsets of blood cells, the inventive sampling device is specially adapted to perform volumetric counting of white blood cells. The reagent contains a hemolytic agent that removes red blood cells in the blood sample. This prevents the possibility of counting red blood cells in the sample. On the other hand, lysis of red blood cells simplifies the differentiation and identification of white blood cells in a blood sample.

[0015] The coloring agent ensures that individual white blood cells are labeled. This allows individual white blood cells to be observed or detected.

EP 1 701 150

White blood cells can, e.g., be detected by scanning the measuring cavity or obtaining an image of the measuring cavity. It is therefore possible to calculate the number of white blood cells by adding up the number of individually detected white blood cells in a given volume.

[0016] The invention also provides a method for volumetric calculation of white blood cells in a blood sample. The method consists in taking a blood sample into the measuring cavity, a sampling device, the blood sample being undiluted whole blood that has been directly introduced into the measuring cavity (20), irradiating the sample, obtaining a digital image of the magnification of the irradiated sample in the measuring cavity and digital digital analysis. an image to identify white blood cells and to determine the number of white blood cells in the sample. In the method according to the invention, the sampling device has a sample inlet which is defined between opposite walls in the sampling device, said walls being so close together that capillary force can be generated in the sample inlet. The measuring cavity in the sampling device is placed in communication with the sample inlet, and the measuring cavity is defined by two flat surfaces, flat surfaces are arranged at a predetermined distance from each other to determine the thickness of the sample for optical measurement, wherein the flat surfaces of said measuring cavity are arranged closer to each other than said sample inlet walls, so that capillary force can collect blood from the sample inlet into the measuring cavity. In addition, the measuring cavity contains a reagent that is placed in a dried form on the surface defining the measuring cavity, comprising a hemolytic agent for lysis of red blood cells and a coloring agent to react with the sample so that the white blood cells are stained, with the white blood cells being distinguished by selective staining. coloring agent.

[0017] The invention further provides a system for volumetric calculation of white blood cells in a blood sample. The system includes a sampling device as described above. The system further includes a measuring apparatus comprising a sample collection device holder arranged to receive a sample collection device that holds the blood sample in a measuring cavity and a light source adapted to irradiate the blood sample. The measuring apparatus further includes an imaging system comprising a magnifying system and a digital camera for acquiring a digital zoom image

EP 1 701 150 an irradiated sample in a measuring cavity, wherein white blood cells are distinguished on a digital image by selective staining of a coloring agent. The measuring apparatus also includes an image analyzer adapted to analyze the obtained digital image for identifying white blood cells and determining the number of white blood cells in the blood sample.

[0018] The method and system of the invention provides a very simple analysis of a blood sample for determining the number of white blood cells. The analysis does not require complicated measuring devices or advanced operations performed by the operator. Therefore, it can be carried out directly in connection with the patient's examination, without the need for a specialist's work. The measuring apparatus uses the properties of the sampling instrument to perform analysis on a sample of undiluted whole blood that has been directly introduced into the measuring cavity. The measuring apparatus is adapted to image the sample volume for calculating the volume of white blood cells from one image.

[0019] The blood sample can be mixed with the reagent in the measuring cavity. In a few minutes or less, the reaction of the blood sample with the reagent will lead to hemolysis of red blood cells and staining of white blood cells so that the sample will be ready for presentation for optical measurement. The blood sample can be mixed with the reagent, e.g. by the dispersion or diffusion of the reagent into the blood sample or by actively vibrating or moving the sampling device such that agitation occurs in the measuring cavity.

[0020] The sampling device comprises a body member having two flat surfaces to define said measuring cavity.

Flat surfaces are spaced at a predetermined distance from each other to determine the thickness of the sample for optical measurement. This means that the sampling device provides the exact thickness for the optical measurement that can be used to accurately determine the number of white blood cells per unit volume of blood sample. The volume of the sample being tested will be well determined by the thickness of the measuring cavity and the imaged area of ​​the sample. Thus, a well-defined volume can be used to combine the number of white blood cells with the volume of the blood sample so that the volume number of white blood cells is determined.

