Counting of white blood cells

(54) Title: (56) Publications cited: (47) Abstract

HemoCue AB, Box 1204,262 23 Ängelholm SE

Stellan Lindberg, Förslöv SE

Johnny Svensson, Ängelholm SE

AWAPATENT AB

Volumetric determination of the number of white blood cells in a blood sample

WO Al 9 702 482, EP A2 0 321 889, WO A2 030 69 421, US Al 4 581 223

Sampling device for volumetric determination of the number of white blood cells in a blood sample comprises a measuring cavity for receiving a blood sample. The measuring cavity has a predetermined, fixed thickness. The sampling device further comprises a reagent arranged in dried form on a surface defining the measurement cavity. The reagent includes a hemolysis agent for lysis of red blood cells in the blood sample and a staining agent for selective staining of white blood cells in the blood sample.

A system comprises the sampling device and a measuring device. The measuring device includes a holder for the sample acquisition device, a light source and an imaging system for recording a digital image of an enlargement of the sample. The measuring device further comprises an image analyzer which is arranged to analyze the recorded digital image to determine the number of white blood cells in the blood sample.

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SUMMARY

Sampling device for volumetric determination of the number of white blood cells in a blood sample comprises a measuring cavity for receiving a blood sample. The measuring cavity has a predetermined, fixed thickness. The sampling device further comprises a reagent arranged in dried form on a surface defining the measurement cavity. The reagent includes a hemolysing agent for lysis of red blood cells in the blood sample and a staining agent for selective staining of white blood cells in the blood sample.

A system comprises the sampling device and a measuring device. The measuring device includes a holder for the sample acquisition device, a light source and an imaging system for recording a digital image of an enlargement of the sample. The measuring device further comprises an image analyzer which is arranged to analyze the recorded digital image for determining the number of white blood cells in the blood sample.

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FIELD OF ART

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

BACKGROUND OF THE INVENTION

Determination of the number of white blood cells is often of importance in the treatment of a patient. This assay may be required for the diagnosis of, for example, leukemia or infectious or inflammatory diseases or for monitoring treatments. It is desirable to enable analysis results to be obtained as quickly as possible to minimize patient waiting times and to enable a physician to make a decision regarding treatment and diagnosis immediately when conducting an initial examination of the patient. It would therefore be desirable to provide an analytical method that can be quickly performed by the doctor or a nurse without having to send a sample to a laboratory.

Today, the number of white blood cells is normally obtained by staining a blood sample and studying the sample under a microscope in a special chamber intended for counting, such as a Burker chamber. The chamber intended for counting is provided with a grid that divides the chamber into well-defined small volumes. The number of white blood cells can then be determined by counting the number of white blood cells per box in the grid. The number of white blood cells is obtained manually by an analyst who must be experienced in performing the analysis in order to perform a reliable analysis.

This analysis is time consuming. Furthermore, the analysis results may vary depending on the person performing the analysis because it is performed manually.

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There are a small number of existing automated assay methods for determining the number of white blood cells. The number of white blood cells can be determined by the Coulter principle, which is based on cell size determination and thus the cell type by sensing an impedance. A method of counting white blood cells by the Coulter principle is described in US 5,262,302.

Another method for determining the number of white blood cells is described in US 5,585,246. Here, a blood sample must be prepared by mixing it with a fluorescent dye and ligand complex that labels the white blood cells. The sample is inserted into a capillary and irradiated with a laser source that sweeps over the sample into the capillary. The fluorescence is measured to determine the number of white blood cells.

WO99 / ​​45384 discloses a chamber intended to accommodate a sample, which chamber has varying thicknesses. The varying thickness separates different blood components. The blood sample is stained with a dye to distinguish at least three different types of white blood cells in the blood sample. The white blood cells can be determined by number using an optical scanning instrument for studying a portion of the chamber.

