Method and apparatus for determining red blood cell indices of a blood sample utilizing the intrinsic pigmentation of hemoglobin contained within the red blood cells

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DESCRIPTION

Method and apparatus for determining red blood cell indices in a blood sample using the intrinsic pigmentation of hemoglobin contained in red blood cells

BACKGROUND OF THE INVENTION

1. Technical field

The present invention relates to apparatus and methods for the analysis of blood samples in general, and for the determination of the volume of a red blood cell as well as the average volume of cells of a particular sample.

2. Background information

Physicians, veterinarians, and scientists have examined human and animal biological fluids, especially blood, to determine their amounts of constituent particles as well as to identify the presence of unusual particles not seen in healthy subjects. Particles generally measured, quantified, and identified include red blood cells (RBC), white blood cells (WBC), and platelets. RBC analyzes can include determinations of the number, size, volume, shape, content, and concentration of RBC hemoglobin, and the hematocrit (also referred to as the cell-filled volume). RBC analysis may also involve the determination of the presence and / or concentration of certain components within red blood cells such as DNA, RNA, including the detection of the presence and / or enumeration of hematoparasites (e.g. malaria parasites ) or in RBCs or trypanosomes that are extracellular or leismaniasis organisms that are in WBCs as well as many other hematoparasites. WBC analyzes may include a determination of the population frequency of WBC subtypes generally referred to as a differential WBC count, as well as the reporting of any unusual cell types not found in healthy individuals. Platelet assays (or in hundreds of animals including birds, reptiles, and fish, thrombocytes that have a similar function to platelets in mammals but are approximately ten times larger and are nucleated) can include number, size, platelet shape, texture and volumetric determinations, including determination of the presence of platelet or thrombocyte clumps within the sample.

Known blood test techniques, described in detail in medical texts such as Wintrobe's Clinical Hematology 12^to Editing, they generally divide examination methods into manual, spin, and impedance type methods. Manual methods for cell enumeration typically involve the creation of an accurately determined volume of a blood or fluid sample that is quantitatively diluted and visually counted in a counting chamber. Manual examination methods include examining a peripheral smear in which the relative amounts of the particle types are determined by visual inspection. Centrifuge examination methods include centrifuging the sample, causing the sample to separate into layers of constituents according to the relative densities of said constituents. Each component layer can be tinted to improve visibility or detection. Impedance methods involve examining an exact volume of blood that is treated according to the particles to be measured, for example, lysing RBC for enumeration of nucleated cells and volumetrically diluting the sample in a conductive fluid. The procedure typically involves monitoring a current or voltage applied to the sample passing through a narrow passage to determine the effect that the particles have on the current / voltage as the particles pass through in a single row. Other techniques involve analyzing the intensity and angle of incident light scattering for particles passing in a single row through a beam of light. Flow cytometric methods may also be used that involve staining the particles of interest in suspension with fluorophores, binding to antibodies directed against surface epitopes present on cells or particle types, exciting the stained particles with light of appropriate wavelengths, and analyzing the emission of individual particles / cells.

All of the above-mentioned methods, apart from peripheral smear or centrifugal separation, require an exact volume of sample to be dispensed. Inaccuracies in the sample volume will result in quantitative errors of the same magnitude in the associated analysis. With the exception of centrifugation methods, all of the above-mentioned methods also require the sample to be mixed with one or more liquid reagents or diluents, and also require instrument calibration to obtain accurate results. In the case of peripheral smears, a high degree of training is required to properly examine the smear. Several of the aforementioned methods generate large volumes of contaminated waste that are expensive to handle. Furthermore, the methods described above are not suitable for determining the complete blood count (CBC) in birds, reptiles and fish in which the red blood cells and thrombocytes are nucleated and certain mammals in which the size of the red blood cells is very small and can be confused with platelets.

The amount of information that can be determined by examining the blood of a human or animal is enormous. It is particularly useful for determining RBS indices, for example, individual cell size, hemoglobin content and concentration in individual cells, and RBC population statistics within

ES 2 398 488 T3 a sample. The statistical mean and dispersion values ​​(for example coefficients of variation) for each of the aforementioned parameters can provide important information, as is evident from their discussion within the previously referenced text by Wintrobe, which has allowed clinicians to better catalog RBC disorders.

WO-99/44593 describes the analysis of quiescent anticoagulated whole blood samples.

Summary of the invention

In accordance with the present invention, a method is provided for determining RBC indices including volume and hemoglobin content and concentration for individual RBCs, as well as RBC population statistics, including the total number of rBc present in the sample. , and the mean values ​​for each of the aforementioned indices within a substantially undiluted blood sample.

