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

Procedure and apparatus for determining the hematocrit of a blood sample using the intrinsic pigmentation of the hemoglobin contained in the red blood cells.

Background of the invention

<dl><dt>1.</dt><dd> Technical field </dd></dl>

The present invention relates to apparatus and methods for the analysis of blood samples in general, and for the determination of the hematocrit of a particular blood sample.

<dl><dt>2.</dt><dd> Background Information </dd></dl>

Doctors, veterinarians and scientists have examined biological fluids of humans and animals, especially blood, to determine their amounts of constituent particles as well as to identify the presence of unusual particles not seen in healthy subjects. Generally measured, quantified and identified particles include red blood cells (RBC), white blood cells (WBC) and platelets. RBC analyzes may include determinations of the number, size, volume, shape, content and concentration of hemoglobin of RBC, and the hematocrit (also called the cell-filled volume). RBC analyzes 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 (eg malaria parasites) or in the RBC or trypanosomes that are extracellular or leismaniasis organisms that are in the WBC as well as 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 notification of any unusual cell type not found in healthy individuals. Platelet analysis (or in hundreds of animals that include birds, reptiles and fish, thrombocytes that have a function similar to that of platelets in mammals but are approximately ten times larger and nucleated) may include the number, size, shape, texture of platelets and volumetric determinations, including the determination of the presence of platelet agglomerations or thrombocytes within the sample.

Known blood test techniques, described in detail in medical texts such as Wintrobe's Clinical Hematology 12th Edition, generally divide the test methods into manual, centrifugal and impedance methods. Manual methods for cell enumeration typically involve the creation of a precisely 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 particle types are determined by visual inspection. Centrifugal examination methods include centrifugation of the sample, causing separation of the sample into layers of constituents according to the relative densities of said constituents. Each component layer can be dyed to improve visibility or detection. The impedance methods involve examining an exact amount of blood that is treated according to the particles to be measured, for example, by lysing RBC for the enumeration of the nucleated cells and volumetrically diluting the sample in a conductive fluid. The procedure typically involves monitoring a current or voltage applied to the sample that passes 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 scattering of incident light for particles that pass in a single row through a beam of light. Cytometric flow methods can also be used that involve staining the particles of interest in suspension with fluorophores, exciting the light-stained particles of appropriate wavelengths, and analyzing the emission of the individual particles / cells. US-6235536-B describes the analysis of quiescent anticoagulated whole blood samples.

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

Despite the complex amount of hematological information that is obtained from the complete blood count, an assay is often the most necessary: ​​the hematocrit. It is the hematocrit that tells the doctor if the patient has anemia due to bleeding or nutritional causes such as iron deficiency relatively common in growing children and women of reproductive age, processes associated with disorders such as chronic infections, metabolic disorders such uremia or neoplastic diseases as well as pharmacological effects. A high hematocrit value indicates the presence of too many red blood cells due to processes of disorders such as dehydration in which the blood is concentrated. A high hematocrit value may also be indicative of real increases in the mass of red blood cells that occur as a result of processes of disorders such as polycythemia, or pharmacological effects such as the administration of too many anabolic steroids or chronic hypoxia due to disorders of the lungs or certain types of congenital heart disorders. The importance and usefulness of hematocrit make it one of the most frequently requested blood tests. As a consequence, easy, accurate, economical and rapidly available hematocrit determinations are very desirable and will benefit patients. An instrument that can use a disposable analysis chamber, one that can operate without internal fluids apart from the capillary flow (that is, one that can function independently of gravity and orientation), and one that can be used as a portable device would be of great benefit.

Summary of the invention

According to a first and broad aspect of the present invention, a method is provided for determining the hematocrit of a blood sample according to claim 1. The blood sample may be a substantially undiluted blood sample. The height of said chamber is such that at least some of the RBCs within the sample, either individually or in aggregate form, are in contact with both interior surfaces of the panels either individually or in the form of aggregate.

