HK1123873B - Apparatus and method for performing counts within a biologic fluid sample - Google Patents

Description

Apparatus and method for performing counts within a biological fluid sample

Applicants hereby claim priority from U.S. provisional patent application No. 60/728,058, filed on 19/2005 and U.S. patent application No. 11/257,757, filed on 25/10/2005, the disclosures of both of which are incorporated herein by reference.

Technical Field

The present invention relates generally to chambers for analyzing biological fluids, and more particularly to chambers that allow counting of particulate matter within biological fluids. Background

Complete Blood Count (CBC) is the most frequently performed test set for whole blood and includes many individual analyses, such as white blood cell count (WBC), red blood cell count (RBC), and platelet count, among others. The methods used vary in the integrity of the analyte collection, the complexity and cost of the apparatus, and the cost of a single test. The least complex method, such as QBC described in U.S. Pat. No. 4,156,570The method has the cheapest capital cost and is simple to perform, but generally has a higher single test cost. QBCThe method is best suited for point-of-care situations where operator training is minimal and where little testing is performed daily. At the other end of the range, large volume blood analyzers used by hospitals or benchmark laboratories can have a capital cost twenty times greater, but have a relatively low single test cost when used on a large basis, which makes them more cost effective in these settings.

One of the simplest and oldest methods of counting cells involves the use of a hemocytometer. In a hemocytometer, a precise dilution of the blood is performed. An approximate amount of the dilution is then placed into a counting chamber having a height sufficient to maintain the diluted sample at the same uniformity as the cells found in the diluted sample as it flows into the chamber. That is, the chamber must not selectively concentrate or dilute any cells or other components as a result of the sample flowing into and through the chamber. This is because only a representative fraction of the cells in a known region of the chamber are counted. If the distribution of cells is skewed, such a count will falsely reflect the count of the entire sample.

Such as Abbot Cell-DynOr Bayer AdviaAre based on certain variables of Flow Cytometry (FC), where a precise amount of blood is precisely diluted and mixed with reagents in multiple steps. The fluidic valves route the diluted sample into the multiple test zones. As with a hemocytometer, the distribution of cells within the diluent must remain relatively uniform so that the count of a representative portion of the diluted sample can represent the count in the original sample. The complexity of the instruments required for this approach is such that the reliability of these instruments is relatively low. In practice, with these larger systems, it is often the case thatThere is a need for weekly, or more often preventative, maintenance or repair that requires the skill of a specially trained laboratory technician or service technician, all of which substantially increase the cost of the operation. Another stealth cost of operation is the washing, cleaning and calibration procedures required for the system to perform properly.

At QBCIn the system, an approximate amount of blood is placed in a capillary tube, separated and examined. This method, while not requiring an exact sample, does not produce a true cell count and does not give an accurate estimate of the number of cells when there are very few.

Intermediate systems are described in U.S. patent nos. 6,723,290, 6,866,823, 6,869,570, and 6,929,953, in which blood is placed into a single-use disposable for analysis. These patents describe reliable, low cost, and easy to use methods and instruments that can provide the same range of analytical data as the flow cytometer systems described above. In this system, a similar amount of undiluted sample is placed into a disposable whose properties allow the distribution of cells within the sample to remain substantially uniform. The cells in a given imaged region are counted, the volume of the region is determined, and the cell count per volume is then calculated. In this system, just a portion of the sample added to the chamber needs to be counted, as with a hemocytometer, because the distribution of cells is substantially uniform. This method requires a single use disposable item, which is advantageous for small tests, but is not particularly suited for large test use.