There is still a need for speeding up and simplification of existing automated methods for determining the number of white blood cells so that the analysis can be done with accuracy.

SUMMARY OF THE INVENTION

An object of the invention is to provide a simple assay for determining a volumetric determination of the number of white blood cells. It is further an object of the invention to provide a quick analysis without the need for complicated devices or extensive sample preparations.

These objects are achieved in whole or in part by means of a sampling device, a method and a system according to

528 697 the independent requirements. Preferred embodiments are apparent from the dependent claims.

Thus, a sampling device for volumetric determination of the number of white blood cells in a blood sample is provided. The sampling device comprises a measuring cavity for receiving a blood sample. The measuring cavity has a predetermined fixed thickness. The sampling device further comprises a reagent arranged in a dried form on a surface defining the measurement cavity, which reagent comprises a hemolysing agent for lysis of red blood cells in the blood sample, and a dye for selective staining of white blood cells in the blood sample.

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

Although many existing methods can enumerate various blood cells and even subgroups of blood cells, the sampling device of the invention is particularly suitable for performing volumetric determination of the number of white blood cells. The reagent includes a hemolysing agent that will lyse the red blood cells in the blood sample. This destroys the ability to determine the red blood cells in the sample. On the other hand, the lysis of the red blood cells facilitates the differentiation and identification of the white blood cells in the blood sample.

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The invention also provides a method for volumetric determination of the number of white blood cells in a blood sample. The method comprises taking a blood sample into a measuring cavity of a sampling device, which measuring cavity contains a reagent comprising a hemolysing agent and a dye to react with the sample so that the white blood cells are stained, to irradiate the sample containing the stained white blood cells, image of an enlargement of the irradiated sample arranged in the measurement cavity; wherein the white blood cells are distinguished by selective staining by the staining agent, and digitally analyzing the digital image for identification of white blood cells and determining the number of white blood cells in the sample.

The invention further provides a system for volumetric determination of the number of white blood cells in a blood sample. The system includes a sampling device as described above. The system further comprises a measuring device comprising a holder for a sample collection device, which holder is arranged to receive the sample collection device containing a blood sample arranged in the measuring cavity, and a light source arranged to irradiate the blood sample. The measuring device further comprises an imaging system comprising an enlargement system and a means for receiving a digital image of an enlarged sample of the irradiated sample arranged in the measuring cavity, wherein white blood cells are distinguished in the digital image by selective staining by the dye. The measuring device further comprises an image analyzer which is arranged to analyze the recorded digital image for identification of white blood cells and for determining the number of white blood cells in the blood sample.

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 any complicated measuring device or advanced steps such as

528 697 must be performed by an operator. Therefore, it can be performed directly in connection with examination of a patient, without the need for a qualified technician. The measuring apparatus uses the characteristics of the sampling device to perform an analysis on a sample of non-thinned whole blood that has been absorbed directly into the measuring cavity. The measuring device is arranged to image a volume of the sample to provide a volumetric determination of the number of white blood cells from the single image.

The blood sample is allowed to mix with the reagent in the measuring cavity. Within a few minutes, the reaction of the blood sample with the reagent will have hemolyzed the red blood cells and stained the white blood cells so that the sample is ready to be presented for the optical measurement. The blood sample may be mixed with the reagent by, for example, dispersion or diffusion of the reagent into the blood sample or by active vibration or movement of the sample collection device so that agitation is caused in the measuring cavity.

The sampling device may comprise a body member having two flat surfaces for defining said measuring cavity. The planar surfaces may be arranged at a predetermined distance from each other for determining a sample thickness for an optical measurement. This means that the sampling device provides a well-defined thickness for the optical measurement that can be used to accurately determine the number of white blood cells per volumetric unit of the blood sample. A volume of an analyzed sample will be well defined by the thickness of the measuring cavity and by imaging an area of ​​the sample. The well-defined volume can thus be used to relate the number of white blood cells to the volume of the blood sample so that the volumetric number of white blood cells is determined.

