APPARATUS FOR PERFORMING COUNTS WITHIN A BIOLOGIC FLUID SAMPLE

Abstract

A method and an apparatus for enumerating one or more specific elements within a biologic fluid sample are provided. An embodiment of the method includes the steps of: a) providing a chamber formed between a first planar member that is transparent and a second planar member, which members are separated from one another by a substantially uniform height; b) introducing the biologic fluid sample into the chamber, wherein the chamber height is sized such that the sample extends between the first and second members, and sized relative to the specific elements within the sample such that the specific elements non-uniformly distribute within the sample upon introduction into the chamber; c) examining substantially all of the sample within the chamber and enumerating all of at least one of the specific elements; d) determining the volume of sample contained within the chamber, and e) determining the number of the at least one of the specific elements per unit volume.

Claims

1. An apparatus for enumerating elements within a biological fluid sample, comprising: a chamber defined by a first planar member that is transparent, a second planar member, and a plurality of separator elements disposed between the first planar member and the second planar member, which separator elements are independent of the first planar member and the second planar member and each of which has a height, and wherein at least one of the first planar member and separator elements are deformable relative to the other by capillary force resultant from the biological fluid sample residing within the chamber in an amount that the chamber assumes a mean chamber height substantially equal to a mean separator height.

2. The apparatus of claim 1, further comprising one or more sidewalls extending between the first planar member and the second planar member.

3. The apparatus of claim 2, wherein the one or more sidewalls include a bonding material.

4. The apparatus of claim 3, wherein the sidewalls are formed into a shape that substantially or completely encloses the chamber.

5. The apparatus of claim 1, wherein the separator elements are spherical beads having a height equal to or less than twenty microns.

6. The apparatus of claim 5, wherein the separator elements are spherical beads having a height of about four microns.

7. The apparatus of claim 1, wherein the biological fluid sample is an anticoagulated human whole blood sample, and the at least one of the first planar member and separator elements are deformable relative to the other by capillary force resultant from the anticoagulated whole blood sample biological fluid sample residing within the chamber in the amount that the chamber assumes the mean chamber height substantially equal to the mean separator height.

8. An apparatus for enumerating elements within a biological fluid sample, comprising: a chamber defined by a first planar member that is transparent, a second planar member, and a plurality of separator elements disposed between the first planar member and the second planar member, which separator elements are independent of the first planar member and the second planar member and each of which has a height, and wherein the first planar member is deformable by capillary force resultant from the biological fluid sample residing within the chamber in an amount that the chamber assumes a mean chamber height substantially equal to a mean separator height.

9. The apparatus of claim 8, further comprising one or more sidewalls extending between the first planar member and the second planar member.

10. The apparatus of claim 9, wherein the one or more sidewalls include a bonding material.

11. The apparatus of claim 10, wherein the sidewalls are formed into a shape that substantially or completely encloses the chamber.

12. The apparatus of claim 8, wherein the separator elements are spherical beads having a height equal to or less than twenty microns.

13. The apparatus of claim 12, wherein the separator elements are spherical beads having a height of about four microns.

14. The apparatus of claim 1, wherein the biological fluid sample is an anticoagulated human whole blood sample, and the first planar member is deformable by capillary force resultant from the anticoagulated whole blood sample biological fluid sample residing within the chamber in the amount that the chamber assumes the mean chamber height substantially equal to the mean separator height.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The principles of the invention are further clarified by referring to the following figures.

[0017] FIG. 1 is a diagram of a chamber according to the teachings of this invention having two transparent surfaces separated by a known and relatively uniform space.

[0018] FIG. 2 is a cross section of the chamber diagram of FIG. 1 after a volume of blood has been introduced into the chamber.

[0019] FIG. 3 is a diagrammatic top planar view of a chamber showing a filled and an unfilled chamber.

[0020] FIG. 4 is an enlarged diagrammatic view of a central region of a chamber.

[0021] FIG. 5 is an enlarged diagrammatic view of a peripheral region of a chamber.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Referring to FIGS. 1-5, the present apparatus for analyzing biologic fluids includes one or more chambers 2 defined by a first planar member and a second planar member, separated from one another by a distance referred to hereinafter as the chamber height 16. At least one of the first planar member and the second planar member is sufficiently transparent so that a biologic fluid sample disposed within a chamber 2 may be imaged. To facilitate the description of the present invention, the planar members are referred to hereinafter as the top planar member 4 and the bottom planar member 3. The top planar member 4 hereinafter is also described as being transparent. In alternative embodiments, the bottom planar member 3 may be transparent rather than the top planar member 4, or in addition to the top planar member 4.

[0023] The planar members 3, 4 can be formed from a variety of materials, having different or identical properties. Patent Cooperation Treaty Patent Application Serial No. PCT/2005/011602, commonly owned with the present application and hereby incorporated by reference in its entirety, discloses examples of acceptable planar members 3, 4. As a further example, the top planar member 4 may be formed from a polyethylene terphthalate (PET) tape having a thickness and width of approximately 25 and one inch, respectively. The bottom planar member 3 can be similarly formed from PET tape of similar width, having a thickness of approximately 128. Present invention embodiments wherein the planar members 3, 4 are flexible, permit the chambers 2 to be wound on a reel.