According to a first broad aspect of the present invention, there is provided a method according to claim 1. The blood sample may be a substantially undiluted blood sample. In this method, the hemoglobin concentration of the red blood cell in contact with the inner surfaces can be determined using the determined optical density value which is that of the pixel optically aligned with the part of the red blood cell that extends between the inner surfaces. .

A method for determining a cell volume of a red blood cell within a substantially undiluted blood sample is described. The method includes the steps of: 1) providing a substantially undiluted blood sample; 2) depositing the sample in an analysis chamber adapted to quiescently support the sample for analysis, the chamber being defined by an inner surface of a first panel, and an inner surface of a second panel, in which both panels are transparent, and the chamber has a height that extends between the interior surfaces of the panels, and said height is such that at least one red blood cell within the sample is in contact with both interior surfaces; 3) imaging at least one red blood cell in contact with the interior surfaces, including a part of the red blood cell in contact with both interior surfaces; 4) determine the mean optical density of the part of the red blood cell in contact with both interior surfaces; 5) determining the total optical density of the whole red blood cell; and 6) determining the cell volume of at least one red blood cell using the height of the camera, the average optical density determined for the part of the red blood cell that has been imaged in contact with both interior surfaces, and the optical density determined for the entire imaged red blood cell.

An advantage of the present invention is that it can be used to determine the characteristics of a blood sample using an extremely small sample volume that can be obtained directly from the patient by piercing a capillary, making it more useful from the point of view. view of a careful application or a venous blood sample, if desired.

Another advantage of the present invention is that it can function to determine the characteristics of a blood sample using the intrinsic pigmentation of hemoglobin, and therefore it is not necessary to add any colorant or dye. The high molar extinction coefficient of hemoglobin allows accurate determinations of its relative or absolute concentration within very small optimal path distances, as small as a few microns.

Another advantage of the present invention is that it is possible to determine individual RBC indices in a particular cell, so that associations between indices can be identified.

Another advantage of the present method is that it works without external and internal fluids, and is independent of gravity or orientation, and is therefore adaptable for use in a portable device and under conditions of microgravity.

Another advantage of the method of the present invention is that unlike impedance counters, the apparatus of the present invention does not need to be calibrated each time it is used. The apparatus of the present invention is also not subject to variations such as the shape of the cells, the orientation of the cells as they flow through an orifice of the impedance-type cell counters for their measurement, or the effects of osmolality. diluent fluids required for impedance counting.

The present method and the advantages associated therewith will be more readily apparent in view of the detailed description provided below, including the accompanying drawings.

Brief description of the drawings

Figures 1 to 4 are schematic representations of the cross-sections of the analysis chambers that can be used in the present method.

Figure 5 is a schematic plan view of a tape having a plurality of analysis chambers.

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Figure 6 is a schematic plan view of a disposable container having an analysis chamber.

Figure 7 is a schematic cross-sectional view of a disposable container having an analysis chamber.

Figure 8 is a schematic of an analysis device that can be used with the present method.

Figure 9 is an enlarged view of a part of the analysis chamber shown in Figure 1.

Figure 10 is a block diagram illustrating the steps of the method for determining the hemoglobin concentration within a red blood cell, and the average hemoglobin concentration within a plurality of red blood cells according to one aspect of the present invention.

Figure 11 is a block diagram illustrating the steps of the method for determining the cell volume of a red blood cell, and the mean cell volume of a population of red blood cells according to one aspect of the present invention.

Figure 12 is a block diagram illustrating the steps of the method for determining the hemoglobin content of a red blood cell, and the mean hemoglobin content of a population of red blood cells according to one aspect of the present invention.

Detailed description of the embodiments of the invention

The method and apparatus of the present invention for analyzing a substantially undiluted whole blood sample allows the determination of the cell volume (CV) of a red blood cell (RBC), the mean cell volume (MCV), the concentration of hemoglobin of a cell (HCC), the mean hemoglobin concentration of cells (MCHC), and the mean hemoglobin content of cells (MCH), as well as their statistical population values, without adding any dye, reagent (other than anticoagulants in some embodiments) or diluents to the sample.

The present method uses an analysis chamber that is operable to quiescently contain a substantially undiluted anticoagulated whole blood sample for analysis. The chamber is typically sized to hold about 0.2 to 1.0 µl of sample, but the chamber is not limited to any particular volume capacity, and the capacity can vary to suit the application of the analysis. The phrase "substantially undiluted" as used herein describes a blood sample that is either not diluted at all or has not been determinedly diluted, but has some reagent added to it for testing purposes. In the event that the addition of the reagents dilutes the sample, at most, such dilution does not have a significant impact from a clinical point of view on the analysis performed. Typically, the only reagents that will be used in performing the present method are anticoagulants (eg, EDTA, heparin) and, in some cases, an isovolumic spherical agent. These reagents are generally added in dry form and are not intended to dilute the sample. In certain circumstances (eg very rapid tests), it may not be necessary to add the anticoagulant agent, but it is preferable in most cases to ensure that the sample is in a form acceptable for testing. The term "quiescent" is used to describe that the sample is deposited within the chamber for analysis, and said sample does not move purposefully relative to the chamber during analysis; that is, the sample is quiescent within the chamber. In the event that movement occurs within the blood sample, it will be mainly due to the Brownian movement of the constituents formed from the blood sample, and such movement is not disabling the use of the device of this invention.