The measuring cavity preferably has a uniform thickness of 50-200 microns, and more preferably 100-150 microns. This means that the measuring cavity does not force

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the blood sample to be smeared into a single layer, allowing a larger blood volume to be analyzed over a small cross-sectional area. Thus, a sufficiently large volume of the blood sample can be analyzed to obtain reliable values ​​for the number of white blood cells using a relatively small image of the blood sample. For most tests, the number of white blood cells is so low that there will only be minor anomalies caused by white blood cells being arranged on top of one another.

The digital image can be recorded with a depth of field corresponding to at least one third of the thickness of the measuring cavity. If the focus of the imaging system is centered within the thickness of the measuring cavity, the depth of field will be arranged so that the entire thickness of the measuring cavity can be imaged with a sufficient sharpness to identify the white blood cells. This means that a sufficient focus is obtained through the entire sample thickness, whereby the entire thickness of the measuring cavity can be analyzed simultaneously in the digital image of the sample. By choosing not to focus really sharply on a specific part of the sample, a sufficient focus is obtained on the entire sample thickness to allow identification of the number of white blood cells in the sample. The depth of field should more preferably correspond to at least half the thickness of the measuring cavity or even at least the entire thickness of the measuring cavity. This would further facilitate the identification of the white blood cells in the blood sample.

The dye may be provided for selective staining of the cores of the white blood cells. This means that the white blood cells can be identified as colored dots and thus are easily counted in a digital image.

The dye may be slightly in the group hematoxylin, methylene blue, methylene green, toluidine blue, gentian violet, Sudan analogues, gallocyanine and Fuksin analogues. However, it should be understood that the dye is not

528 697 limited to this group without other substances being relevant.

The hemolyzing agent may be a quaternary ammonium salt, a saponin, a bile acid such as deoxycholate, a digitoxin, a snake poison, a glucopyranoside or a non-ionic Triton type detergent. However, it should be understood that the hemolysing agent is not limited to this group without other substances being possible.

The sampling device may further comprise a sample inlet which communicates the measuring cavity with the exterior of the sampling device, which inlet is arranged to receive a blood sample. The test inlet may be arranged to draw a blood sample through a capillary force, and the measuring cavity may further draw blood from the inlet and into the cavity. As a result, the blood sample can easily be absorbed into the measuring cavity by simply passing the sample inlet into contact with blood. Then, the capillary forces of the sample inlet and the measuring cavity will draw a well-defined amount of blood into the measuring cavity. Alternatively, the blood sample may be sucked or pressed into the measuring cavity by applying an external pumping force to the sampling device.

The sample may be irradiated with light having a wavelength corresponding to a peak of the absorbent of the dye. Accordingly, the stained white blood cells containing an accumulation of staining substance will be detected by a low light transmission.

The irradiation can be done by a laser source. The laser source can provide light with a well-defined wavelength to match the absorbance of the dye. Furthermore, the laser source provides collimated light, which minimizes interference from the jet light, whereby a point of low light transmittance will be sharply discerned.

Alternatively, the irradiation can be carried out by means of an LED. This light source can still provide adequate irradiation conditions for proper operation

528 697 distinction of white blood cells from other matter in the sample.

The digital image can be obtained using a magnification of 10-200x, and more preferably 4010Ox. Within these magnification ranges, the white blood cells are sufficiently enlarged to be detectable while the depth of field may be arranged to cover the sample thickness.

The analysis involves identifying areas of high light absorbance in the digital image. The analysis may further include identifying black or dark dots in the digital image. Since the dyes can accumulate in the cores of the white blood cells, the light absorbance may have peaks at separate points. These dots will form black dots in the digital image.

The analysis may further include electronic magnification of the recorded digital image. Although the sample is enlarged to obtain an enlarged digital image of the sample, the recorded digital image can itself be enlarged electronically to simplify the distinction between objects that are imaged very close to each other in the recorded digital image.