According to a second and broad aspect of the invention, an apparatus is provided for determining the hematocrit of a substantially diluted blood sample that is quiescently contained within an analysis chamber, according to claim 11. The height of the chamber is such that at least some of the RBCs within the sample individually or as an aggregate, are in contact with both interior surfaces either individually or as an aggregate.

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

Another advantage of the present invention is that a hematocrit value can be determined independently of the knowledge of the magnification factor of the instrument (image size / image unit) and without knowing the height of the camera. Therefore, the method of the present invention has great versatility with respect to the type of instrument and analysis chamber that can be used.

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

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

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 show 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.

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

Figure 7 is a schematic view of a cross section 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 a block diagram illustrating a method according to the present invention.

Detailed description of the embodiments of the invention

The method and apparatus of the present invention for determining the hematocrit of a blood sample allows the determination of said hematocrit without adding dyes, reagents (apart from anticoagulants in some embodiments) or diluents to the sample, or without the need to know exactly the volume of the sample or the height or volume of the analysis chamber. In some embodiments, the method and apparatus of the present invention includes the addition of an agent that causes aggregation of the RBCs. Agents such as polybrene, and anti-glycoforin antibodies, or the like cause almost instantaneous aggregation of the RBCs within the sample. At least some of these RBC aggregates will be in contact with the opposite interior surfaces of the chamber. The optical density of an RCB aggregate that extends between the surfaces can be used in the same way as the optical density of a single cell to calculate the hematocrit.

The present method uses an analysis chamber that is operable to quiescently contain a sample of substantially undiluted whole blood for analysis. The chamber typically has a size to contain approximately 0.2 to 1.0 µl of sample, but said chamber is not limited to any particular volume capacity, and said capacity may 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 diluted with determination, but some reagent has been added for analysis purposes. In the event that the addition of the reagents dilutes the sample, at most, said dilution does not have a clinically important impact on the analysis performed. Typically, the only reagents that will be used in the performance of the present method are anticoagulants (eg, EDTA, heparin) and, in some cases, an isovolumetric sphere forming agent or an aggregate forming agent and are not intended to dilute the sample. In certain circumstances (for example, very rapid analyzes), it may not be necessary to add the anticoagulant agent, but it is preferable to do so in most cases to ensure that the sample is in a

Acceptable form for analysis. The term "quiescent" is used to describe that the sample is deposited within

of the camera for analysis, and said sample does not move resolutely with respect to the camera during the analysis; that is, the sample is quiescently inside 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 of the blood sample, which 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 having an inner surface 14, and a second panel 16 having an inner surface 18. Both panels 12 and 16 are transparent enough to allow light transmission of certain wavelengths therethrough 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 with each one individually with both inner surfaces 14, 18, and / or one or more RBC aggregates 23 within the sample are each in contact with both inner surfaces 14, 18 of the chamber panels 12, 16 and one or more empty areas of RBC 24 (eg, deficiencies) 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 cameras that have the aforementioned characteristics, and, therefore, is not limited to any particular type of analysis camera. An analysis chamber having parallel panels 12, 16 simplifies the analysis and is therefore preferred, but not required for the present invention; for example, a camera having a panel arranged at a known non-parallel angle with respect to the other panel could be used.

