ES2648978T3 - White blood cell count - Google Patents

Abstract

Sample acquisition device (10) for the volumetric enumeration of white blood cells in a blood sample, said sample acquisition device (10) comprising: a measuring cavity (20) for accommodating a blood sample in which the sample of blood is undiluted whole blood that has been acquired directly within the measurement cavity, said measurement cavity (20) having a predetermined fixed thickness, measured such that said thickness, when the device is in use, in combination with a area of the sample from which images are being obtained, defines an analyzed sample volume; wherein the sample acquisition device (10) further comprises: a reagent (22), which is disposed dry on a surface defining the measuring cavity (20), said reagent (22) comprising a hemolyzing agent for lyse red blood cells in the blood sample, and a staining agent to selectively stain white blood cells in the blood sample; an inlet (18) of samples, which is defined between opposite walls within the sample acquisition device (10), said walls being arranged so close to each other that a capillary force can be created at the inlet (18) of samples; said measuring cavity (20) being arranged in communication with said sample inlet (18); and a body element (12) having two flat surfaces to define said measuring cavity (20), in which the flat surfaces are arranged at a predetermined distance from each other to determine a sample thickness for an optical measurement, and in wherein the flat surfaces of said measuring cavity (20) are arranged closer together than said walls of the sample inlet (18), so that a capillary force can introduce blood from the sample inlet (18) into the cavity (20) of measurement.

Description

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DESCRIPTION

White blood cell count Technical field

The present invention relates to a sample acquisition device, a method and a system for the volumetric enumeration of white blood cells in a blood sample.

Background of the invention

The determination of white blood cell count is often important in relation to the treatment of a patient. This analysis may be necessary to diagnose for example leukemia, or infectious or inflammatory diseases or to monitor treatments. It is desirable to allow the results of the analysis to be obtained as quickly as possible in order to minimize waiting times for patients and allow a physician to make a treatment decision and diagnose directly when performing a first examination of the patient. Therefore, it would be preferable to provide a method of analysis that the doctor or a nurse can perform quickly without having to send the test to a laboratory.

Currently, the white blood cell count is usually obtained through a manual procedure by staining a blood sample and microscopic observation of the sample in a special counting chamber, for example a Burker chamber. The counting chamber is equipped with a grid that divides the chamber into small, well-defined volumes. The white blood cells are allowed to settle at the bottom of the counting chamber in order to allow the microscope to focus all the cells in the chamber and, therefore, to facilitate counting. Therefore, it is necessary for the sample to settle for several minutes before counting. Then the white blood cell count can be determined by counting the number of blood cells per box in the lattice. The white blood cell count is obtained manually by an analyst, who needs to have experience in performing the analysis in order to perform a reliable analysis.

This analysis takes a lot of time. Also, since it is performed manually, the results of the analysis may vary depending on the person performing the analysis.

There are a limited number of existing automated analysis methods to determine the white blood cell count. The white blood cell count can be determined by means of the Coulter principle, which is based on determining the cell size and thereby the cell type by detecting an impedance. A method for counting white blood cells by the Coulter principle is described in US 5,262,302.

The Coulter principle is the predominant automated analysis method that is currently being used. However, some other methods have been described. One such method for determining the white blood cell count is disclosed in US 5,585,246. In this case, a blood sample has to be prepared by mixing with a fluorescent dye and a ligand complex marking white blood cells. The sample is inserted into a capillary and irradiated by a laser source that scans the sample in the capillary. Fluorescence is measured in order to determine the number of white blood cells. A similar method is disclosed in WO 97/02482, using a fluorescent dye and a laser source that explores a capillary. This method is adapted for the enumeration of white blood cells in aferesis products that contain a limited number of white blood cells. In this case, the capillary is quite thick and it is necessary to wait until the white blood cells have settled in the bottom of the capillary before the capillary can be explored.

In WO 99/45384, a sample containing a camera having a variable thickness is shown. The variable thickness separates different compounds from the blood. The blood sample is dyed with a dye to differentially highlight at least three different types of white blood cells in the blood sample. White blood cells can be listed using an optical scanning instrument to visualize a part of the camera.

In EP 1 161 994 A2, a cuvette for inserting blood products and reagents into it is disclosed. The cuvette has a hollow main body that contains the blood product and the fluorescent staining reagent in hemolysis. The bucket is composed of a colorless and transparent plastic and has a lid made of rubber. The hemolysis fluorescent staining reagent is added to the cuvette when the white blood cell count is being performed. Then the blood product is added to the cuvette and the reagent and the blood product are mixed and the reagent is reacted with the blood product. The cuvette is centrifuged and after exposure to centrifugal force, it is adjusted on a micro-leukocytometer to count the number of leukocytes in the blood product.

US Patent 5,948,686 discloses a sample chamber that includes a first wall and a second transparent wall. The walls are separated from each other by a thickness across the plane. The thickness across the plane may vary depending on the hematocrit and the target constituents. A whole blood sample is mixed with an amount of a sensitive dye that may be in liquid or dry form. After

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Mixing the sample is introduced into the sample chamber where the sample is kept quiescent for a short period of time to allow the formation of cylinders and gaps. The lagoons are open areas that remain between the cylinders formed, which are clusters of red blood cells. White blood cells are found in the lagoons along with platelets. White blood cells are detected by a microscope in the transparent lagoons.