It would be advantageous to have a system in which the components in an undiluted whole blood sample can be counted in a chamber that is sufficiently thin to enable cell count and cell morphology to be obtained from the sample, and in which the effects of uneven distribution can be mitigated. Such an analysis system would reduce or eliminate fluid handling and accurate measurement or dilution of the sample, resulting in a much simpler and less expensive method for such analysis. Disclosure of Invention

A method and apparatus for counting components within a fluid medium is provided which is simple, accurate and relatively low cost. The method and apparatus are particularly well suited for performing blood cell counts (i.e., WBCs (white blood count), RBCs (red blood cell count), etc.) within anticoagulated whole blood samples. In this method, an approximate amount of sample is placed into a chamber of very small height, typically less than 20 microns in height and preferably about four microns for counting blood. Upon entering the chamber, the distribution of certain types of components within the sample changes significantly. The change in distribution for certain components within the sample may be attributed to the size of the components within the sample relative to the height of the chamber. If a blood sample is introduced into the chamber, for example, red blood cells within the sample will concentrate at the periphery of the chamber and white blood cells within the sample will concentrate near the sample inlet of the chamber. Red blood cells are typically dispersed within the sample at a distance from the inlet that is farther than the white blood cells, which are larger and relatively rigid compared to the red blood cells, because red blood cells are smaller and typically have highly variable membranes and can fit into compact spaces. Although the relatively thin height of the chamber allows for easy visualization of the components, the distribution of the components within the sample is such that there are generally no localized areas of the sample that represent the entire sample. Thus, there are no localized regions representing the entire sample that can be counted to give an accurate count of the entire sample. In this method, in contrast to all other counting methods we know, the whole sample added to the chamber is examined and all the unevenly distributed cells within the specific type of sample to be examined are counted. Once the total number of unevenly distributed cell types to be examined within the sample is known, the count per unit volume of that type of unevenly distributed cell of the sample can be calculated by dividing the number of counted cells by the volume contained within the chamber. The phenomenon of non-uniformity of cell distribution within small chambers has been well known since cell counting and is always avoided as highly undesirable because it is nearly impossible to manually count all components within a chamber to get an accurate total count. In addition, the minute sample size used by such chambers precludes accurate initial measurement of the sample volume within such chambers, or subsequent calculation of the sample volume of an irregularly spread sample. However, with the recent advent of accurate and fast digital imaging systems that allow these counts to be made and the entire area of the chamber sample to be calculated, thin film chambers can now be advantageously used for simple and accurate methods of obtaining blood cell or other counts.

In certain embodiments, the present methods for enumerating one or more specific elements within a biological fluid sample comprise the steps of: a) providing a chamber formed between a first planar member and a second planar member, the first planar member being transparent, the first and second planar members being spaced apart from one another at a substantially uniform height; b) introducing a biological fluid sample into a chamber, wherein the chamber has a height such that the sample extends between the first and second members to at least a portion of the chamber, and wherein the chamber height is sized relative to one or more specific components within the sample such that the one or more specific components are non-uniformly distributed within the sample when introduced into the chamber; c) examining substantially all of the sample within the chamber and counting all of the at least one specific component; d) determining a volume of a sample contained within the chamber; and e) determining the amount of at least one specific component per unit volume.

In contrast to all the prior art we know, the present invention examines the entire biological fluid sample (e.g. undiluted whole blood) present in a thin film confined in a chamber defined by two relatively flat substrates, wherein the total volume of the sample added to the chamber can be determined. In contrast to all other methods in which only a portion of the sample is examined, all of the at least one specific component within the sample is counted. The term "all of at least one particular ingredient" means all of the particular types of the particular ingredients. For example, if one or more specific components include components A, B, and C, "at least one specific component" refers to component A, then counting "all of the at least one specific component" means counting all of the components A within the sample.

Any chamber formed by at least one transparent wall may be used. The chamber can be created by techniques such as micro-machining, etching, substrate deposition. The techniques described in co-pending U.S. patent application Ser. Nos. 09/885,193 and 09/366,881, which use layers of separator elements to achieve a uniform thickness of the chamber, are examples of acceptable techniques.