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 arranged between panels 12, 16. Spacers 26 may have any structure that can be arranged between panels 12, 16 that acts to separate said panels 12, 16 from one another. The dimension 28 of a separator 26 extending between the panels 12, 16 is referred to herein as the height 28 of the separator 26. The heights 28 of the separators 26 are not typically exactly the same as each other (eg manufacturing tolerances) , but are within the commercially acceptable tolerance for spacing means in similar analysis apparatus. Spherical accounts are an example of an acceptable separator 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 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 inner surfaces of the panels 12, 16, thereby making the chamber height only slightly lower 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 first 12 and second 16 panels. In Figure 4, the first panel 12 is formed of one more material flexible than the spherical spacers 26 and the second panel 16, and will cover the spacers 26 in an arrangement similar to a tent. In this embodiment, although there are small local regions of the chamber 10 that can deviate from the desired height 20 of the chamber, the average height 20 of the chamber 10 will be very close to that of the average diameter of the spacers 26. The analysis indicates that the average camera height 20 can be controlled up to one percent (1%) or better at camera heights less than four micrometers using this embodiment. Subject to the flexibility characteristics described above (as well as other factors such as the distribution density of the separators), the spacers 26 and the panels 12, 16 can be made of a variety of materials as long as said loaves 12, 16 Be transparent enough. Examples of acceptable panels 12, 16 are transparent plastic films consisting of acrylic or polystyrene materials, and spherical beads 26 made of polystyrene, polycarbonate, silicone and similar materials are acceptable separators 26. A specific example of an acceptable separator are spheres made of polystyrene that are commercially available, for example, in Thermo Scientific of Fremont, California, USA, catalog number 4204 A, with a diameter of four micrometers (4 μm) . With reference to Figure 5, the panel 12 to be placed 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 in some points. In some embodiments, the bonding material 32 forms an operable external chamber wall to laterally contain the sample 34 within the 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.

Another example of an acceptable chamber 10 is placed in a disposable container 36 as shown in Figures 6 and 7. The 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 light to pass through the chamber 10. At least a part of the first panel 12 and the second panel 16 are placed parallel, and within that part the interior surfaces 14, 18 are separated from each other by a height 20. This embodiment of chamber 10 is described in more detail in US Pat. No. 6,723,290. The analysis chambers shown in Figures 2 to 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 in individual contact with both inner surfaces of the camera panels, and / or one or more RBC aggregates are in contact with both surfaces interiors of the chamber panels, and one or more empty areas of RCB (for example, gaps) within the quiescent sample extend between the interior surfaces. Because the size of the RBCs within a blood sample is a function of the type of blood sample to be analyzed (for example, human, monkey, horse, goat, fish, bird, etc. .), the acceptable height of the camera will vary depending on the individual to be tested. A chamber height of approximately two to six micrometers (2-6 μm) is acceptable for individual RCBs for most animal species based on typical RBC sizes and the fact that such RBCs can be deformed to some degree (for example, partially compressed spheres discussed above). A hematocrit analysis of an animal species having RBCs substantially greater or less than human RBCs can be performed in a chamber that has respectively a greater or lesser chamber height, respectively. In addition, a hematocrit analysis using RBC aggregates may have a chamber height that is imposed by the height of the RBC aggregates.

In some applications, an isovolumetric sphere 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 acquire substantially spherical geometry . A specific example of a sphere forming agent is Zwittergent® 3-16 detergent, which is an amphoteric detergent produced by Caliobrichem, an entity of EMD Chemicals, Inc. From New Jersey, USA The amount of sphere forming agent added to the sample is an amount suitable for forming spheres in at least one number of RBC required to perform the hematocrit analysis of the present invention. The specific amount will depend on the particular agent and the circumstances of the test, which can be determined by a person skilled in the art without undue experimentation. RBCs in their natural state often have a biconcave disk shape 38 (see Figure 1) instead of spherical shape 40. As a result, in the absence of the effect of the isovolumic sphere forming agent, some percentage of RBCs shaped Disk will not be in contact with both camera panels. Increasing the number of RBCs that have a substantially spherical geometry will increase the number of RBCs in contact with both panels including some cells 42 that are contained by the camera panels, but would otherwise be spherical.

The analysis of the quiescently arranged sample within the chamber is performed using an analysis device that is operable to capture images of at least a portion of the sample and perform an image analysis. The image is produced in a way that allows to determine the optical density of the sample with a criterion

per unit. The expression "criteria per unit" or "image unit" means an incremental unit of which

You can dissect the sample image. A pixel, which is generally defined as the smallest element of an image that can be treated individually within a particular image capture system, is an example of an image unit, and an image unit can also include a small number of pixels in a collective unit. The method of the present invention, however, is not limited for use with any particular analysis device.