There is still a need to accelerate and simplify existing automated methods to determine the white blood cell count so that the analysis can be provided at the point of attention. In addition, since white blood cell count is a frequently performed analysis of this type, any improvement in the method of analysis would have a great impact on patient care. An analysis method that provides the possibility of obtaining results at the point of attention would be particularly advantageous.

Summary of the invention

An object of the invention is to provide a simple analysis to determine the volumetric enumeration of white blood cells. A further object of the invention is to provide a quick analysis without the need for complicated devices or large sample preparations.

These objects are achieved partially or completely by means of a sample acquisition device, a method and a system according to the independent claims. Preferred embodiments are apparent from the dependent claims.

Therefore, a sample acquisition device is provided for the volumetric enumeration of white blood cells in a blood sample. The sample acquisition device comprises a measurement cavity to accommodate a blood sample, in which the blood sample is undiluted whole blood that has been acquired directly within the measurement cavity. The measurement cavity has a predetermined fixed thickness measured so that said thickness, when the device is in use, in combination with an area of the sample from which images are being obtained, defines an analyzed sample volume. The sample acquisition device further comprises a reagent, which is disposed dry on a surface that defines the measurement cavity, said reagent comprising a hemolyzing agent to lyse red blood cells in the blood sample, and a staining agent to have selectively white blood cells in the blood sample. The sample acquisition device further comprises a sample inlet, which is defined between opposite walls within the sample acquisition device, said walls being arranged so close to each other that a capillary force can be created at the sample inlet. The measurement cavity in the sample acquisition device is arranged in communication with the sample inlet, and the measurement cavity is defined by two flat surfaces, in which the flat surfaces are arranged at a predetermined distance from each other to determine a sample thickness for an optical measurement, and in which the flat surfaces of said measurement cavity are arranged closer together than said walls of the sample inlet, so that a capillary force can introduce blood from the sample inlet into The measuring cavity.

The sample acquisition device provides the possibility to directly obtain a whole blood sample within the measurement cavity and provide it for analysis. There is no need for sample preparation. In fact, the blood sample can be aspirated into the measurement cavity directly from a patient's finger in which a puncture has been performed. Providing the sample acquisition device with a reagent allows a reaction within the sample acquisition device that makes the sample ready for analysis. The reaction starts when the blood sample comes in contact with the reagent. Therefore, there is no need to manually prepare the sample, which makes the analysis especially suitable to be performed directly in an examination room while the patient is waiting.

Since the reagent is provided in dry form, the sample acquisition device can be transported and stored for a long time without affecting the usability of the sample acquisition device. Therefore, the sample acquisition device with the reagent can be manufactured and prepared long before the analysis of a blood sample.

Although many existing methods can count different blood cells and even subgroups of blood cells, the sample acquisition device according to the invention is specifically adapted to perform the volumetric enumeration of white blood cells. The reagent comprises a hemolyzing agent that lyse the red blood cells in the blood sample. This destroys the possibility of enumerating the red blood cells in the sample. On the other hand, the lysate of the red blood cells simplifies the distinction and identification of the white blood cells within the blood sample.

The staining agent provides the marking of the individual white blood cells. This allows white blood cells to be visualized or detected individually. For example, white blood cells can be detected by scanning the measurement cavity or obtaining an image of the measurement cavity. The white blood cell count can therefore be obtained by adding the number of white blood cells detected individually in a defined volume.

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The invention also provides a method for the volumetric enumeration of white blood cells in a blood sample. The method comprises acquiring a blood sample within a measurement cavity of a sample acquisition device, in which the blood sample is undiluted whole blood that is acquired directly within the measurement cavity (20), irradiating the sample, acquire a digital image of an enlarged sample of the irradiated sample in the measurement cavity and digitally analyze the digital image to identify white blood cells and determine the number of white blood cells in the sample.

In the method according to the invention, the sample acquisition device has a sample inlet, which is defined between opposite walls within the sample acquisition device, in which said walls are arranged so close to each other that a capillary force can be created at the sample inlet, and the measurement cavity is arranged in communication with said sample inlet. The measurement cavity is defined by two flat surfaces, in which said flat surfaces are arranged at a predetermined distance from each other to determine a sample thickness for an optical measurement, and in which the flat surfaces of said measurement cavity are arranged closer to each other than said walls of the sample inlet, so that a capillary force can introduce blood from the sample inlet into the measurement cavity. In addition, the measurement cavity contains a reagent, which is arranged dry on a surface that defines the measurement cavity, which comprises a hemolyzing agent to lyse red blood cells and a staining agent to react with the sample so that the globules whites are tined, in which the white blood cells are distinguished by selective staining of the staining agent.