The method requires that the volume of sample introduced into the chamber is known or determinable with substantial accuracy. The term "substantially accurate" is defined as being suitable for the volumetric accuracy of the upcoming test. The volumetric determination of the sample can be performed using a number of different techniques, including but not limited to: 1) the sample volume is calculated when first deposited by interferometric imaging using optical techniques available from sources such as Zygo corporation of Middlefield, CT; or 2) calculating the sample volume by measuring the area of the sample film after the thin film is formed (by the sample expanding in the chamber to form a film) and multiplying the area by the average height of the sample film; or 3) utilize or manufacture a chamber (i.e., thickness and length) with a precisely known volume, wherein the added blood sample will flow into the chamber until the chamber cannot contain more blood (i.e., since the total amount of blood contained is known in advance, the total number of components counted can be divided by the known chamber volume to give a count/volume).

For purposes of the present invention, the reading or cell counting instrument may be similar in function to that shown in co-pending U.S. patent application Ser. Nos. 09/981,581 and 10/023,405.

These and other objects, features and advantages of the present invention will become apparent from the detailed description of the invention provided below and as illustrated in the accompanying drawings. Drawings

The principles of the present invention are further elucidated by reference to the following figures.

FIG. 1 is a schematic view of a chamber having two transparent surfaces separated by a known and relatively uniform space according to the teachings of the present invention.

Fig. 2 is a cross-section of the schematic chamber of fig. 1 after a volume of blood has been introduced into the chamber.

Fig. 3 is a diagrammatic top plan view showing the chamber so as to show the filled and unfilled chambers.

Fig. 4 is an enlarged diagrammatic view of the central region of the chamber.

Fig. 5 is an enlarged diagrammatic view of the surrounding area of the chamber. Detailed Description

With reference to fig. 1 to 5, the present apparatus for analyzing biological fluids comprises one or more chambers 2, said chambers 2 being defined by a first and a second planar member separated from each other by a distance hereinafter referred to as chamber height 16. At least one of the first planar member and the second planar member is sufficiently transparent so that a biological fluid sample disposed within the chamber 2 can be imaged. For the purposes of describing the present invention, the planar members are hereinafter referred to as "top" planar member 4 and "bottom" planar member 3. The top planar member 4 is also described below as transparent. In alternative embodiments, the bottom planar member 3 may be transparent instead of, or in addition to, the top planar member 4.

The planar members 3,4 can be formed of various materials having different or identical characteristics. Patent co-pending patent application No. PCT/2005/011602, which is a common owner of the present application and is hereby incorporated by reference in its entirety, discloses examples of acceptable planar members 3, 4. As another example, the top planar member 4 may be formed from polyethylene terephthalate (PET) tape having a thickness and width of approximately 25 μ and one inch, respectively. The bottom planar member 3 can similarly be formed from a PET strip of similar width and having a thickness of approximately 128 μ. The embodiment of the invention in which the planar members 3,4 are flexible allows the chamber 2 to be wound on a reel.

Although no side walls are required to practice the invention, in certain embodiments, the chamber 2 is further defined by one or more side walls 7. In a preferred embodiment, the side walls 7 are constructed of an adhesive material (bonding material) extending between the top planar member 4 and the bottom planar member 3. The side walls 7 may be positioned to create different chamber configurations. For example, in certain embodiments, the bonding material may be applied such that the one or more side walls 7 extend substantially across the width of the planar members 3, 4. In other embodiments, the sidewall 7 may be formed in a shape that substantially or completely surrounds the chamber 2. The embodiment shown for example in fig. 3 shows an enclosure of an oval side wall 7 formed by an adhesive material. The side wall 7 may be made of a material other than the adhesive material.

For embodiments using sidewalls 7 of adhesive material, the adhesive material may be comprised of any of a variety of different materials that adhere to the planar members 3,4 or interact with the planar members 3,4 sufficiently to create a seal suitable for holding a sample within the chamber 2. In a preferred embodiment, the bonding material is a material having adhesive characteristics that attach the planar members 3,4 to each other. Bonding materials including light-curing adhesives, many examples of which are readily available, are particularly useful.

In certain embodiments, the invention includes one or more isolator elements 5 disposed within the chamber. Examples of acceptable isolator elements 5 are disclosed in co-pending patent application Ser. Nos. 09/885,193 and 09/366,881, and PCT patent application PCT/2005/011602, which are hereby incorporated by reference in their entirety. An example of an acceptable isolator element 5 is a spherical bead made of polystyrene with a well-known and precisely controlled diameter. In embodiments where the planar members 3,4 are formed of a substantially rigid material, the spacer element 5 may not be required, depending on the actual configuration of the chamber.