BRIEF DESCRIPTION OF DRAWINGS

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

Fig. 1 is a schematic view of a sample collection device in the form of a cuvette according to an embodiment of the invention.

Fig. 2 is a schematic view of a sampling device according to another embodiment of the invention.

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

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

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Fig. 5 is a digital image of a blood sample to be used for volumetric determination of the number of white blood cells.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

With reference to Fig. 1, a sampling device in the form of a cuvette 10 according to an embodiment of the invention will be described. The cuvette 10 is for single use and is intended to be discarded after being used for analysis. This means that the cuvette 10 does not require complicated handling. The cuvette 10 is preferably formed in a plastic material and can be produced by injection molding. This makes the production of the cuvette 10 simple and inexpensive, whereby the cost of cuvettes 10 can be kept down.

The cuvette 10 comprises a body member 12 having a body 14 which can be touched by a user without causing any disturbance to the analysis results. The body 14 may also have projections 16 which can fit a cuvette holder in an analysis device. The projections 16 may be arranged so that the cuvette 10 will be correctly positioned in the analyzer.

The cuvette 10 further comprises a sample inlet 18. The sample inlet 18 is defined between opposing walls within the cuvette 10, which walls are 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 exterior of the cuvette 10 to allow blood to be drawn into the cuvette. 10. The cuvette 10 further comprises a measuring cavity 20 disposed between opposing walls within the cuvette 10. The measuring cavity 20 is arranged to be in communication with the sample inlet 18. The walls defining the measuring cavity 20 are arranged closer together than the walls of the sample inlet 18, whereby a capillary force can draw blood from the sample inlet 18 and into the measuring cavity 20.

The walls of the measuring cavity 20 are spaced 50-200 microns apart, and more preferably

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100-150 microns. The distance is uniform over the entire measuring cavity 20. The thickness of the measuring cavity 20 defines the volume of blood being examined. Since the analysis result is to be compared with the volume of the blood sample being examined, the thickness of the measuring cavity 20 must be very accurate, ie only very small variations in the thickness are allowed inside the measuring cavity 20 and between the measuring cavities of different cuvettes 10. The thickness allows analysis of a relatively large sample volume in a small amount. area of ​​the cavity. The thickness theoretically allows white blood cells to be arranged on top of one another inside the measurement cavity 20. However, the amount of white blood cells in blood is so small that the probability of this happening is very small.

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

Reagent 22 is applied by introducing reagent 22 into the measurement cavity 20 using a pipette or dispenser. The reagent is dissolved in water or an organic solvent when introduced into the measurement cavity 20. The solvent with the reagent 22 can fill the measurement cavity 20. Thereafter, drying is performed so that the solvent will evaporate and the reagent 22 adheres to the surfaces of the measuring cavity 20.

Reagent 22 comprises a hemolysing agent and a dye. The hemolysing agent may be a quaternary ammonium salt, a saponin, a bile acid such as deoxycholate, a digitoxin, a snake poison, a glucopyranosive or a non-ionic Triton type detergent. The dye may be hematoxylin, methylene blue, methylene green, toluidine blue, gentian violet, a Sudan analogue, gallocyanin or a Fuksin analogue. When a blood sample makes contact with the reagent 22, the hemolysis agent will lyse the red blood cells so that the red blood cells are mixed with the blood plasma. Furthermore, the dye will accumulate in the cores of the white blood cells. Reagent 22 should contain different amounts of staining agent to clearly stain all of the white blood cell cores. Thus, there will often be an excess of dye that will be mixed together in the blood plasma. The excess of dye will give a homogeneous low background level with dye in the blood plasma. The accumulated dye in the white blood cells will be distinguishable relative to the background level of the dye.