The invention also provides a system for the volumetric enumeration of white blood cells in a blood sample. The system comprises a sample acquisition device as described above. The system further comprises a measuring apparatus comprising a sample acquisition device holder arranged to accommodate the sample acquisition device containing a blood sample in the measurement cavity, and a light source arranged to irradiate the sample of blood. The measuring apparatus further comprises an image obtaining system, which comprises an enlargement system and a means of acquiring digital images to acquire a digital image of an extension of the irradiated sample in the measurement cavity, in which the globules Whites are distinguished in the digital image by selective staining of the staining agent. The measuring apparatus also comprises an image analyzer arranged to analyze the acquired digital image to identify white blood cells and determine the number of white blood cells in the blood sample.

The method and system of the invention provide a very simple analysis of a blood sample to determine the white blood cell count. The analysis does not require a complicated measuring device or an operator to perform advanced stages. Therefore, it can be done in direct connection with the examination of a patient, without the need of a qualified technician. The measuring device uses the properties of the sample acquisition device to perform an analysis on a sample of undiluted whole blood that has been acquired directly within the measurement cavity. The measuring device is arranged to obtain an image of a sample volume to make a volumetric enumeration of the white blood cells from the image.

The blood sample is allowed to mix with the reagent in the measurement cavity. Within a few minutes or less, the reaction of the blood sample with the reagent will have hemolyzed the red blood cells and had the white blood cells so that the sample is ready to be presented for optical measurement. The blood sample can be mixed with the reagent for example by dispersing or diffusing the reagent within the blood sample or by actively vibrating or moving the sample acquisition device so that agitation occurs in the measurement cavity.

The sample acquisition device comprises a body element that has two flat surfaces to define said measurement cavity. The flat surfaces are arranged at a predetermined distance from each other to determine a sample thickness for an optical measurement. This implies that the sample acquisition device provides a well defined thickness for optical measurement, which can be used to accurately determine the white blood cell count per volumetric unit of the blood sample. A volume of an analyzed sample will be well defined by the thickness of the measurement cavity and an area of the sample from which images are being obtained. Therefore, the well-defined volume could be used to associate the number of white blood cells with the volume of the blood sample so that the volumetric white blood cell count is determined.

The measurement cavity preferably has a uniform thickness of 50-170 micrometers. A thickness of at least 50 micrometers implies that the measurement cavity does not force a smear of the blood sample to give rise to a monolayer which allows a larger volume to be analyzed along a small cross-sectional area. Therefore, a sufficiently large volume of the blood sample can be analyzed in order to obtain reliable white blood cell count values using a relatively small image of the blood sample. The thickness is more preferably at least 100 micrometers, which allows an even smaller cross-sectional area or a larger sample volume to be analyzed. In addition, the thickness of at least 50 micrometers and more preferably 100 micrometers also simplifies the manufacture of the measurement cavity.

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which has a well defined thickness between two flat surfaces.

For most of the samples arranged in a cavity that is no more than 170 micrometers thick, the white blood cell count is so low that there will only be minor deviations due to the white blood cells that are arranged overlapping each other. However, the effect of such deviations will be related to the white blood cell count and therefore, at least to some extent, can be managed by means of the statistical correction of the results at least for the large values of the white blood cell count. This statistical correction can be based on measurements of the measuring device. The deviations will be even smaller for a measurement cavity that has a thickness of no more than 150 micrometers, so a simpler calibration can be used. This thickness may not even require any calibration for overlapping blood cells.

In addition, the thickness of the measurement cavity is small enough to allow the measuring device to obtain a digital image so that the entire depth of the measurement cavity can be analyzed simultaneously. Since an extension system is to be used in the measuring device, it is not easy to obtain a large depth of field. Therefore, the thickness of the measurement cavity will preferably not exceed 150 micrometers in order to simultaneously analyze the entire thickness in a digital image. The depth of field can be arranged to handle a thickness of the measurement cavity of 170 micrometers.

The digital image can be acquired with a depth of field corresponding at least to the thickness of the measurement cavity. This implies that a sufficient approach to the entire thickness of the sample is obtained so that the entire thickness of the measurement cavity can be analyzed simultaneously in the digital image of the sample. Therefore, there is no need to wait for white blood cells to settle in the measurement cavity, so the time to obtain the analysis is reduced. By choosing not to focus very sharply on a specific part of the sample, a sufficient focus of the entire thickness of the sample is obtained to identify the number of white blood cells in the sample. This implies that a white balloon may be somewhat blurred and still considered to be in focus at depth of field.

The sample acquisition device may be provided with a reagent that has been applied to the surface dissolved in a volatile liquid that has evaporated to leave the reagent in dry form.

It has been achieved that the reagent is advantageously dissolved in a volatile liquid before being inserted into the measurement cavity. This implies that the liquid can evaporate effectively from the narrow space of the measurement cavity during the manufacture and preparation of the sample acquisition device.

The reagent may preferably be dissolved in an organic solvent and more preferably may be dissolved in methanol. Such solvents are volatile and can be used appropriately to dry the reagent on a surface of the measurement cavity.

The staining agent can be arranged to selectively have the white blood cell nucleus. This implies that white blood cells can be identified as colored dots and therefore can easily be counted in a digital image.

The staining agent can be any one of the group of hematoxylin, methylene blue, methylene green, methylene azure, cresyl violet acetate, toluidine blue, gentian violet, Sudan analogs, galocyanine and fuchsin analogs, or any combination thereof. However, it should be appreciated that the staining agent is not limited to this group, but many other substances can be contemplated.