[0021] The measuring cavity has a uniform thickness of 50170 micrometers. A thickness of at least 50 microns suggests a recess

Measuring EP 1 701 150 does not force the blood sample to smear into a single layer, which allows analysis of a larger volume of blood in an area with a small cross-section. Thus, a large enough blood sample volume can be observed to obtain reliable white blood cell counts using a relatively small blood sample image. The thickness is more preferably at least 100 microns, which allows analysis of surfaces with an even smaller cross-section or larger sample volume. In addition, a thickness of at least 50 microns, and even more preferably 100 microns simplifies the production of a measuring cavity with a precisely defined thickness between two flat surfaces.

[0022] For most samples placed in a cavity not more than 170 micrometers thick, the number of white blood cells is so small that only small deviations due to white blood cells are superimposed. However, the effects of such deviations will be related to the number of white blood cells, and therefore at least to some extent can be treated by statistically correcting results, at least for high values ​​of white blood cells. This statistical correction may be based on calibrations of the measuring apparatus. The deviations will be even smaller for a measuring cavity with a thickness of no more than 150 micrometers, but simpler calibration can be used. This thickness may not even require calibration for overlapping blood cells.

[0023] In addition, the thickness of the measuring cavity is small enough to allow the measuring apparatus to obtain a digital image so that the entire depth of the measuring cavity can be simultaneously analyzed. Because the measuring apparatus has a magnifying system, it is not easy to obtain a large depth of field. Therefore, the thickness of the measuring cavity should not exceed 150 microns so that the entire thickness is simultaneously analyzed on a digital image. The depth of field can be adapted to the thickness of the measuring cavity of 170 microns.

[0024] The digital image can be obtained with a depth of field at least corresponding to the thickness of the measuring cavity. This means that sufficient sharpness over the entire thickness of the sample is obtained, so that the entire thickness of the measuring cavity can be simultaneously analyzed on the digital image of the sample. So there is no need to wait for the white blood cells to settle in the measuring cavity, thanks

EP 1 701 150 so that the time for analysis is reduced. By choosing no sharp focus on a particular portion of the sample, sufficient sharpness was obtained over the entire thickness of the sample to allow identification of the number of white blood cells in the sample. This means that white blood cells may be a little blurred and are still considered to be focused at depth of field.

[0025] The sampling device may be provided with a reagent that has been applied to a surface dissolved in a volatile liquid that has evaporated to leave the reagent in a dried form.

[0026] He realized that the reagent is preferably dissolved in a volatile liquid before entering the measuring cavity. This means that the liquid can be effectively evaporated from the narrow space of the measuring cavity during the manufacture and preparation of the sampling device.

[0027] The reagent may preferably be dissolved in an organic solvent, and more preferably dissolved in methanol. Such solvents are volatile and can be appropriately used for drying the reagent on the surface of the measuring cavity.

[0028] The staining agent may be prepared for the selective staining of white blood cell nuclei. This means that white blood cells can be identified as colored spots, making them easier to count.

[0029] The coloring agent can be any of the group of hematoxylin, methylene blue, methylene green, methylene blue, cresyl violet, toluidine blue, gerberian violet, sudan analogues, galocyanine and fuchsin analogues or combinations thereof. However, it should be noted that the coloring agent is not limited to this group, but many other substances can be considered.

[0030] The hemolyzing agent may be a quaternary ammonium salt, saponin, bile acid such as deoxycholic acid, digitoxin, snake venom, glucopyranoside or non-ionic Triton type detergent. However, it should be noted that the hemolytic agent is not limited to this group, but many other substances can be considered.

[0031] The sampling device further includes a sample inlet connecting the measuring cavity to the outer surface of the sample device, said inlet being adapted to collect a blood sample.

EP 1 701 150

The sample inlet can be prepared for collecting a blood sample by capillary force, and the measuring cavity can further draw blood from the inlet into the cavity. As a result, the blood sample can easily be inserted into the measuring cavity by simply shifting the sample inlet to contact with the blood. Then, the capillary forces of the sample inlet and measuring cavity will introduce a well-defined amount of blood into the measuring cavity.