Reagent 22 may also include other constituents which may be active, ie, participate in the chemical reaction with the blood sample, or be inactive, ie not participate in the chemical reaction with the blood sample. For example, the active ingredients may be arranged to catalyze the hemolysis or staining action. For example, the inactive ingredients may be arranged to improve the adhesion of the reagent 22 to a wall surface in the measuring cavity 20.

Within a few minutes, the blood sample will have reacted with reagent 22, whereby the red blood cells have been lysed and the dye has accumulated in the nuclei of the white blood cells.

Referring now to Fig. 2, another embodiment of the sampling device will be described. The sampling device 110 includes a chamber 120 which forms the measuring cavity. The sampling device 110 has an inlet 110 for the chamber 120 for transporting blood into the chamber 120. The chamber 120 is connected to a pump (not shown) via a suction tube 121. The pump can apply a suction force in the chamber 120 via the suction tube 121 so that blood can be sucked. into chamber 120 through inlet 118. The sampling device 110 can be disengaged from the pump before the measurement is performed. Like the cuvette's measuring cavity, chamber 120 has a well-defined thickness that defines the thickness

528 697 of the sample to be examined. Further, a reagent is applied to the walls of the chamber 120 for reaction with the blood sample.

Referring now to Fig. 3, a device 30 for volumetric determination of the number of white blood cells will be described. The device 30 includes a sample holder 32 for receiving a cuvette 10 containing a blood sample. The sample holder 32 is arranged to receive the cuvette 10 so that the measuring cavity 20 of the cuvette 10 is correctly positioned within the device 30. The device 30 includes a light source 34 for irradiating the blood sample within the cuvette 10. The light source 34 can be a light bulb that radiates light throughout the visible spectrum. The dye accumulated in the cores of the white blood cells will absorb light at specific wavelengths, whereby the cores of the white blood cells will appear in a digital image of the sample. If a color image is recorded, the white blood cells will appear as specially colored dots. If a black and white image is recorded, the white blood cells will appear as black dots against a lighter background.

Alternatively, the light source 34 may be a laser or an LED. This can be used for increased contrast in the image so that the white blood cells can be more easily detected. In this case, the light source 34 is arranged to emit electromagnetic radiation with a wavelength corresponding to an absorption peak of the dye. Furthermore, the wavelength should be chosen so that the absorption of the blood cells is relatively small. Furthermore, the walls of the cuvette should be substantially transparent to the wavelength. For example, where methylene blue is used as a dye, light source 34 may be arranged to emit light having a wavelength of 667 nm.

The device 30 further comprises an imaging system 36 arranged on one side opposite the sample holder 32 relative to the light source 34. The imaging system 36 is thus arranged to receive radiation having

528 697 transmitted through the blood sample. The imaging system 36 comprises a magnification system 38 and an image acquisition means 40. The magnification system 38 is arranged to provide a magnification of 10-200x, and more preferably 40-100x. Within these magnification ranges, it is possible to distinguish the white blood cells. Furthermore, the depth of field of the enlargement system 38 can still be arranged to at least correspond to the thickness of the measuring cavity 20.

The magnification system 38 includes an objective lens or lens system 42 disposed close to the sample holder 32, and an ocular lens or lens system 44 disposed at a distance from the objective lens 42. The objective lens 42 provides a first magnification of the sample, which is further enlarged by the eyepiece lens 44 The magnification system 38 may include additional lenses to obtain a suitable magnification and imaging of the sample. The magnification system 38 is arranged so that the sample in the measurement cavity 20, when disposed in the sample holder 32, will be focused on an image plane of the image acquisition means 40.