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

The sample acquisition device further comprises a sample inlet that communicates the measurement cavity with the outside of the sample acquisition device, said entry being arranged to acquire a blood sample. The sample inlet may be arranged to introduce a blood sample by a capillary force and the measurement cavity may also introduce the blood from the entrance into the cavity. As a result, the blood sample can be easily acquired within the measurement cavity by simply moving the sample inlet in contact with the blood. Then, the capillary forces of the sample inlet and the measurement cavity will introduce a well defined amount of blood into the measurement cavity.

The sample acquisition device may be disposable, that is, it is arranged for use only once. The sample acquisition device provides a kit to perform a white blood cell count, since the sample acquisition device can accommodate a blood sample and contains all the necessary reagents in order to present the sample for cell count. This is particularly allowed since the sample acquisition device is adapted to be used only once and can be formed without

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Consider cleaning the sample acquisition device and reapplying a reagent. In addition, the sample acquisition device can be molded into plastic material and thereby manufactured at a low price. Therefore, it can still be cost effective to use a disposable sample acquisition device.

The sample can be irradiated by light of a wavelength corresponding to a peak in the absorbance of the staining agent. Therefore, white globules that contain a staining agent accumulation will be detected by low light transmittance.

Irradiation can be done by means of a laser source. The laser source can provide light of a well-defined wavelength that adjusts the absorbance of the staining agent. In addition, the laser source provides collimated light, which minimizes alterations of the parasitic light, so that a point of low light transmittance will be clearly distinguished.

The irradiation can be performed alternatively by means of a light emitting diode. This light source can still provide sufficient irradiation conditions to properly distinguish white blood cells from other matter in the sample.

The digital image can be acquired using a magnification power of 3-200x, more preferably 3-10x. Within these ranges of magnification power, the white blood cells are sufficiently enlarged in order to be detected, while the depth of field can be arranged to cover the sample thickness. A low magnification power implies that a great depth of field can be obtained. However, if a low magnification power is used, white blood cells can be difficult to detect. A smaller magnification power can be used by increasing the number of pixels in the acquired image, that is, by improving the resolution of the digital image. Thus, it has become possible to use a 3-4x magnification power, while white globes are still allowed to be detected.

The analysis includes identifying areas of high light absorbance in the digital image. The analysis may also include identifying black or dark spots in the digital image. Since staining agents can accumulate in the nucleus of white blood cells, the absorbance of light can have spikes at separate points. These dots will form black dots in the digital image.

The analysis may also include electronically expanding the acquired digital image. While the sample is being enlarged to acquire an enlarged digital image of the sample, the acquired digital image itself can be electronically enlarged to distinguish in a simplified way between objects from which images are being obtained very close to each other in the acquired digital image.

Brief description of the drawings

The invention will now be described in additional detail by way of example with reference to the attached drawings.

Figure 1 is a schematic view of a sample acquisition device according to an embodiment of the invention.

Figure 2 is a schematic view of a sample acquisition device according to an embodiment that is not part of the invention.

Figure 3 is a schematic view of a measuring device according to an embodiment of the invention.

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

Figure 5 is a digital image of a blood sample to be used for the volumetric enumeration of white blood cells.

Detailed description of a preferred embodiment

Referring now to Fig. 1, a sample acquisition device 10 according to an embodiment of the invention will be described. The sample acquisition device 10 is disposable and will be discarded after being used for analysis. This implies that the sample acquisition device 10 does not require complicated handling. The sample acquisition device 10 is preferably formed of plastic material and can be manufactured by injection molding. This makes the manufacture of the sample acquisition device 10 simple and economical, while the cost of the sample acquisition device 10 can be kept low.

The sample acquisition device 10 comprises a body element 12, which has a base 14, which an operator can touch without causing any interference in the analysis results. The base 14 may also have projections 16 that can adjust a support in the analysis apparatus. The projections 16 can be provided

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so that the sample acquisition device 10 is correctly placed in the analysis apparatus.

The sample acquisition device 10 further comprises an input 18 of samples. The sample inlet 18 is defined between opposite walls within the sample acquisition device 10, the walls being arranged so close to each other that a capillary force can be created in the sample inlet 18. The sample inlet 18 communicates with the outside of the sample acquisition device 10 to allow blood to enter the sample acquisition device 10. The sample acquisition device 10 further comprises a chamber for counting white blood cells in the form of a measuring cavity 20 disposed between opposite walls within the sample acquisition device 10. The measuring cavity 20 is arranged in communication with the sample inlet 18. The walls defining the measurement cavity 20 are arranged closer together than the walls of the sample inlet 18, so that a capillary force can introduce blood from the sample inlet 18 into the measurement cavity 20.