Referring now to Figure 1, the analysis chamber 10 is defined by a first panel 12 that has an interior surface 14, and a second panel 16 that has an interior surface 18. Both panels 12 and 16 are transparent enough to allow the transmission of light of certain wavelengths through them in an amount sufficient to perform the optical density analysis described below. At least a part of the panels 12, 16 are parallel to each other, and in that part the inner surfaces 14, 18 are separated from each other by a height 20 such that at least some individual RCBs 22 within a sample are in contact each one individually with both interior surfaces 14, 18, and / or one or more RBC aggregates 23 within the sample are each in contact with both interior surfaces 14, 18 of the chamber panels 12, 16 and one or more voids of RBC 24 (eg, gaps) within the quiescent sample extend between the interior surfaces, as will be discussed in more detail below. The present method can use a variety of different types of analysis chambers having the aforementioned characteristics, and is therefore not limited to any particular type of analysis chamber. An analysis chamber having parallel panels 12, 16 simplifies analysis and is therefore preferred, but not required for the present invention; for example, a camera could be used having one panel arranged at a known non-parallel angle to the other panel.

Referring now to Figures 2-5, an example of an acceptable chamber 10 is shown that includes a first panel 12, a second panel 16, and at least three spacers 26 disposed between panels 12, 16. The spacers 26 may have any structure that can be arranged between the panels 12, 16 that acts to

ES 2 398 488 T3 separate said panels 12, 16 from each other. The dimension 28 of a spacer 26 extending between the panels 12, 16 is referred to herein as the height 28 of the spacer 26. The heights 28 of the spacer 26 are typically not exactly equal to each other (eg, manufacturing tolerances) , but are within the commercially acceptable tolerance for spacing media in similar analytical apparatus. Spherical beads are an example of an acceptable spacer 26 and are commercially available from, for example, Bangs Laboratories of Fishers, Indiana, USA.

In the embodiment of the chamber shown in Figure 3, the spacers 26 consist of a material that has a greater flexibility than one or both of the first panel 12 and the second panel 16. As can be seen in Figure 3, the larger spacers 26 are compressed to such an extent that most of the spacers 26 are touching the interior surfaces 14, 18 of the panels 12, 16, thus making the height of the chamber is only slightly less than the mean diameters of the spacers 26. In the embodiment of the chamber shown in Figure 4, the spacers 26 consist of a material that has less flexibility than one or both of the first 12 and second panels 16. In Figure 4, the first panel 12 is formed of a material more flexible than spherical spacers 26 and second panel 16, and will cover spacers 26 in a tent-like arrangement. In this embodiment, although there are small local regions of chamber 10 that may deviate from the desired height 20 of the chamber, the mean height 20 of chamber 10 will be very close to that of the mean diameter of spacers 26. Analysis indicates that average chamber height 20 can be controlled to one percent (1%) or better at chamber heights less than four microns using this embodiment. Subject to the flexibility characteristics described above (as well as other factors such as the distribution density of the spacers), the spacers 26 and panels 12, 16 can be made of a variety of materials as long as said loaves 12, 16 are sufficiently transparent. Examples of acceptable panels 12, 16 are transparent plastic films consisting of acrylic or polystyrene materials, and spherical beads made of polystyrene, polycarbonate, silicone, and the like are acceptable spacers 26. A specific example of an acceptable spacer are spheres made of polystyrene that are commercially available, for example, from Thermo Scientific of Fremont, California, USA, catalog number 4204A, with a diameter of four microns (4 pm). Referring to Figure 5, the panel 12 to be positioned vertically on top of the other includes a plurality of ports 30 arranged at regular intervals (for example acting as air vents), and the panels 12, 16 are joined together at some points. In some embodiments, bonding material 32 forms an outer chamber wall operable to laterally contain sample 34 within analysis chamber 10. This example of an acceptable analysis chamber is described in more detail in US Patent Application Publication No. 2007/0243117, and No. 2007/0087442, and US Provisional Patent Application Nos. 61 / 041,783, filed April 2, 2008; and 61 / 110,341, filed October 31, 2008.