[0032] The sampling device may be disposable, i.e. it is intended for use only once. The sampling device provides a kit for counting the number of white blood cells because the sampling device can take a blood sample and contains all the reagents needed to count the cells in the sample. This is especially possible because the sampling device is designed to be used only once and can be formed without considering the possibility of cleaning the sampling device and re-applying the reagent. Also the sampling device can be molded in plastic and therefore manufactured at a low price. Thus, it may be cost-effective to use a disposable sampling device.

[0033] The sample can be irradiated with light at a wavelength corresponding to the absorbance peak of the colorant. Consequently, stained white blood cells that contain accumulation of a coloring agent will be detected by low light transmission.

[0034] Irradiation can be done with a laser source. The laser source can provide light with a well-defined wavelength matching the absorbance of the colorant. In addition, the laser source provides collimated light, minimizing scattered light interference, so that a point with low light transmission will be clearly highlighted.

[0035] The irradiation can alternatively be carried out by means of a light emitting diode. This light source can still provide sufficient irradiation conditions to correctly distinguish white blood cells from another substance in the sample.

[0036] A digital image can be obtained using a magnification power of 3-200x, more preferably 3-10x. Within these magnification power ranges, white blood cells are enlarged enough to be detected and the depth of field can be adjusted to cover the thickness of the sample. A low magnification factor means that you can get a large depth of field. If

However, low magnification power was used, white blood cells may be difficult to detect. Lower magnification can be used by increasing the number of pixels in the obtained images, i.e. improving the resolution of the digital image. In this way, it is possible to use a magnification force of 3-4x, which also allows the detection of white blood cells.

[0037] The analysis involves identifying areas with high light absorbance in the digital image. The analysis may further include identifying black or dark dots in the digital image. Since the coloring agents can be accumulated in the nucleus of white blood cells, the absorbance of light can have peaks at separate points. These points form black dots in the digital image. [0038] The analysis may further comprise electronic zooming of the obtained digital images. Although the sample is enlarged to obtain an enlarged digital image of the sample, the obtained digital image can be electronically enlarged to simplify the distinction between objects that are photographed very closely together in the obtained digital images.

Brief description of the figures [0039] The invention will now be described in more detail by way of example with reference to the accompanying drawings.

[0040] Fig. 1 is a schematic view of a sampling device according to an embodiment of the invention.

[0041] Fig. 2 is a schematic view of a sampling device according to an embodiment that is not part of the invention.

[0042] Fig. 3 is a schematic view of a sampling device according to an embodiment of the invention.

[0043] Fig. 4 is a block diagram of a method according to an embodiment of the invention.

[0044] Fig. 5 is a digital image of the blood sample for volumetric calculation of white blood cells.

Detailed Description of Preferred Embodiments [0045] With reference to Fig. 1, an exemplary sampling device 10 according to an embodiment of the invention will be described. The sampling device 10 is disposable and should be discarded after use

EP 1 701 150 analysis. This means that the sampling device 10 does not require complicated operation. The sampling device 10 is preferably formed in a plastic material and can be manufactured by injection molding. This makes the production of the sampling instrument 10 simple and cheap, so that the costs of the sampling instrument 10 can be reduced.

[0046] The sampling device 10 comprises a body member 12 that has a base 14 that can be touched by the operator without causing any interference with the results of the analysis. The base 14 may also have projections 16 that may match the handle on the analysis apparatus. The projections 16 can be arranged such that the sampling device 10 will be correctly positioned in the analysis apparatus.

[0047] The sampling device 10 further includes a sample inlet 18. The sample inlet 18 is formed between opposite walls inside the sampling device 10, the walls being so close together that capillary force can be generated in the sample inlet 18. The sample inlet 18 connects to the outside of the sampling device 10, allowing blood to be 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 disposed between opposite walls inside the sampling device 10. The measuring cavity 20 is located in connection with the sample inlet 18. The walls defining the measuring cavity 20 are located closer than the sample inlet walls 18, so that capillary force can draw blood from the sample inlet 18 into the measuring cavity 20.

[0048] The walls of the measuring cavity 20 are spaced 50170 micrometers. The measuring cavity 20 more preferably has a thickness of at least 100 microns. Furthermore, the measuring cavity 20 is more preferably larger than 150 microns. The distance is uniform throughout the measuring cavity 20. The thickness of the measuring cavity 20 determines the volume of blood being tested.