The walls of the measuring cavity 20 are arranged at a distance of 50-170 micrometers from each other. The measurement cavity 20 is more preferably at least 100 micrometers thick. In addition, the measurement cavity 20 is more preferably not more than 150 micrometers thick. The distance is uniform throughout the entire measuring cavity 20. The thickness of the measurement cavity 20 defines the volume of blood being examined. Since the result of the analysis is to be compared with the volume of the blood sample being examined, it is necessary that the thickness of the measurement cavity 20 be very precise, that is, only very small variations in the thickness within the thickness of the sample are allowed. Measuring cavity 20 and between measuring cavities 20 of different sample acquisition devices 10. The thickness allows a relatively large sample volume to be analyzed in a small area of the cavity. The thickness theoretically allows the white blood cells to be arranged one above the other within the measurement cavity 20. However, the amount of white blood cells in the blood is so low that the probability of this happening is very small.

The sample acquisition device 10 is normally adapted to measure white blood cell counts 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 cells to be counted. In addition, when the white blood cell count exceeds 12 x 109 cells / liter of blood, the effect of the blood cells that are arranged overlapping each other will begin to be significant in the measured white blood cell count. At this white blood cell count, the white blood cells will cover approximately 8% of the cross-section of the sample that is being irradiated, if the thickness of the measurement cavity is 140 micrometers. Therefore, in order to obtain corrected white blood cell counts, it will be necessary to take this effect into account. Therefore, a statistical correction of white blood cell count values above 12 x 109 cells / liter of blood can be used. This statistical correction will increase for increasing white blood cell counts, since the effect of overlapping blood cells will be greater for larger white blood cell counts. The statistical correction can be determined by means of the calibration of a measuring device. Alternatively, the statistical correction can be determined at a general level to configure the measuring devices to be used in relation to the sample acquisition device 10. This statistical correction is of similar magnitude to the statistical corrections that are currently made in the analysis apparatus that use the Coulter principle. It is contemplated that the sample acquisition device 10 can be used to analyze white blood cell counts of up to 50 x 109 cells / liter of blood.

The surface of a wall of the measurement cavity 20 is at least partially coated with a reagent 22. The reagent 22 may be lyophilized, heat dried or vacuum dried and may be applied to the surface of the measurement cavity 20. When a blood sample is acquired within the measurement cavity 20, the blood will come into contact with the dry reagent 22 and initiate a reaction between the reagent 22 and the blood.

Reagent 22 is applied by inserting reagent 22 into the measurement cavity 20 using a pipette or dispenser. Reagent 22 is dissolved in a volatile liquid, for example an organic solvent such as methanol, when inserted into the measurement cavity 20. The solvent with reagent 22 can fill the measuring cavity 20. Then, drying is performed so that the solvent will evaporate and reagent 22 will bind to the surfaces of the measurement cavity 20.

Since the reagent is going to dry on a surface of a narrow space, the liquid will have a very small surface in contact with the ambient atmosphere, so evaporation of the liquid becomes more difficult. Therefore, it is advantageous to use a volatile liquid, such as methanol, which allows the liquid to evaporate effectively from the narrow space of the measurement cavity.

According to an alternative manufacturing method, the sample acquisition device 10 can be formed by joining two pieces together, whereby one piece forms the lower wall of the measurement cavity 20 and the other piece forms the upper wall of the cavity 20 of measurement. This allows a reagent 22 to dry on an open surface before the two pieces join together. Therefore, reagent 22 can be dissolved in water, since it is not necessary for the solvent to be volatile.

Reagent 22 comprises a hemolyzing agent and a staining agent. The hemolyzing agent may be a salt of

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quaternary ammonium, a saponin, a bile acid, such as deoxycholic acid, a digitoxin, a snake venom, a glucopyranoside or a non-ionic detergent of the Triton type. The staining agent may be hematoxylin, methylene blue, methylene green, methylene azure, cresyl violet acetate, toluidine blue, gentian violet, a Sudan analogue, galocyanine or a fuchsin analogue, or any combination of the same. When a blood sample comes into 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. In addition, the staining agent will accumulate in the white blood cell nuclei. Reagent 22 must contain sufficient amounts of staining agent to clearly have all the white blood cell nuclei. Therefore, there will often be an excess of staining agent, which intermingles in the blood plasma. Excess staining agent will give a low background level of homogeneous staining agent in the blood plasma. The staining agent accumulated in the white blood cells may be distinguished from the background level of the staining agent.

Reagent 22 may also comprise other constituents, which may be active, that is, that take part in the chemical reaction with the blood sample, or not active, that is to say that they do not take part in the chemical reaction with the blood sample. The active constituents can be arranged for example to catalyze the action of hemolysis or staining. Non-active constituents can be arranged, for example, to improve the binding of reagent 22 to the surface of a wall of the measuring cavity 20.

Within a few minutes, the blood sample will have reacted with reagent 22, so that the red blood cells will have lysed and the staining agent will have accumulated in the white blood cell nuclei.

Referring to Figure 2, an embodiment of a sample acquisition device that is not part of the present invention will be described. The sample acquisition device 110 comprises a chamber 120 that forms the measurement cavity. The sample acquisition device 110 has an inlet 118 into the chamber 120 for transporting blood into the chamber 120. The chamber 120 is connected to a pump (not shown) through a suction tube 121. The pump can apply a suction force in the chamber 120 through the suction tube 121 so that blood can be sucked into the chamber 120 through the inlet 118. The sample acquisition device 110 can be disconnected from the pump before of the measurement being made. Like the measuring cavity 20 of the sample acquisition device 10 according to the first embodiment, the chamber 120 has a well defined thickness that defines the thickness of the sample to be examined. In addition, a reagent 122 is applied to the walls of chamber 120 so that it reacts with the blood sample.