Another example of an acceptable chamber 10 is placed in a disposable container 36 as shown in Figures 6 and 7. Chamber 10 is formed between a first panel 12 and a second panel 26. Both first 12 and second 16 panels are transparent to allow the passage of light through the chamber 10. At least a part of the first panel 12 and the second panel 16 are placed in parallel, and within that part the inner surfaces 14, 18 are separated from each other by a height 20. This embodiment of chamber 10 is described in more detail in US Patent No. 6,723,290. The analysis chambers shown in Figures 2-7 represent chambers that are acceptable for use in the present method. However, the present method is not limited to these particular embodiments.

A suitable chamber height is one in which at least some of the RBCs within the sample are individually in contact with both interior surfaces of the chamber panels, and / or one or more RBC aggregates are in contact with both surfaces. interiors of the chamber panels, and one or more RCB voids (eg, gaps) within the quiescent sample extend between the interior surfaces. Because the size of RBCs 22 within a blood sample is a function of the type of blood sample to be tested (e.g. human, monkey, horse, goat, fish, bird, etc.), the acceptable height of the chamber will vary depending on the individual to be tested. A chamber height of approximately two to six microns (2-6 pm) is acceptable for individual RCBs for most animal species based on typical RBC sizes and the fact that these RBCs can be deformed to some degree. (for example, the partially compressed spheres discussed above). A hematocrit analysis of an animal species having RBCs substantially larger or smaller than human RBCs can be performed in a chamber that has respectively a larger or smaller chamber height, respectively. In addition, a hematocrit test using RBC aggregates may have a chamber height that is dictated by the height of the RBC aggregates.

In those chamber embodiments that do not use spacers 26, the height 20 of chamber 10 may be determined as a part of the chamber manufacturing process and provided with the chamber. Alternatively, the height 20 of chamber 10 can be determined using a variety of techniques including the use of a known amount of sensitive colorant, or the use of geometric features arranged within the chamber, which can be used to determine the sample volume for a known field area, and consequently the height of the chamber. These techniques and others are described in US Patent Nos. 6,723,290 and 6,929,953. However, the present invention is not limited to these techniques.

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In some applications, an isovolumic spherical forming agent (for example an amphoteric detergent or a reagent that acts similarly) is mixed with at least a portion of the sample to make at least some of the RBCs substantially spherical. . RBCs 22 in their natural state are often shaped like a biconcave disc 38 (see Figure 1) rather than spherical 40. As a consequence, in the absence of the effect of the isovolumic spherical forming agent, a high percentage of the disc-shaped RBCs 22 will not be in contact with both panels 12,16 of the chamber. Increasing the number of RBCs 22 that have a substantially spherical geometry will increase the number of RBCs 22 in contact with both panels 12, 16, including some cells 42 that are contracted by the chamber panels, but would otherwise be spherical. The isovolumic spherical forming agent may be disposed in a single region of a chamber 10 (eg, by placing a particular portion of an interior surface). In the absence of sample mixing within chamber 10, the agent will mix only with the part of the sample proximate to said agent, thereby leaving other parts of the sample untreated by said spherical-forming agent. This selective failure of a portion of the RBC 22 to form isovolumic spheres allows, as will be described below, the qualitative morphology of the RBC 22 to be examined by image analysis as well as the presentation of images to a physician to inspect characteristics such as roundness. , their shape, and the presence of bumps on the cells. The formation of isovolumic spheres did not disturb any of the quantitative analyzes of the RBCs 22.

Analysis of the quiescently disposed sample within chamber 10 is performed using an analysis device that is operable to image at least a portion of the sample and perform image analysis. The image is produced in a way that allows the optical density of the sample to be determined on a per-unit basis. The term "criterion per unit" or "image unit" means a defined incremental unit from which the sample image can be dissected. A pixel, which is generally defined as the smallest element of an image that can be individually processed within a particular imaging system, is an example of an imaging unit, and an imaging unit may also include a small number of pixels in a collective unit. The magnification of an imaging device can also be described in linear terms (eg, micrometers per pixel in the focal plane), where the linear dimension is along a particular axis of an orthogonal grating applied to the image. The actual area of ​​the sample captured by pixels (or other imaging unit) of the sensor at the focal plane is therefore a function of the magnification factor applied by the imaging device. Image acquisition device must be known or determinable. The volume associated with that pixel is, therefore, the image area per pixel times the known height of the camera, since the point in the camera that was detected is one where the RCB extends through the entire the camera. For example, if the magnification were 0.5 microns per pixel, an image occupying 200 pixels would have an area of ​​50 square microns, and a volume of 50 square microns times the height of the camera.