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 (e.g., about 400-670 nm; light of about 413 nm and about 540 nm is particularly effective in determining the optical density (OD) of the RBCs within a human blood sample) and typically includes the optics for manipulating 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 inside the chamber 10, directing the light through the quiescently arranged sample between the panels from the camera, and then captured the light using an image dissector. An example of an acceptable image dissector 48 is an image sensor of the type charge coupling device (CCD) that transforms an image of the light passing through the sample into an electronic data format. Image sensors of the complementary metal oxide semiconductor type ("CMOS") are another example of a sensor that can be used, and the present invention is not limited to any of these examples. The programmable analyzer 50 includes a central processing unit (CPU) and is connected to the sample illuminator 46 and the image dissector 48. The CPU is adapted (for example, programmed) to selectively perform the functions necessary to perform 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 its combinations. A person skilled in the art would be able to program the treatment units to perform the functionality described herein without excessive experimentation. The patent of

USA No. 6,866,823 entitled "Apparatus for Analyzing Biologic Fluids" and issued on August 15, 2005, describes

said analysis device 44.

The analysis device is adapted to determine an OD value associated with the light signal detected with a unit criterion for a part of the sample from which images have been captured. The detected light signal (ie, OD values) can be used by an edge determination algorithm to identify the positions and boundaries of the RBC. RBCs that are in contact with both inner surfaces of the chamber have an OD profile similar to that of a partially compressed sphere. The lateral edges of the cells that are not in contact with the surfaces will have an OD that (in relative terms) can be considered to be close to zero. The value of the determined OD: 1) increases when moving in one direction towards the center of the RBC (for example, as the light transmission path through the cell increases); 2) reaches a maximum value and remains substantially constant when the RBC is in contact with the upper and lower surfaces (that is, when the path of the light transmitted through the RBC is constant); and 3) decreases when moving in a direction that moves away from the center of the RBC (for example, as the path of light transmission through the cell decreases). This characterization of the OD profile of a RBC is particularly uniform for RBCs that have a spherical shape.

The analysis device is further adapted to determine a maximum average OD value for a group of RBC and / or RBC aggregates 23 in contact with both interior surfaces. The determination of what constitutes an acceptable size of RBC group and / or RBC aggregates in contact with the interior surfaces can be made

with a criterion by sample analysis, or it can be done periodically for a number "n" of sample analysis

the same type; for example, human blood samples. For example, a group of RBC identified as being in contact with both interior surfaces can be comparatively evaluated to determine the average maximum OD value and the statistical deviation of the OD within the group. It is desirable to determine the average maximum OD value because the OD of hemoglobin within cells can vary from one cell to another even within a particular sample. If the standard deviation is greater than a predetermined threshold, a new RBC group in contact with both panels can be selected, or the existing group can be extended, until the aforementioned analysis establishes an RBC group that has an average value of Maximum OD with an acceptable standard deviation. An average maximum OD value of the RBCs within a group that is approximately plus or minus one percent (1%) of the average maximum OD value of all the RBCs that are in contact with both surfaces within the sample would be , for example, within the acceptable standard deviation values. However, what constitutes an acceptable standard deviation value may vary depending on the application in the handling and the specific statistical analysis being used (for example, standard error, etc.). Current statistical data regarding the OD of the RBCs are available and can be used in determining acceptable statistical values ​​of OD. The determination of whether RBCs within a particular group have a maximum mean OD value that is within a clinically acceptable standard deviation can also be adapted since, as indicated above, it is well known that the population of RBC within an individual typically has small variations in the hemoglobin concentration and a continuous standard deviation of the results can be used to determine how many cells should be examined before obtaining an average value of acceptable accuracy; For example, for samples from an individual that has normal blood parameters, an acceptable group size may be as few as 100 RBC, while samples from an individual that has abnormal blood parameters may require analysis of 1000 or more RBC. The specific number of RBC and / or RBC aggregates in contact with both interior surfaces that is used to establish an acceptable average maximum OD value is not limited to any particular number or percentage of the RBCs within a sample, and may include all (for example thousands) of the RBCs in contact with both surfaces.