Referring now to Figure 3, an apparatus 30 for the volumetric enumeration of white blood cells will be described. The apparatus 30 comprises a sample holder 32 for housing a sample acquisition device 10 with a blood sample. The sample holder 32 is arranged to accommodate the sample acquisition device 10 so that the measurement cavity 20 of the sample acquisition device 10 is correctly placed inside the apparatus 30. The apparatus 30 comprises a light source 34 for illuminating the blood sample inside the sample acquisition device 10. The light source 34 may be an incandescent lamp, which radiates light throughout the visible spectrum. The staining agent that accumulates in the white blood cell nuclei will absorb light of specific wavelengths, so that the white blood cell nuclei will appear in a digital image of the sample. If a color image is acquired, the white blood cells will appear as color points specifically. If a black and white image is acquired, the white blood cells will appear as black dots against a lighter background.

The light source 34 may alternatively be a laser or a light emitting diode. This can be used to increase the contrast in the image so that white blood cells can be detected more easily. In this case, the light source 34 is arranged to irradiate electromagnetic radiation of a wavelength that corresponds to a absorption peak of the staining agent. The wavelength must also be chosen so that the absorption of blood compounds is relatively low. In addition, the walls of the sample acquisition device 10 must be essentially transparent to the wavelength. For example, when methylene blue is used as a staining agent, the light source 34 can be arranged to radiate light having a wavelength of 667 nm.

The apparatus 30 further comprises an imaging system 36, which is disposed on the opposite side of the sample holder 32 in relation to the light source 34. Therefore, the imaging system 36 is arranged to receive the radiation that has been transmitted through the blood sample. The image obtaining system 36 comprises an enlargement system 38 and an image acquisition means 40. The expansion system 38 is arranged to provide an extension power of 3-200x, more preferably 3-100x, and most preferably 3-4x. Within these intervals of magnification power, it is possible to distinguish white blood cells. The image can be acquired with an improved resolution in order to allow a lower magnification power to be used. In addition, the depth of field of the expansion system 38 is still arranged to correspond at least to the thickness of the measurement cavity 20.

The magnification system 38 comprises a lens or objective lens system 42, which is arranged near the sample holder 32, and an eyeglass lens or system 44, which is disposed at a distance from the lens 42

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of objective. The objective lens 42 provides a first enlargement of the sample, which is further enlarged by the ocular lens 44. The enlargement system 38 may comprise additional lenses to achieve proper enlargement and imaging of the sample. The enlargement system 38 is arranged so that the sample in the measurement cavity 20 when placed in the sample holder 32 will focus on an image plane in the image acquisition medium 40.

The image acquisition means 40 is arranged to acquire a digital image of the sample. The image acquisition means 40 can be any type of digital camera, such as a CCD camera. The pixel size of the digital camera establishes a restriction in the imaging system 36 so that the circle of confusion in the image plane may not exceed the pixel size within the depth of field. However, white blood cells can still be detected even if they are somewhat blurred and, therefore, the confusion circle can be allowed to exceed the pixel size although it is considered to be within the depth of field. The digital camera 40 will acquire a digital image of the sample in the measurement cavity 20, in which the entire thickness of the sample is sufficiently focused on the digital image to count the white blood cells. The imaging system 36 will define an area of the measurement cavity 20, from which images will be obtained in the digital image. The area from which images are being obtained along with the thickness of the measurement cavity 20 define the volume of the sample from which images are being obtained. The imaging system 36 is configured to adjust the imaging of blood samples in the sample acquisition devices 10. There is no need to change the configuration of the imaging system 36. Preferably, the imaging system 36 is disposed within a housing so that the configuration is not accidentally changed.

The apparatus 30 further comprises an image analyzer 46. The image analyzer 46 is connected to the digital camera 40 to receive digital images acquired by the digital camera 40. The image analyzer 46 is arranged to identify patterns in the digital image that correspond to a white balloon for counting the number of white blood cells that are present in the digital image. Therefore, the image analyzer 46 can be arranged to identify black spots on a lighter background. The image analyzer 46 may be arranged to electronically enlarge the digital image first before analyzing the digital image. This implies that the image analyzer 46 can more easily distinguish white globules from those that are obtaining images that are close to each other, even though the electronic enlargement of the digital image will make the digital image somewhat 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 that are identified in the digital image by the volume of the blood sample, which is well defined as described above. The volumetric white blood cell count may be presented on a screen of the apparatus 30.

The image analyzer 46 can be performed as a processing unit, comprising codes for performing the image analysis.