Referring now to Figure 8, an example of an analysis device 44 that can be adapted for use with the method of the present invention includes a sample illuminator 46, an image dissector 48, and a programmable analyzer 50. Sample illuminator 46 includes a light source that selectively produces light of a wide range of wavelengths sufficient to be useful for hematocrit analysis (eg, about 400-670 nm; light of about 413 nm and about 540 nm is particularly effective in determining the optical density of RBCs within a human blood sample in view of the high absorption of light that occurs within hemoglobin at the aforementioned wavelengths, which is reflected in the high molar extinction coefficient (ε) at the aforementioned wavelengths) and typically includes optics to manipulate light. Sample illuminator 46 uses transmittances to produce an image. The light transmission properties of the sample can be measured, for example, by placing a light source on one side of the sample that is within chamber 10, directing the light through the sample arranged quiescently between the panels. from the camera, and then captured the light using an image dissector. An example of an acceptable image dissector 48 is a charge-coupled device (CCD) type image sensor that transforms an image of light passing through the sample into an electronic data format. Complementary metal oxide semiconductor ("CMOS") image sensors are another example of an image sensor that can be used, and the present invention is not limited to any of these examples. Programmable analyzer 50 includes a central processing unit (CPU) and is connected to sample illuminator 46 and image dissector 48. The CPU is adapted (eg, programmed) to selectively perform the functions necessary to carry out the method. of the present invention. It should be noted that the functionality of the programmable analyzer 50 can be implemented using hardware, software, firmware, or one of their combinations. A person skilled in the art would be able to program the treatment units to perform the functionality described herein without undue experimentation. US Patent No. 6,866,823 entitled "Apparatus for Analyzing Biologic Fluids" and issued August 15, 2005, describes such an analysis device 44.

The analysis device 44 is adapted to determine a DO value associated with the detected light signal on a per image unit criterion for an imaged portion of the sample. The OD of an RBC 22 is determined by the concentration of hemoglobin within the cell, the molar extinction coefficient (also called molar absorptivity) for hemoglobin at a given wavelength, and the distance of the optical path traveled through the hemoglobin, and can be represented by the following relationship:

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OD = scL where ε = molar extinction coefficient of hemoglobin, c = hemoglobin concentration, and L = the distance traveled through the RBC 22 (ie, the hemoglobin disposed within the cell). The molar extinction coefficient is an intrinsic property of hemoglobin that can be obtained from experimentation, or by commonly available empirical data. In embodiments of the analysis device that use light sources that have a margin of error (for example, an LED that has a design considering wavelength, plus or minus a certain amount), it is useful for accuracy purposes to initially calibrate the device. and determining the molar extinction coefficient of hemoglobin, which can then be used with that particular device until the light source is replaced, at which time the device can be recalibrated.

The detected light signal (ie, the OD values) can be used by an edge determination algorithm to identify the positions and boundaries of the RBCs. The RBCs 22 that are in contact with both inner surfaces of chamber 10 have an OD profile similar to that of a partially compressed sphere. The lateral edges of cells 22 that are not in contact with surfaces 14, 18 will have an OD that (in relative terms) can be considered to be approaching zero. The determined OD value: 1) increases as it moves in a direction toward the center of the RBC 22 (eg, as the light transmission path through the cell increases); 2) it reaches a maximum value and remains substantially constant when the RBC is in contact with the upper and lower surfaces 14, 18 (that is, when the path of the light transmitted through the RBC is constant); and 3) decreases as it moves in a direction away from the center of RBC 22 (eg, as the light transmission path through the cell decreases). This characterization of the OD profile of an RBC is particularly uniform for RBCs that are spherical in shape, and is not limited to RBCs in contact with both inner surfaces.

In some embodiments, the analysis device 44 is further adapted to determine a mean maximum OD value for a group of RBC 22 and / or aggregates of RBC 23 in contact with both interior surfaces. Determination of what constitutes an acceptable RBC pool size and / or RBC aggregates in contact with interior surfaces can be done on a per sample analysis criterion, or it can be done periodically for an "n" number of sample analyzes. the same type; for example, human blood samples. For example, a group of RBCs 22 identified as being in contact with both interior surfaces 14,18 can be benchmarked to determine the mean maximum DO value and the statistical deviation of DO within the group. It is desirable to determine the mean maximum OD value because the OD of hemoglobin within cells 22 can vary from cell to cell even within a particular sample. If the standard deviation is greater than a predetermined threshold, a new pool of RBCs 22 can be selected in contact with both panels 12, 16, or the existing pool can be expanded, until the aforementioned analysis establishes a pool of RBCs 22 that have a mean maximum DO value with an acceptable standard deviation. A mean maximum OD value of the RBCs 22 within a group that is approximately plus or minus one percent (1%) of the mean maximum OD value of all RBCs that are in contact with both surfaces 14,18 within the sample would, for example, be within acceptable standard deviation values. However, what constitutes an acceptable standard deviation value may vary depending on the management application and the specific statistical analysis being used (eg, standard error, etc.). Current statistical data regarding the OD of RBCs 22 are available and can be used in determining acceptable OD statistical values. Determining whether the RBCs within a particular group have a mean maximum OD value that is within a clinically acceptable standard deviation can also be tailored since, as noted above, It is well known that the RBC population within an individual typically has small variations in hemoglobin concentration and a continuous standard deviation of the results can be used to determine how many cells should be examined before obtaining an acceptable mean accuracy value; For example, for samples from an individual who has normal blood parameters, an acceptable group size may be as few as 100 RBC, while samples from an individual who have abnormal blood parameters may require analysis of 1000 or more RBC. The specific number of RBC 22 and / or RBC 23 aggregates in contact with both inner surfaces that is used to establish an acceptable mean maximum OD value is not limited to any particular number or percentage of RBC 22 and / or RBC aggregates. 23 within a sample, and may include all (eg thousands) of the RBC 22 and / or aggregates of RBC 23 in contact with both surfaces 14,18.