In certain embodiments, the top planar member 4 includes one or more inlets 8 and vent apertures 10. The inlet 8 provides access to the chamber for the biological sample. The vent aperture 10 provides a passage through which air can escape when a biological sample is introduced into the chamber 2. In embodiments where at least a portion of the chamber 2 is open (e.g., the sidewalls of the chamber 2 do not form a complete enclosure), the inlet 8 and vent aperture 10 may be omitted.

To illustrate the utility of the apparatus of the present invention, the following examples of methods for using the apparatus are provided. However, the method and apparatus of the present invention are not limited to these specific examples.

Referring to fig. 2, a chamber 2 is shown with an undiluted anticoagulated whole blood sample 6 having been added through a fill hole 8. In some applications, it is not necessary for the sample 6 to fill the entire chamber 2. In those embodiments where one or both of the top planar member 4 and the bottom planar member 3 are relatively flexible, it is preferred that the chamber 2 not be completely filled, but that a small unfilled area 9 be left. The underfill region 9 is advantageous in such embodiments of the chamber 2 because capillary forces from the underfill region exert a strong downward force on the planar members 3,4 of the chamber 2 that is beneficial for maintaining the uniformity of the height 16 of the chamber 2.

In a second embodiment, fig. 3 shows a pair of chambers 2', 2 "adjacent to each other. The chamber 2' arranged on the left shows an unfilled chamber defined in part by a sidewall enclosure 7. The top planar member 4 of the chamber 2' includes an inlet 8 and a pair of vent apertures 10. A sample 6 of biological fluid, for example blood, has been introduced into the chamber 2 "arranged on the right through the inlet 8. The sample 6 spreads from the inlet 8 to fill most of the chamber, leaving a small air space 9 located adjacent the vent aperture 10. Because of the relative values of the chamber height 16 and the average "thickness" (e.g., diameter) of one or more particular components (e.g., white blood cells, red blood cells) within the sample, the distribution of components within the sample typically becomes highly non-uniform. Highly non-uniform distribution is in strong contrast to prior art methods that rely on uniform distribution of components to ensure accuracy.

By means of a diagram showing a microscopic field of view in the vicinity of the inlet, a schematic example of the inhomogeneous distribution of the components within the chamber 2 is shown in fig. 4. In this example, plasma 11 is more prevalent than red blood cells 12. Because of the size of the white blood cells, the white blood cells 13 are also concentrated in this area. Also visible in this figure are the spacer particles 5 and the platelets 14. In this example, the particular component to be counted can be one or more of white blood cells 13 or red blood cells 12, for example. The components to be counted can also be a subset of the identified components; for example, a specific type of white blood cell, or a white blood cell having a surface epitope (surface epitope) that is selectively stained so as to be identifiable and individually counted, or the like.

In contrast, fig. 5 schematically shows a microscopic view to depict the portion of the chamber 2 disposed near the chamber sidewall 7. In this field, a large number of red blood cells 12 are adjacent the sidewall 7 and make up the majority of the field.

It is clear from these examples that with the prior art method, which only considers a small part of the sample, it is practically impossible to have an accurate count. The method and apparatus of the present invention can provide accurate counts in comparison to applications where the components to be counted are not uniformly distributed. At the same time, specific information (e.g., cell morphology of white blood cells) can be obtained regarding certain specific components. In order to obtain accurate counts using the present method, the entire sample is imaged using a digital camera, and the image is analyzed for the detection and counting of each specific target heterogeneously dispersed component disposed within the chamber. Depending on the area of the sample, the analysis can be performed on image frames at a time when the entire area of the sample is imaged, or a series of images can be "stitched" together to form a larger image that is analyzed simultaneously. Suitable instruments and software for imaging are described in U.S. Pat. nos. 6,866,823, 6,869,570, and 6,929,953. The same image analysis then determines the actual volume of the sample within the chamber. Once the count is complete and the volume is determined, the count per unit volume is calculated.