The image acquisition means 40 is arranged to record a digital image of the sample. The image capture means 40 can be any type of digital camera, such as a CCD camera. The pixel size of the digital camera places a limit on the imaging system 36 so that the blur circle in the image plane cannot exceed the pixel size within the depth of field. The digital camera 40 will record a digital image of the sample provided in the measurement cavity 20, the entire sample thickness being sufficiently focused in the digital image to count the white blood cells. The imaging system 36 will define an area of ​​the measurement cavity 20 which will be imaged in the digital image. The area imaged together with the thickness of the measurement cavity 20 defines the volume of the sample being imaged. The imaging system 36 is set to fit imaging of blood samples in cuvettes 10. There are

528 697 no need to change the setting of the imaging system 36. The imaging system 36 is preferably arranged inside a housing so that the setting is not unintentionally altered.

The device 30 further comprises an image analyzer 46. The image analyzer 46 is connected to the digital camera 40 for receiving the digital images recorded with the digital camera 40. The image analyzer 46 is arranged to identify patterns in the digital image corresponding to a white blood cell for counting the number of white blood cells that appear in the digital image. Thus, the image analyzer 46 may be provided for identifying dark spots in a lighter background. The image analyzer 46 may be arranged to first electronically enlarge the digital image before analyzing the digital image. This means that the image analyzer 46 can more easily distinguish white blood cells that are imaged close together, although the electronic magnification of the digital image will blur the digital image somewhat.

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 image by the blood sample volume, which is well defined as described above. The volumetric number of white blood cells can be presented on a display of device 30.

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

Referring now to Fig. 4, a method for volumetric determination of the number of white blood cells will be described. The method includes taking a blood sample into a cuvette, step 102. An undiluted whole blood sample is taken into the cuvette. The sample may be taken from capillary blood or venous blood. A sample of capillary blood can be drawn into the measuring cavity directly from a pricked finger on a patient. The blood sample makes contact with a reagent in the cuvette, which initiates a reaction. The red ones

528 The 697 blood cells will be lysed and a dye will accumulate in the cores of the white blood cells. Within a few minutes of taking the blood sample, the sample is ready to be analyzed. The cuvette is arranged in an analyzer, step 104. An analysis can be initiated by pressing a button on the analyzer. Alternatively, the assay is automatically initiated by the device sensing the presence of the cuvette.

The sample is irradiated, step 106, and a digital image of an enlargement of the sample is recorded, step 108. The sample is irradiated with electromagnetic radiation having a wavelength corresponding to an absorption peak of the dye. This means that the digital image will contain black or dark spots in the positions of the white blood cell nucleus.

The recorded digital image is transferred to an image analyzer that performs image analysis, step 110, to calculate the number of black dots in the digital image.

Fig. 5 shows an example of a digital image showing the possibility of identifying white blood cells in a blood sample that is hemolyzed and stained. This digital image was obtained with a cuvette having a cavity thickness of 140 µm and using 50x magnification. The light source radiates white light, which indicates that the white blood cells can be identified even though the radiation is not very well adapted to an absorption peak of the staining body. The dye used was methylene blue. Distinct black dots are shown in Fig. 5, indicating white blood cells. The image shown in 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 herein. The black dots can easily be counted with an image analyzer.

In manual methods for counting the number of white blood cells, about 200 bodies are usually counted to determine the number of white blood cells in the blood sample.

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For example, the method and apparatus presented here may be arranged to count about 2000 bodies, which provides better statistical certainty regarding the results obtained. A normal healthy adult has a number of white blood cells of 4-5x10⁹ bodies per liter of blood. This means that 2000 bodies are found in samples that have a volume of 0.4-0.5 µl. For example, if an area of ​​1.5 x 1.5 mm in the measuring cavity having a thickness of 140 µm is imaged, the volume imaged amounts to 0.315 µl. Some of the recorded image can be selected for analysis. Thus, the recorded image can first be roughly analyzed so that no anomalies are allowed in the portion used to determine the number of white blood cells. The portion of the recorded image selected for analysis may be selected to be of a suitable size so that a sufficient volume of the blood sample will be analyzed.

It will be appreciated that the preferred embodiments described herein are in no way limiting and that many alternative embodiments are possible within the scope of protection defined by the appended claims.

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