Referring now to Figures 9 and 10, the analysis device 44 is further adapted to determine the hemoglobin concentration ("CHC") of an RBC 22 by examining a portion 25 of an RBC in contact with both interior surfaces 14, 18 of chamber 10. The hemoglobin concentration is uniform within any given RBC. The OD value is determined on a per-pixel basis (or other imaging unit). The DO signal per pixel is representative of the DO signal attributable to the height "L" of the part of the camera "aligned" with said pixel. In the determination of HCC, the DO is detected, at the height of the chamber is either known or can be determined, and the molar extinction coefficient of hemoglobin (ε) is known. Therefore, HCC is determined using the relationship between the optical density (OD), the extinction coefficient of hemoglobin (ε), and the length of the path through the hemoglobin (L), which, for one part 25 of an RBC 22 in contact with the inner surfaces of the chamber is equal to the height of the chamber:

OD = scL, c = OD / sL,

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The mean cell hemoglobin concentration ("MCHC") of an RBC 22 is determined using the same methodology described above to determine the HCC of an individual RBC, repeated for the same number of RBCs in contact with both interior surfaces of the chamber. , using the results to determine a mean value and an acceptable standard deviation.

Referring now to Figures 9 and 11, the analysis device 44 is further adapted to determine the cell volume ("CV") of an individual RBC 22 in contact with both interior surfaces of chamber 10 by integrating the volume of the RBC. as a function of the OD of hemoglobin within the RBC. Volume integration can be performed using a variety of analytical techniques. For example, according to a first technique, the cell volume of an individual RBC 22 in contact with both inner surfaces 14, 18 can be determined using the height of the chamber 20, the mean value of the maximum optical density determined by the part 25 of the RBC 22 imaged in contact with both inner surfaces 14, 18, and the optical density determined for the entire RBC 22 imaged. The total optical density of the imaged RBC is divided by the mean value of the maximum optical density, and the result is corrected for the height of the camera. According to another technique, the cell volume is determined by dividing the individual RBC 22 into different parts: the part 25 that is in contact with both surfaces ("Region I") and a part 27 that is not in contact with both or even one of the interior surfaces 14, 18 ("Region II"). The volume of the part of the cell 25 in contact with the inner surfaces 14, 18 is determined by detecting the OD of said part 25 (ie Region I). OD is detected and defined on a per-pixel (or other imaging unit) criteria. The area of ​​the camera represented by the pixel is determined, as stated above, by the size of the image per pixel which is a function of the magnification factor of the instrument. The volume associated with that pixel is therefore the image area per pixel multiplied by the known height of the camera, since the point on the camera that was detected is one where the RBC 22 extends through of the entire height of the chamber 20. The volume of the part of RBC 25 in contact with both surfaces 14, 18 (that is, Region I) is determined by adding the volumes associated with each pixel within the contact area of ​​the two surfaces . The volume of the portion 27 of the RBC 22 that is not in contact with both surfaces 14, 18 (ie Region II) is determined in a similar manner. The DO value determined by the contact area of ​​the two surfaces is compared to the DO value for each pixel within the part 27 of the RBC 22 that is not in contact with both surfaces 14, 18 (i.e. Region II ). Since the molar extinction coefficient of hemoglobin (ε) is a linear function, the relative OD value of each pixel within Region II also represents the height of the RBC 22 associated with that pixel; For example, if the OD for that pixel is 50% of the OD in Region I, the height of the RBC 22 at that point is 50% of the height of the RBC in Region I (that is, the height camera 20). The volume associated with each pixel in Region II is determined on a per-pixel basis as described above and added to determine the volume in Region II of RBC 22. The volume of RBC 22 is the sum of Regions I and II. Decreasing the image area at each pixel (ie, increasing the resolution) increases the accuracy of the cell volume determination. These techniques are provided as examples of operable techniques, but the method of the present invention is not limited to such techniques.