It can be appreciated that the present invention can also perform most of the functions of a flow cytometer by adding fluorescent or other labels to cell specific ligands and examining the chamber to count cells that have ligand labels tethered to the cell surface.

While the invention has been shown and described with respect to detailed embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims (25)

1. A method for enumerating one or more specific elements within a biological fluid sample, comprising the steps of:

providing a chamber formed between a first planar member and a second planar member, wherein the first planar member is transparent, the first and second planar members being separated from each other by a height;

introducing the biological fluid sample into the chamber, wherein the chamber height is dimensioned such that the sample extends between the first and second members to at least a portion of the chamber, wherein the chamber height is dimensioned relative to the specific component such that: the specific component is non-uniformly distributed within the sample when introduced into the chamber;

examining substantially all of the sample within the chamber and counting all of the at least one specific component;

determining a volume of a sample contained within the chamber; and

determining the amount of said at least one specific component per unit volume.

2. The method of claim 1, wherein the biological fluid sample is anticoagulated whole blood.

3. The method of claim 2, wherein the steps of examining, determining a volume, and determining a number utilize digital image analysis.

4. The method of claim 3, wherein the at least one specific component being counted comprises white blood cells.

5. The method of claim 4, wherein the at least one specific component is a subset of leukocytes having surface epitopes that are selectively stained identifiable and individually enumerated.

6. The method of claim 1, wherein the steps of examining, determining a volume, and determining a number utilize digital image analysis.

7. The method of claim 1, wherein all specific components that are not uniformly distributed within the sample are counted.

8. A method for enumerating one or more specific elements within a biological fluid sample, comprising the steps of:

providing a chamber formed between a first planar member and a second planar member, the first planar member being transparent, the first and second planar members being separated from one another by a height, the chamber having a known volume;

introducing the biological fluid sample into a chamber, wherein the chamber has a height such that the sample extends between the first and second members to substantially the entire extent of the chamber, wherein the chamber height is sized relative to a particular component such that the particular component is non-uniformly distributed within the sample when introduced into the chamber;

examining substantially all of said sample within the chamber and counting all of at least one of said specific components; and

determining the amount of said at least one specific component per unit volume.

9. The method of claim 8, wherein the biological fluid sample is anticoagulated whole blood.

10. The method of claim 9, wherein the steps of examining, determining a volume, and determining a number utilize digital image analysis.

11. The method of claim 10, wherein the specific components being counted comprise white blood cells.

12. The method of claim 11, wherein the specific component is a subset of leukocytes having surface epitopes that are selectively stained identifiable and individually enumerated.

13. The method of claim 8, wherein the steps of examining, determining a volume, and determining a number utilize digital image analysis.

14. An apparatus for enumerating one or more specific elements within a biological fluid sample, comprising:

a transparent first planar member; and

a second planar member;

wherein the first and second planar members are separated from each other by a height, and the height is set with respect to the specific component within the sample such that the specific component is unevenly distributed within the sample when introduced into the chamber.

15. The apparatus of claim 14, further comprising one or more sidewalls extending between the first and second planar members.

16. The apparatus of claim 15, further comprising an inlet disposed in the first planar member.

17. The apparatus of claim 16, further comprising one or more vent apertures.

18. The apparatus of claim 15, wherein the one or more sidewalls comprise an adhesive material.

19. The apparatus of claim 18, wherein the sidewall is formed in a shape that substantially or completely surrounds the chamber region.

20. The apparatus of claim 19, further comprising an inlet disposed in the first planar member.

21. The apparatus of claim 20, further comprising one or more vent apertures.

22. The apparatus of claim 15, wherein the one or more sidewalls consist essentially of a bonding material.

23. The apparatus of claim 22, wherein the sidewall is formed in a shape that substantially or completely surrounds the chamber region.

24. The apparatus of claim 23, further comprising an inlet disposed in the first planar member.

25. The apparatus of claim 24, further comprising one or more vent apertures.