With a method for determining the hematocrit of a biological sample according to the present invention, the steps of said method are shown in the block diagram of Figure 9, a sample of substantially undiluted whole blood is placed in a chamber, as described. previously. An anticoagulant agent is mixed with the sample, and in some cases an isovolumetric sphere forming agent and / or an aggregating agent, either before introducing it into the chamber or when introducing it into the chamber. Reagents added in dry or semi-dry form, for example, by means of surface coating, are particularly easy to use. However, the present invention is not limited to reagents in dry form, and liquid reagents that do not significantly dilute the sample can be used, for example. The sample is quiescent in the chamber. In certain circumstances (for example, very rapid analyzes), it may not be necessary to add the anticoagulant agent, but it is preferable to do so in most cases to ensure that the sample is in an acceptable form for analysis.

Images of at least a portion of the sample that is quiescent in the chamber are obtained using the analysis device by transmitting light through the sample and detecting the transmitted light. The sample part from which the image is obtained includes a number of RBC and / or RBC aggregates that are in contact with the inner surface of each camera panel, and at least one empty sample area of ​​any RBC (zone RBC vacuum), which extends between the inner surfaces of the camera panels. Although it is not a requirement that images of the entire sample within the chamber be captured, it is preferable to do so since it typically provides a more complete analysis of said sample (and all its constituents), and an additional increase in accuracy, since that the distribution of RBC and deficiencies within a chamber is not typically homogeneous for a substantially undiluted whole blood sample.

A group of individual RBCs or aggregates of RBCs in contact with the inner surfaces is determined by the analyzer using the image of the sample part, and an average maximum OD value having an acceptable standard deviation from that group is determined . As indicated above, the group size may vary depending on the analysis, and may include iterations to determine the above-mentioned maximum OD average value that has an acceptable standard deviation. An arbitrary upper limit value of one hundred percent (100%) is assigned to determine the average maximum OD value of the individual RBCs and / or the RBC aggregates in contact with the interior surfaces.

Similarly, the analysis device is adapted to identify where one or more empty areas of RBC (for example, deficiencies) that extend between both interior surfaces are located within the chamber. The OD value of the empty RBC zone or zones is determined, or if more than one empty RBC zone is present and analyzed, the average OD value of the empty RBC zones can be determined. An arbitrary lower limit value of zero percent (0%) is assigned to the CD value of the RBC empty zone or zones.

The hematocrit of the sample is determined by assigning a relative value of the OD value of each unit of the sample part from which images are obtained based on the upper and lower limit values ​​(that is, depending on the regions in which RBCs extend completely through the height of the chamber and regions where there is no RBC). An average value of the relative values ​​of the percentages for each unit is determined. The relative mean value is a percentage of the RBC volume of the sample between 100% (ie, all RBCs) and 0% (no RBCs). The percentage is, by definition, equal to the hematocrit of the sample, that is, the filled volume of red blood cells in the sample.

An advantage of the method of the present invention is that it is not necessary to have all the RBCs within the sample in contact with each panel of the chamber. The method can be performed with only one of the RBCs and / or RBC aggregates in contact with both interior surfaces of the chamber. 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 hematocrit of the sample can be determined without knowing the total area or volume of said sample within the chamber. Therefore, it is not necessary to use a camera that has a precisely defined height, which makes the manufacturing of the cameras more economical.

Although this invention has been shown and described with respect to its detailed embodiments, those skilled in the art will understand that various changes in form and detail can be made without departing from the scope of the invention. For example, the invention was described above from the point of view of hematocrit determination for a substantially undiluted blood sample. Actually, one of the advantages of the present invention is its ability to analyze blood without using diluents. That is, in alternative embodiments, the present invention can be used in blood that has been diluted for several reasons as long as the sample dilution factor is known or determined.