The aforementioned techniques for determining cell volume are operable to determine the volume of the particular RBC 22 detected for OD. Because the OD of hemoglobin within RBCs 22 can vary from RBC to RBC even within a particular sample, determining the volume of cell 22 using the OD detected for that particular cell increases the accuracy of the determination of the volume. However, many RBC 22 are not in contact with both surfaces 14, 18 of chamber 10. For those RBC 22 that are not in contact with both surfaces, the cell volume can be determined using the mean value of the optical density. maximum obtained previously from the RBCs that are in contact with both surfaces 14, 18. The mean value of the maximum optical density obtained as described is sufficiently accurate to provide an exact volume of the other cells 22. As a further alternative, for those embodiments of the present invention that utilize an isovolumic spherical forming agent, the cell volume for an RBC fragment or an RBC 22 that is not in contact with both inner surfaces14, 18 can also be determined assuming the above-mentioned RBC or RBC fragment is spherical. If the perimeter of the RBC 22 can be determined using a profiling technique as described above, the circular area can be used to determine the size of the sphere and therefore the volume of the RBC 22.

The analysis device 44 is further adapted to determine the mean cell volume ("MCV") for the RBCs 22 within the sample using the same methodology described above, repeated for a number of RBCs 22, using the results to determine a value. mean and a measure of the accuracy or confidence of the mean value, for example, an acceptable standard error of the mean value. The number of RBCs 22 required to determine an MCV with an acceptable measure of accuracy will depend on the population of RBCs analyzed, and this number can range from about a few hundred to several thousand RCBs 22. One way to determine whether the number of RBCs, whose cell volumes have been determined, is an acceptable population for determining an MCV, is to iteratively determine the mean cell volumes of the sample within the population and determine the standard error for those mean values, that is, the standard deviation of the mean values. Once the measure of accuracy, (eg, standard error) is within a predefined acceptable range, then the MCV value is accepted.

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Referring now to Figures 9 and 12, the analysis device 44 is further adapted to determine the cell hemoglobin content ("CH") of an RBC 22 by integrating the hemoglobin concentration over the determined volume occupied by the RBC 22. individual. For RBCs 22 in contact with both interior surfaces 14, 18 of chamber 10, the CH is determined using the concentration (CHC) and volume (CV) determined for that particular RBC 22, as described above. If the CH is determined based on an individual RBC 22, then it can be done using the CHC determined for that particular RBC 22 instead of a mean CHC value (MCHC), thereby achieving a higher degree of accuracy. For RBCs 22 that are not in contact with both interior surfaces 14,18 of chamber 10, the CH is determined using the mean value of the maximum optical density to determine the concentration and volume for that particular RBC 22.

The analysis device 44 is further adapted to determine the mean cell hemoglobin content ("MCH") for RCB 22 within the sample using the same methodology described above, repeated for a number of RBC 22, using the results to determine a mean value and an acceptable measure of accuracy (for example, the standard error of the mean value). The number of RCB 22 required to determine an MCH with an acceptable measure of accuracy will depend on the population of RBC analyzed, the number of which can range from a few hundred to several thousand RBC 22. Once the accuracy measure (for example, the standard error) are within an acceptable range, then the MCH value is accepted.

The methodologies described above for determining CHC, MCHC, CV, MCV, CH and MCH values ​​are examples of how these parameters can be determined from a substantially undiluted blood sample using the chamber and testing device described with the present invention. . The present invention is not limited to these specific examples.

With the method of the present invention, a substantially undiluted whole blood sample is placed in chamber 10, as described above. An anticoagulant agent, and in some cases, an isovolumic spherical forming agent and / or an aggregating agent, is mixed with the sample, either before it is introduced into the chamber or at the time it is introduced into the chamber. Reagents added in dry or semi-dry form, for example by surface coating, are particularly easy to use. However, the present invention is not limited to reagents in dry form, and, for example, liquid reagents that do not significantly dilute the sample can be used. The sample is quiescent within the chamber. In certain circumstances (eg very rapid tests), it may not be necessary to add the anticoagulant agent, but it is preferable in most cases to ensure that the sample is in a form acceptable for testing. In certain analyzes (for example, those that provide information on individual RCBs), it may be preferable not to include the aggregating agent.

At least a portion of the sample that is quiescent within chamber 10 is imaged using analysis device 44 by transmitting light through the sample and detecting the transmitted light. Although it is not a requirement that images of the entire sample within chamber 10 be imaged, it is preferable to do so as it typically provides a more complete analysis of that sample (and all of its constituents), and a further increase in accuracy. as the distribution of RBC 22 and RBC 24 voids within a chamber is typically not homogeneous for a substantially undiluted whole blood sample.

A group of RBCs 22 in contact with the inner surfaces 12, 16 of the chamber 10 is determined by the analysis device 44 using the image of the part of the sample. Depending on what parameter of the blood sample is required, the analysis device 44 will determine one or more values ​​of the parameter values ​​to arrive at the required parameter value.