[0024] Although sidewalls are not required for the practice of this invention, in some embodiments, the chambers 2 are further defined by one or more sidewalls 7. In preferred embodiments, the sidewalls 7 consist of bonding material that extends between the top planar member 4 and the bottom planar member 3. The sidewalls 7 may be positioned to create different chamber configurations. For example, in some embodiments bonding material may be applied so that one or more sidewalls 7 extend substantially across the width of the planar members 3, 4. In other embodiments, the sidewalls 7 may be formed in a shape that substantially or completely encloses the chamber 2. The embodiment shown in FIG. 3, for example, shows an elliptical shaped side wall 7 enclosure formed by bonding material. The sidewalls 7 may be made of material other than bonding material.

[0025] For sidewall 7 embodiments that use bonding material, the bonding material may consist 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 adequate to retain the sample within the chamber 2. In preferred embodiments, the bonding material is a material with adhesive properties that attaches the planar members 3, 4 to one another. Bonding materials that include a light-curing adhesive, of which numerous examples are readily available, are particularly useful.

[0026] In some embodiments, the present invention includes one or more separator elements 5 disposed within the chamber. Examples of acceptable separator elements 5 are disclosed in co-pending U.S. patent application Ser. Nos. 09/885,193 and 09/366,881, both of which are hereby incorporated by reference in their entirety, and PCT Patent Application No. PCT/2005/011602. An example of an acceptable separator element 5 is a spherical bead made of polystyrene, of known and precisely controlled diameter. In embodiments wherein the planar members 3, 4 are formed from substantially rigid material, there may be no need for the separator elements 5, depending upon the actual configuration of the chamber.

[0027] In some embodiments, the top planar member 4 includes one or more of an inlet port 8 and a vent aperture 10. The inlet port 8 provides access to the chamber for the biologic sample. The vent aperture 10 provides a passage through which air may escape as the biologic sample is introduced into the chamber 2. In embodiments where at least a portion of the chamber 2 is open (e.g., where the side walls of the chamber 2 do not form a complete enclosure), the inlet port 8 and vent aperture 10 may be omitted

[0028] To illustrate the utility of the present invention apparatus, the following examples of methods for using the apparatus are provided. The present invention method and apparatus are not, however, limited to these particular examples.

[0029] Referring to FIG. 2, a chamber 2 is shown after a sample 6 of undiluted anticoagulated whole blood has been added through fill hole 8. In some applications, it is not necessary that the sample 6 fill the entirety of the 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 preferable that the chamber 2 not be completely filled, leaving small unfilled areas 9. The unfilled areas 9 are advantageous in such chamber 2 embodiments, because the capillary force from the unfilled areas exerts a strong downward force on the planar members 3, 4 of the chamber 2, which force is helpful in keeping the height 16 of the chamber 2 uniform.

[0030] In a second embodiment, FIG. 3 illustrates a pair of chambers 2, 2 adjacent one another. The chamber 2 disposed 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 port 8 and a pair of vent apertures 10. A biologic fluid sample 6 (e.g., blood) has been introduced into the chamber 2 disposed on the right through the inlet port 8. The sample 6 has spread from the inlet port 8 to fill the majority of the chamber, leaving small air spaces 9 disposed adjacent the vent apertures 10. Because of the relative magnitudes of the chamber height 16 and the average thickness (e.g., diameter) of one or more specific elements (e.g., WBCs, RBCs) present within the sample, the distribution of elements within the sample typically becomes highly non-uniform. A highly non-uniform distribution contrasts strongly with prior art methods that rely upon a uniform distribution of elements to ensure accuracy.

[0031] An example of a non-uniform distribution of elements within a chamber 2 is illustrated in FIG. 4, by showing a diagrammatic representation of a microscopic field near the inlet port. In this representation, the plasma 11 is more prevalent than the RBCs 12. Because of their size, WBCs 13 are also concentrated in this area. Also seen in this figure are the separator particles 5 and platelets 14. In this example, the specific elements to be enumerated, for example, could be one or more of the WBCs 13 or RBCs 12. The elements to be enumerated could also be subsets of the identified elements; e.g., specific types of WBCs, or WBCs having surface epitopes which are selectively stained to be identifiable and separately enumerated, etc.

[0032] In contrast, a microscopic field is diagrammatically illustrated in FIG. 5, depicting a portion of the chamber 2 disposed near the chamber sidewall 7. In that field, masses of RBCs 12 are disposed adjacent the side wall 7 and make up the majority of the field.

[0033] It is clear from these examples that an accurate enumeration is not practically possible using prior art methods that only consider a fraction of the sample. The present invention method and apparatus, in contrast can provide an accurate enumeration in applications where the elements to be enumerated are not uniformly distributed. At the same time, specific information regarding certain of the specific elements can be obtained (e.g., WBC cell morphology). To obtain an accurate enumeration using the present method, the entirety of the sample is imaged using a digital camera and the image is subject to an analysis which detects and enumerates every one of the specifically targeted non-uniformly dispersed elements disposed within the chamber. Depending upon the area of the sample, this analysis can be performed an image frame at a time as the entire area of the sample is imaged, or a series of images can be stitched together to create a larger image which is analyzed at once. A suitable instrument and software for this 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 sample within the chamber. Once the count has been completed and the volume determined, the count per unit volume is calculated.

[0034] It can be appreciated that this invention can also perform most of the functions of a flow-cytometer by adding fluorescent or other markers to cell-specific ligands and examining the chamber to enumerate which cells have the ligand-marker bound to their surfaces.

[0035] Although this invention has been shown and described with respect to the detailed embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail thereof may be made without departing from the spirit and the scope of the invention.