Abstract:
A method for analyzing a biologic fluid sample includes the steps of: a) providing a spatially mapped chamber; b) providing a predetermined repeatable non-uniform spatial distribution of one or more constituents within the sample, which distribution indicates the presence or absence of a statistically significant number of constituents within the sample in each chamber sub-region; c) selecting one or more image techniques for each sub-region based on the presence or absence of the statistically significant number of one or more constituents in that sub-region as indicated by the distribution; d) creating image data representative of the biologic fluid sample in each sub-region, using the one or more image techniques selected for that sub-region; and e) analyzing the sample.

Description:
The present application claims priority to PCT Patent Appln. No. PCT/US2013/073636 filed Dec. 6, 2013, which is entitled to the benefit of and incorporates by reference essential subject matter disclosed in the U.S. Patent Appln. No. 61/734,179, filed Dec. 6, 2012 and U.S. Patent Appln. No. 61/896,432 filed Oct. 28, 2013. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Technical Field 
     The present invention generally relates to methods for imaging a biologic fluid sample, and more specifically relates to methods and apparatuses for imaging a biologic fluid sample at more than one resolution and in some instances less than the entire sample. 
     2. Background Information 
     Historically, biologic fluid samples such as whole blood, urine, cerebrospinal fluid, body cavity fluids, etc., have had their particulate or cellular contents evaluated by smearing a small undiluted amount of the fluid on a slide and evaluating that smear under a manually operated microscope. Reasonable results are attainable using these techniques, but they rely heavily upon the technician&#39;s experience and technique. These techniques are also labor-intensive and thus not practically feasible for commercial laboratory applications. 
     Automated apparatuses for analyzing biologic fluid samples are known, including some that are adapted to image a sample of biologic fluid quiescently residing within a chamber. Automated analysis devices can produce results that are as accurate as manual examination methods in a substantially reduced period of time. Nonetheless, the speed at which automated devices operate can be significantly limited by high resolution imaging. High resolution imaging produces substantial volumes of electronic data that must be processed by the apparatus. It would be desirable to provide an automated device and methodology that reduced the time required to consistently provide accurate results. 
     SUMMARY OF THE DISCLOSURE 
     According to an aspect of the present invention, a method for analyzing a biologic fluid sample is provided. The method includes the steps of: a) providing a chamber for holding the biologic fluid sample, which chamber is spatially mapped to divide the chamber into a plurality of sub-regions; b) providing a predetermined repeatable non-uniform spatial distribution of one or more constituents within the sample when the sample is disposed within the chamber, wherein the distribution indicates the presence or absence of a statistically significant number of at least one of the constituents within the sample in each sub-region of the chamber; c) disposing the biologic fluid sample within the chamber; d) selecting one or more image techniques for each sub-region based on the presence or absence of the statistically significant number of one or more constituents in that sub-region as indicated by the distribution; e) creating image data representative of the biologic fluid sample in each sub-region, using the one or more image techniques selected for that sub-region; and f) analyzing the sample, using the image data representative of the biologic fluid sample in each sub-region. 
     According to another aspect of the present invention an apparatus for imaging a biologic fluid sample is provided. The apparatus includes a chamber, a sample illuminator, at least one image dissector, and a processor. The chamber is operable to hold the biologic fluid sample. The chamber is spatially mapped to divide the chamber into a plurality of sub-regions. The image dissector is operable to produce image signals representative of the sample residing within the chamber. The processor is adapted to include a predetermined repeatable non-uniform spatial distribution of one or more constituents within the sample disposed within the chamber. The distribution indicates the presence or absence of a statistically significant number of at least one of the constituents within the sample in each sub-region of the chamber. The processor is further adapted to select one or more image techniques for each chamber sub-region based on the presence or absence of the statistically significant number of one or more constituents in that sub-region as indicated by the distribution. The processor is further adapted to communicate with the sample illuminator and the image dissector to create image data representative of the biologic fluid sample in select chamber sub-regions, using the one or more image techniques selected for each of the select chamber sub-regions. 
     The present method and advantages associated therewith will become apparent in light of the detailed description of the invention provided below, and as illustrated in the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of a biological fluid sample analysis cartridge. 
         FIG. 2  is an exploded, perspective view of the biological fluid sample analysis cartridge shown in  FIG. 1 . 
         FIG. 3  is a planar view of a tray holding an analysis chamber. 
         FIG. 4  is a sectional view of an analysis chamber. 
         FIG. 5  is a diagrammatic view of an analysis device. 
         FIG. 6  is a diagrammatic illustration of a mapped analysis chamber. 
         FIG. 7  is a diagrammatic illustration of a mapped analysis chamber, including a repeatable, predetermined distribution of sample constituents disposed within the sample chamber. 
         FIG. 8  is a diagrammatic illustration of a mapped analysis chamber, illustrating an example of the number of tile images captured in mapped chamber. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Referring to  FIGS. 1 and 5 , the present invention includes a method and an apparatus for an automated analysis of a biological fluid sample (e.g., whole blood) by an analysis device  12 . The sample deposited in or disposed on a chamber  10  is imaged, and the image of the sample is analyzed using the analysis device  12 . 