An advantage of the method of the present invention is that it is not necessary to have all of the RBCs 22 within the sample in contact with each panel of the chamber. The method can be performed with only some of the RBC 22 in contact with both inner surfaces 14, 18 of chamber 10. Smaller RBCs and RBC fragments are not used to calibrate the analysis, but are measured to determine their contribution. to the hematocrit. Furthermore, with the method of the present invention, the CHC, MCHC, CV, MCV, CH and MCH values ​​of the sample can be determined without knowing the total area or volume of said sample within chamber 10.

The RBCs 22 identified and analyzed by the present invention include reticulocytes (immature red blood cells), which develop in the spinal cord as nucleated cells. Before reticulocytes are released into the circulation, they shed their nuclei. Reticulocytes circulate for about a day in the bloodstream before losing their reticular tincture (which is dependent on the stained remnants of cytoplasmic RNA and nuclear DNA) and develop into mature RBCs 22 containing essentially only hemoglobin. The relative number of reticulocytes in the blood sample can be an important indicator of various disorders. For example, reticulocyte number is a good indicator of spinal cord red cell production activity, since it represents recent production. An abnormally low absolute number of reticulocytes can be indicative of aplastic anemia, pernicious anemia, malignant conditions in the spinal cord, problems in the production of erythropoietin, various deficiencies of vitamins or minerals (B9, B12, and iron), etc. An abnormally high absolute reticulocyte count may indicate rapid production due to the body's replacement of blood losses caused by hemorrhage or hemolysis. As a consequence, there are important reasons for being able to detect and enumerate reticulocytes. In the present invention reticulocytes are

ES 2 398 488 T3 are identified as an RBC 22 because they contain hemoglobin, and because they can be stained to identify remaining RNA and DNA.

Reticulocytes can also be distinguished from other RBC 22s and enumerated by mixing the sample with a dye such as a supervital dye such as acridine orange, "astrozone orange", or similar compounds. The dyes cause the reticulin naturally present in reticulocytes to emit fluorescent radiation when excited with ultraviolet light of approximately 470 nm. The location of the RBCs 22 within the quiescent sample can be determined by means of the optical density of the RBC due to the hemoglobin contained in all reticulocytes and determined from the images of the sample. Reticulocytes can be distinguished from RBC22 that do not contain reticulin and from white blood cells by imaging and examining the sample under fluorescence at one year or more selected wavelengths (eg 470 nm) associated with the supravital dye. For RBCs identified as reticulocytes by the presence of hemoglobin and reticulin fluorescence, the methodologies described above for determining cell volume, cell hemoglobin content, and cell hemoglobin concentration can be used to determine the same parameters for individual reticulocytes. In addition, statistical information (eg mean values ​​and accuracy measures) can be determined. The relative amount of reticulin within each reticulocyte, which varies inversely with the maturity of the reticulocyte, can also be determined by the intensity of the fluorescent signal.As a consequence, even more specific information can be determined regarding individual reticulocytes as well as the reticulocyte population. The relative amount of reticulin in an individual reticulocyte can be determined using either the area or intensity of the fluorescence peak, and can be calculated as a function of the volume of an individual reticulocyte.

Using the present invention, RBC indices including or excluding reticulocytes can also be determined. For example, the MCV of a sample can be represented both including and not including the contribution to volume due to reticulocytes. The determination of MCV without reticulocytes can mask the microcytosis of the population that is affected by the high size of the reticulocytes. Multiple other mathematical relationships between RBCs and reticulocytes can be determined.

As indicated above, individual indices can be determined by the present invention for RBCs 22 that are not in contact with both inner surfaces of loaves 14, 18. RBC fragments can be analyzed in the same way, allowing fragments to be distinguished of RBCs of other constituents found within a blood sample, eg platelets, clumps of platelets, white cell fragments, debris, etc. The ability to detect RBC fragments using the present invention is particularly useful because RBC fragments can be indicative of disorders such as microangiopathic anemia, severe inflammation, and widespread malignancies. Analysis of RBC fragments within a blood sample that has not been treated with an isovolumic spherical forming agent may include, for example, a morphological analysis to determine undisturbed morphological characteristics such as size determinations, deviation from roundness, perimeter to area ratio, and similar characteristics. The OD image produced using the present invention allows the determination of parameters such as sharpness, ellipticity, bumps, etc., for each RBC fragment, which facilitate the aforementioned morphological analysis. In addition, the volume of red cell fragments can be determined by measuring their diameters or circumferences and calculating the volume of a sphere with those dimensions.

Although this invention has been shown and described with respect to its detailed embodiments, those skilled in the art will understand that various changes can be made in shape and details without departing from the scope of the invention.