Referring to Figure 4, a method for the volumetric enumeration of white blood cells will be described. The method comprises acquiring a blood sample in a sample acquisition device, step 102. An undiluted whole blood sample is acquired in the sample acquisition device. The sample can be acquired from capillary blood or venous blood. A sample of capillary blood can be inserted into the measurement cavity directly from a patient's finger in which a puncture has been performed. The blood sample comes into contact with a reagent in the sample acquisition device that initiates a reaction. Red blood cells can be lysed and a staining agent accumulates in the nuclei of white blood cells. Within a few minutes of acquiring the blood sample, the sample is ready for analysis. The sample acquisition device is placed in an analysis apparatus, step 104. An analysis can be initiated by pressing a button of the analysis apparatus. Alternatively, the apparatus automatically starts the analysis by detecting the presence of the sample acquisition device.

The sample is irradiated, step 106, and a digital image of an enlargement of the sample is acquired, step 108. The sample is being irradiated with electromagnetic radiation of a wavelength corresponding to a absorption peak of the staining agent. This implies that the digital image will contain black or darker dots at the positions of the white blood cell nuclei.

The acquired digital image is transferred to an image analyzer, which performs the analysis of the images, step 110, in order to count the number of black dots in the digital image.

In Figure 5, an example of a digital image is shown to indicate the possibility of identifying white blood cells in a blood sample that is hemolyzed and thrown away. This digital image was obtained from a sample acquisition device that had a cavity thickness of 140 pm and using a 50-fold magnification. The light source radiates white light, indicating that white blood cells can be identified even when the irradiation is not specifically adapted to a absorption peak of the staining agent. The staining agent used was methylene blue. The figure shows different black dots indicating white blood cells. The image shown in the figure

5 is a black and white version of a color image. The contrast between the white blood cells and the background appears more clearly in the color image than in the black and white image reproduced in this case. Blackheads can easily be counted by an image analyzer.

5 In manual white blood cell counting methods, approximately 200 cells are normally counted to determine the white blood cell count in the blood sample. The method and apparatus presented in this case can be arranged, for example, to count approximately 2000 cells, which gives a better statistical certainty of the results obtained. A normal healthy adult has a white blood cell count of 4-5 x 109 cells / liter of blood. This implies that 2000 cells are found in samples that have a volume of 0.410 0.5 pl. For example, if an image of an area of 1.5 x 1.5 mm is obtained in the measurement cavity that has a

thickness of 140 mm, the volume from which images are being obtained is 0.315 pl. A part of the acquired image can be selected for analysis. Therefore, the acquired image can be roughly analyzed so that anomalies are not allowed in the part being used to determine the white blood cell count. It can be selected that the part of the acquired image selected for analysis has an appropriate size so that a sufficient volume of the blood sample is analyzed.

It should be emphasized that the preferred embodiments described herein are by no means limiting and that many alternative embodiments are possible within the scope of protection defined by the appended claims.

twenty

Claims (27)