Since the result of the analysis is to be compared with the volume of the blood sample being tested, the thickness of the measuring cavity 20 must be very accurate, i.e. only slight differences in thickness are allowed inside the measuring cavity 20 and between the measuring cavities 20 different sampling devices 10. The thickness allows relatively analysis large sample volume in a small cavity area. Thickness

EP 1 701 150 theoretically allows the placement of white blood cells inside the measuring cavity 20. However, the number of white blood cells in the blood is so low that the probability of this phenomenon is very low.

[0049] 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 will be too small to allow statistically significant amounts of white blood cell counts. In addition, when the number of white blood cells exceeds 12 x 10⁹ cells / liter of blood, the effect of overlapping blood cells begins to be significant in the measured number of white blood cells.

With this number of white blood cells, white blood cells will cover about 8% of the cross-section of the irradiated sample if the thickness of the measuring cavity is 140 micrometers. So, to get the correct white blood cell count, this effect will have to be taken into account. Therefore, statistical correction of white blood cell values ​​above 12 x 10 can be used⁹ cells / liter of blood.

This statistical correction will increase to increase the number of white blood cells because the overlapping effect will be greater to increase the number of white blood cells. Statistical correction can be determined by calibrating the measuring apparatus. Alternatively, statistical correction can be determined at an overall level when setting up the measuring apparatus to be used in conjunction with the sampling device 10. This statistical correction is similar to the statistical corrections that are currently carried out in analysis apparatus using the Coulter principle. It is believed that the 10 sample taking device can be used to analyze the number of white blood cells up to 50 x 10⁹ cells / liter of blood.

[0050] The wall surface of the measuring cavity 20 is at least partially covered with reagent 22. The reagent 22 can be freeze dried, dried or vacuum dried and applied to the surface of the measuring cavity 20. When a sample of blood is drawn into the measuring cavity 20, the blood comes into contact with dried reagent 22 and the reaction between reagent 22 and blood begins.

[0051] Reagent 22 is used by introducing reagent 22 into measuring cavity 20 by means of a pipette or dispenser. Reagent 22 dissolves in a volatile liquid, e.g. an organic solvent such as methanol, when introduced into the measuring cavity 20. The solvent with reagent 22 can

EP 1 701 150 fill the measuring cavity 20. Then drying is carried out so that the solvent is evaporated and the reagent 22 adheres to the surface of the measuring cavity 20.

[0052] The reagent is to be dried on the surface of a narrow space, the liquid has a very small surface in contact with the atmosphere, making evaporation of the liquid more difficult. Thus, it is preferable to use a volatile liquid, such as methanol, that allows the liquid to evaporate effectively from the narrow space of the measuring cavity.

[0053] According to an alternative manufacturing method, the sampling device 10 can be formed by attaching 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 to dry. 22 on an open surface before joining the two parts. Thus, reagent 22 can be dissolved in water because the solvent need not be volatile.

[0054] Reagent 22 contains a hemolyzing agent and a coloring agent. The hemolyzing agent may be a quaternary ammonium salt, saponin, bile acid such as deoxycholic acid, digitoxin, snake venom, glucopyranoside or a Triton nonionic detergent. The coloring agent may be hematoxylin, methylene blue, methylene green, methylene blue, cresyl violet, toluidine blue, gerberian violet, sudan analog, galocyanine or fuchsin analogues or any combination thereof. When the blood sample comes into contact with reagent 22, the haemolysing agent will act on the spillage of red blood cells so that the lyzed red blood cells mix with the blood plasma. In addition, the coloring agent accumulates in the nuclei of white blood cells. Reagent 22 should contain sufficient amounts of coloring agent to clearly stain all white blood cell nuclei. In this way, there is often an excess of coloring agent that will be mixed in the blood plasma. Excess coloring agent ensures a homogeneous, low level of coloring agent background in the blood plasma. The accumulated coloring agent in the white blood cells will differ from the background level of the coloring agent.

[0055] Reagent 22 may also contain other components that may be active, i.e. those that are involved in the chemical reaction with the blood sample, or that may be inactive, i.e. not involved in the chemical reaction with the blood sample. The active ingredients can e.g. be prepared to catalyze action

EP 1 701 150 hemolyzing or coloring. Inactive ingredients may e.g. be used to improve the adhesion of reagent 22 to the wall surface of measuring cavity 20.

[0056] Within a few minutes, the blood sample will react with reagent 22 so that the red blood cells are lysed and the coloring agent accumulates in the nuclei of the white blood cells.

[0057] With reference to Fig. 2, an embodiment of the sampling device that is not part of the invention will be described. The sampling device 110 includes a chamber 120 forming a measuring cavity. The sampling device 110 has an inlet 118 into the chamber 120 for transporting blood to the chamber 120. The chamber 120 is connected to the pump (not shown) via a suction tube 121. The pump may apply suction to chamber 120 through suction hose 121, so that blood can be sucked into chamber 120 through inlet 118. The sampling device 110 can be disconnected from the pump before measurement. Like the measuring cavity of the sampling device 10 of the first embodiment, the chamber 120 has a well-defined thickness that determines the thickness of the sample to be tested. In addition, reagent 122 adhering to the walls of chamber 120 is used to react with the blood sample.

[0058] With reference to Fig. 3, an apparatus 30 for volumetric calculation of white blood cells will be described. The apparatus 30 comprises a sample holder 32 for receiving a sample device 10 with a blood sample. The sample holder 32 is positioned to receive the sampling device 10 such that the measuring cavity 20 of the sampling device 10 is correctly positioned inside the apparatus 30. The apparatus 30 includes a light source 34 for illuminating the blood sample in the sample in the sampling device 10. The light source 34 may be an incandescent lamp that irradiates light throughout the visible spectrum. The colorant accumulated in the nuclei of white blood cells absorbs light at specific wavelengths, so that the nuclei of white blood cells appear in the digital image of the sample. If a colored image is obtained, white blood cells will appear as specifically colored dots. If a black and white image is obtained, white blood cells will appear as dark dots on a lighter background.

[0059] The light source 34 may alternatively be a laser or light-emitting diode. It can be used to increase the contrast of the image so that white blood cells can be more easily detected. In this case, the light source 34 is

EP 1 701 150 adapted to generate electromagnetic radiation with a wavelength corresponding to the colorant absorption peak. The wavelength should be chosen so that the absorption of blood compounds is relatively low. In addition, the walls of the sampling device 10 should be substantially transparent to the wavelength. For example, when a methylene dye is used as the colorant, the light source 34 may be placed to produce light radiation at a wavelength of 667 nm.

[0060] The apparatus 30 further includes an imaging system 36 that is disposed on the opposite side of the sample holder 32 relative to the light source

34. In this way, the imaging system 36 is adapted to receive radiation that was transmitted by the blood sample. The imaging system 36 includes an enlarging system 38 and image acquisition means 40. The magnifying system 38 is adapted to obtain a magnification of 3200x, more preferably 3-100x, and most preferably 3-4x. Within these magnification ranges it is possible to distinguish between white blood cells. The image can be obtained with improved resolution to allow a smaller magnification to be used. Furthermore, the depth of field of the magnifying system 38 can be set to at least correspond to the thickness of the measuring cavity 20.

[0061] The magnifying system 38 comprises an objective lens or lens system 42 that is positioned close to the sample holder 32 and an eyepiece lens or lens system 44 that is positioned at a distance from the objective lens 42. The objective lens 42 provides the first magnification of the sample, which is further enlarged by the eyepiece lens 44. The magnifying system 38 may include further lenses to obtain appropriate magnification and imaging of the sample. The magnifying system 38 is arranged such that the sample in the measuring cavity 20 placed in the sample holder 32 will be focused on the image plane of the image acquisition means 40.

[0062] The image acquisition means 40 are adapted to obtain a digital image of the sample. The image acquisition means 40 can be any digital camera, for example a CCD camera. The pixel size of the digital camera limits the imaging system 36 so that the indistinct circle on the image plane cannot exceed the pixel depth in the field. However, white blood cells can still be detected, even if they are slightly smudged, and therefore the indistinct circle may be larger than the pixel size considered in depth

EP 1 701 150 fields. The digital camera 40 takes a digital image of the sample in the measuring cavity 20, wherein the entire thickness of the sample is sufficiently focused on the digital image to count white blood cells. The imaging system 36 determines the area of ​​the measuring cavity 20 that will be imaged on the digital image.