10 fifteen twenty 25 30 2. 3. 35 Four. 40 5. Four. Five 6. 7. fifty 8. 55 9. 60 10. Sample acquisition device (10) for the volumetric enumeration of white blood cells in a blood sample, said sample acquisition device (10) comprising: a measurement cavity (20) to accommodate a blood sample in which the blood sample is undiluted whole blood that has been acquired directly within the measurement cavity, said measurement cavity (20) having a predetermined fixed thickness, measured so that said thickness, when the device is in use, in combination with an area of the sample from which images are being obtained, defines a volume of analyzed sample; wherein the sample acquisition device (10) further comprises: a reagent (22), which is disposed in dry form on a surface that defines the measurement cavity (20), said reagent (22) comprising a hemolyzing agent to lyse red blood cells in the blood sample, and a staining agent for selectively have white blood cells in the blood sample; an inlet (18) of samples, which is defined between opposite walls within the sample acquisition device (10), said walls being arranged so close to each other that a capillary force can be created at the inlet (18) of samples; said measurement cavity (20) being arranged in communication with said sample inlet (18); Y a body element (12) having two flat surfaces to define said measuring cavity (20), in which the flat surfaces are arranged at a predetermined distance from each other to determine a sample thickness for an optical measurement, and in the that the flat surfaces of said measuring cavity (20) are arranged closer to each other than said sample inlet walls (18), so that a capillary force can introduce blood from the sample inlet (18) into the cavity (20) of measurement. Sample acquisition device (10) according to claim 1, wherein the flat surfaces are arranged at a distance from each other to determine a uniform thickness of 50-170 micrometers for optical measurement. Sample acquisition device (10) according to claim 2, wherein the flat surfaces are arranged at a distance from each other to determine a uniform thickness of at least 100 micrometers for optical measurement. Sample acquisition device (10) according to claim 2 or 3, wherein the flat surfaces are arranged at a distance from each other to determine a uniform thickness of no more than 150 micrometers for optical measurement. Sample acquisition device (10) according to any one of the preceding claims, wherein the reagent (22) has been applied to the surface dissolved in a volatile liquid that has evaporated to leave the reagent (22) in dry form. Sample acquisition device (10) according to any one of the preceding claims, wherein the staining agent is arranged to selectively have the nucleus of the white blood cells. Sample acquisition device (10) according to any one of the preceding claims, wherein the staining agent is any one of the group of hematoxylin, methylene blue, methylene green, methylene azure, cresyl violet acetate, blue of toluidine, gentian violet, Sudan analogues, galocyanine and fuchsin analogs, or any combination thereof. Sample acquisition device (10) according to any one of the preceding claims, wherein the hemolyzing agent is a quaternary ammonium salt, a saponin, a bile acid, a digitoxin, a snake venom, a glucopyranoside or a non-detergent. Triton ionic. Sample acquisition device (10) according to any one of the preceding claims, wherein said sample inlet (18) is arranged to acquire a blood sample. Sample acquisition device (10) according to any one of the preceding claims, wherein the sample acquisition device (10) is disposable. Method for the volumetric enumeration of white blood cells in a blood sample, said method comprising: 10 fifteen twenty 25 30 35 40 Four. Five fifty 55 60 acquiring a blood sample within a measuring cavity (20) of a sample acquisition device (10), in which the blood sample is undiluted whole blood that is acquired directly into the measurement cavity (20), irradiating the sample, acquire a digital image of an enlargement of the irradiated sample in the measurement cavity (20), and digitally analyze the digital image to identify white blood cells and determine the number of white blood cells in the sample; said sample acquisition device having a sample inlet (18), which is defined between opposite walls within the sample acquisition device (10), wherein said walls are arranged so close to each other that a capillary force can be created in the entry (18) of samples; said measurement cavity (20) being arranged in communication with said sample inlet (18); said measuring cavity (20) being defined by two flat surfaces, in which said flat surfaces are arranged at a predetermined distance from each other to determine a sample thickness for an optical measurement, and in which the flat surfaces of said cavity ( 20) of measurement are arranged closer together than said walls of the inlet (18) of samples, so that a capillary force can introduce blood from the inlet (18) of samples into the cavity (20) of measurement; Y said measuring cavity (20) contains a reagent (22), which is arranged in dry form on a surface defining the measuring cavity (20), which comprises a hemolyzing agent to lyse red blood cells and a staining agent to react with the sample so that the white blood cells are tined, in which the white blood cells are distinguished by selective staining of the staining agent. 12. Method according to claim 11, wherein the blood sample is mixed with the reagent (22) in the measurement cavity (20). 13. Method according to claim 11 or 12, wherein the measuring cavity (20) has a thickness of 50-170 micrometers, and said digital image is acquired with a depth of field corresponding at least to the thickness of the cavity (20 ) measurement. 14. Method according to claim 13, wherein a volume of an analyzed sample is well defined by the thickness of the measurement cavity (20) and an area of the sample from which images are being obtained. 15. Method according to any one of claims 11-14, wherein the sample is irradiated by light of a wavelength corresponding to a peak in the absorbance of the staining agent. 16. Method according to any one of claims 11-15, wherein said irradiation is performed by means of a laser source. 17. Method according to any one of claims 11-15, wherein said irradiation is carried out by means of a light emitting diode. 18. Method according to any one of claims 11-17, wherein the digital image is acquired using a magnification power of 3-200x, more preferably 3-10x. 19. Method according to any one of claims 11-18, wherein said analysis comprises identifying areas of high light absorbance in the digital image. 20. Method according to claim 19, wherein said analysis comprises identifying black spots in the digital image. 21. Method according to any one of claims 11-20, wherein said analysis comprises electronically enlarging the acquired digital image. 22. System for the volumetric enumeration of white blood cells in a blood sample, said system comprising: a sample acquisition device (10), and a measuring device (30) comprising: a sample acquisition device holder (32) arranged to accommodate the sample acquisition device (10) containing a blood sample in a 10 2. 3. fifteen 24. twenty 25. 25 26. 27. 30 28. 29. 35 30 40 31. cavity (20) of measurement, a source (34) of light arranged to irradiate the blood sample, an image obtaining system (36), comprising a magnification system (38) and a means (40) for acquiring digital images to acquire a digital image of an enlargement of the irradiated sample in the measurement cavity (20), in which the white blood cells are distinguished in the digital image by selective staining of a staining agent, and an image analyzer (46) arranged to analyze the acquired digital image to identify white blood cells and determine the number of white blood cells in the blood sample; wherein said sample acquisition device (10) is a device according to any one of claims 1-10. System according to claim 22, wherein the expansion system is provided with a depth of field of at least the thickness of the measuring cavity (20) of the sample acquisition device (10). System according to claim 22 or 23, wherein a volume of an analyzed sample is well defined by the thickness of the measurement cavity (20) and an area of the sample from which images are being obtained. System according to any one of claims 22-24, wherein the light source is arranged to radiate light of a wavelength corresponding to a peak in the absorbance of the staining agent. System according to any one of claims 22-25, wherein said light source comprises a laser source. System according to any one of claims 22-25, wherein said light source comprises a light emitting diode. System according to any one of claims 22-27, wherein the expansion system has an extension power of 3-200x, more preferably 3-10x. System according to any one of claims 22-28, wherein the image analyzer is arranged to identify areas of high light absorbance in the digital image. System according to claim 29, wherein the image analyzer is arranged to identify black spots in the digital image. System according to any one of claims 22-30, wherein the image analyzer is arranged to electronically enlarge the acquired digital image.