The area to be imaged together with the thickness of the measuring cavity 20 determines the volume of the sample made. The imaging system 36 is configured to match the imaging of blood samples in the sampling device 10. There is no need to change the configuration of the imaging system 36. Preferably, the imaging system 36 is positioned within the housing so that the setting cannot be accidentally changed.

[0063] The apparatus 30 further includes an image analyzer 46. The image analyzer 46 is connected to a digital camera 40 for receiving digital images received by the digital camera 40. The image analyzer 46 is adapted to identify digital image patterns corresponding to white blood cells for counting the number of white blood cells present in the digital image. In this way, the image analyzer 46 can be arranged to identify dark spots in a lighter background. The image analyzer 46 can be set to first electronically enlarge the digital image before analyzing the digital image. This means that the image analyzer 46 can more easily distinguish between white blood cells that are imaged closely together, even if the digital zoom of the digital image causes the digital image to be slightly blurred.

[0064] 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 digital picture by the volume of the blood sample which is well defined as described above. A volumetric calculation of the number of white blood cells can be shown on the display of the 30 device.

[0065] The image analyzer 46 may be implemented as a processing unit that includes codes for performing image analysis.

[0066] With reference to Fig. 4, a method for calculating the volume of white blood cells will be described. The method comprises collecting a blood sample in a sample collection device, step 102. The sample collection device draws the undiluted whole blood sample. The sample can be taken from capillary or venous blood. A capillary blood sample can be obtained

Pull EP 1 701 150 into the measuring cavity directly from the patient's finger. The blood sample is contacted with the reagent in the sampling device initiating the reaction. Red blood cells are lysed and the coloring agent accumulates in the nuclei of white blood cells. Within minutes of taking the blood sample, the sample is ready for analysis.

The sampling device is placed in the analysis apparatus, step 104. Analysis can be initiated by pressing the analysis apparatus button. Alternatively, the analysis is automatically initiated by a device that senses the presence of a sampling device.

[0067] The sample is irradiated, step 106 and an enlarged digital image of the sample is obtained, step 108. The sample is irradiated with electromagnetic radiation at a wavelength corresponding to the absorption peak of the coloring agent. This means that the digital image will contain black or darker dots at the positions of the white blood cell nucleus.

[0068] The obtained digital image is transferred to an image analyzer that performs image analysis, step 110, to count the number of black dots in the digital image.

[0069] In Fig. 5 an example of a digital image is shown indicating the possibility of identifying white blood cells in a blood sample that is hemolyzed and stained. This digital image was obtained from a sampling device with a cavity thickness of 140 μm and using a 50x magnification. The light source emits white light, indicating that white blood cells can be identified even if irradiation is not specifically tailored to the peak of the colorant absorption level. Methylene blue was used as the dye. In Fig. 5 you can see black dots indicating white blood cells. The image shown in Fig. 5 is the black and white version of the color image. The contrast between white blood cells and the background is clearer in the color image than in the black and white image reproduced here. Black dots can be easily counted by the image analyzer.

[0070] In manual methods for counting white blood cells, about 200 cells are most often counted to determine the number of white blood cells in a blood sample.

For example, the method and apparatus presented herein can be arranged to count about 2,000 cells, which provides better statistical certainty for the results obtained. A normal, healthy adult has a white blood cell count from 4-5 x 10⁹cells / liter of blood. This means that 2000 cells are found in 0.4-0.5 pl samples. For example, if imaging an area of ​​1.5 x 1.5 mm in a cavity

EP 1 701 150 with a thickness of 140 μm, image volume is 0.315 pl. Part of the resulting image can be selected for analysis. Thus, the resulting graphics image can first be analyzed so that no abnormalities are allowed in the portion for determining the number of white blood cells. Part of the obtained images selected for analysis can be selected with the appropriate size to analyze a sufficient volume of blood sample.

[0071] It should be emphasized that the preferred embodiments are not limited in any way, and many possible embodiments are possible within the scope of protection defined by the appended claims.

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