     An example of a chamber  10  that can be used with the present invention is shown in  FIGS. 1-4 . The chamber  10  is formed by a first planar member  14 , a second planar member  16 , and typically has at least three separators  18  disposed between the planar members  14 , 16 . At least one of the planar members  14 , 16  is transparent. The height  20  of the chamber  10  is typically such that sample residing within the chamber  10  will travel laterally within the chamber  10  via capillary forces.  FIG. 4  shows a cross-section of the chamber  10 , including the height  18  of the chamber  10  (e.g., Z-axis).  FIG. 3  shows a top planar view of the chamber  10 , illustrating the area of the chamber  10  (e.g., the X-Y plane). The lateral boundaries of the chamber  10  may be defined, for example, by glue lines  22  extending between the interior surfaces  24 , 26  of the planar members  14 , 16 , or by lines of hydroscopic material disposed on a planar member surface that inhibit lateral travel there across. Sample may be introduced into the sample by engaging an inlet  21  formed between the planar members  14 ,  16  along an edge of the chamber (i.e., the “filling edge  23 ”) with a bolus of sample. Upon contact with the filling edge  23 , the sample is drawn into the chamber inlet by capillary force. 
     The present invention is not limited to use with any particular chamber embodiment. Examples of acceptable chambers are described in U.S. Pat. No. 7,850,916, and U.S. patent application Ser. Nos. 12/971,860; 13/341,618; and 13/594,439, each of which are incorporated herein by reference in its entirety. For purposes of this disclosure, the invention will be described as using the analysis chamber described in U.S. patent application Ser. No. 13/594,439. The analysis chamber  10  disclosed in the &#39;439 Application is mounted on a tray  28  that is removable from a cartridge  30 .  FIG. 1  shows the cartridge  30  in assembled form.  FIG. 2  shows an exploded view of the cartridge  30 , including the analysis chamber  10  and the tray  28 .  FIG. 3  is a top view of the analysis chamber  10  mounted on the tray  28 , depicting a sample residing within the chamber  10 .  FIG. 4  is a diagrammatic cross-section of the chamber  10 . The analysis chamber  10  is typically sized to hold about 0.2 to 1.0 μl of sample, but the chamber  10  is not limited to any particular volume capacity, and the capacity can vary to suit the analysis application. 
     As indicated above, however, the present invention is not limited to use with the aforesaid chamber. An example of another “chamber” type of structure is a slide on which a sample may be smeared. To facilitate the description hereinafter, all such structures operable to hold a sample, and/or on which a sample may be deposited will be referred to hereinafter generically as a “chamber” and is not intended to be limited to the physical characteristics of any such structure unless specifically indicated. 
     The chamber  10  is operable to quiescently hold a liquid sample. The term “quiescent” is used to describe that the sample is deposited within the chamber  10  for analysis, and is not purposefully moved during the analysis. To the extent that motion is present within the blood sample, it will predominantly be due to Brownian motion of formed constituents within the blood sample, which motion is not disabling of the use of this invention. A sample smear may be fixed onto a slide, with no appreciable movement of sample thereafter. 
     Referring to  FIG. 5 , an automated analysis device  12  is shown that controls, processes, images, and analyzes the sample disposed within the cartridge  10 . U.S. Pat. No. 6,866,823 and U.S. patent application Ser. Nos. 13/077,476 and 13/204,415 (each of which is hereby incorporated by reference in its entirety) disclose examples of analysis devices  10  that have optics and a processor for controlling, processing, and analyzing images of the sample, which devices can be modified according to the present invention as will be described below. Except to the extent the analysis device is operable to perform the present methodology (ies), the present invention is not limited to any particular device. The analysis device  12  described below illustrates an example of a device that can be used. 
     The analysis device  12  includes optics including at least one objective lens  32 , a cartridge positioner  34 , a sample illuminator(s)  36 , an image dissector  38 , and a programmable analyzer  40 . The positioner  34  is adapted to selectively change the relative positions of the objective lens  32  and the analysis chamber  10 . One or both of the optics (e.g., the objective lens) and the analysis chamber  10  are moveable relative to the other along all relevant axes (e.g., X, Y, and Z). Relative movement of the chamber  10  in X-Y plane permits the optics to capture all fields of the sample residing within the chamber  10 . Relative movement of the chamber  10  along the Z-axis permits change in the focal position of the optics relative to the sample height. The optics may include hardware that enables the analysis device  12  to capture one or more low resolution images of the sample residing within the chamber  10 , as well as one or more high resolution images of the sample within the chamber  10 . Acceptable optical hardware capable of taking both low and high resolution images of the sample include embodiments that have a plurality of objective lens (e.g., a high resolution objective lens and a low resolution objective lens) and embodiments wherein a single objective lens is used with one or more lenses that can be selectively moved into the optical path and are operable to change the resolution of the device. The present analysis device  12  is not limited to this exemplary optical hardware, however. 
     The sample illuminator  36  illuminates the sample using light at predetermined wavelengths. For example, the sample illuminator  36  can include an epi-fluorescence light source and a transmission light source. The transmission light source is operable to produce light at wavelengths associated with one or more of red, green, and blue light. The red light is typically produced in the range of about 600-700 nm, with red light at about 660 nm preferred. The green light is typically produced in the range of about 515-570 nm, with green light at about 540 nm preferred. The blue light is typically in the range of about 405-425 nm, with blue light at about 413 nm preferred. Light transmitted through the sample, or fluoresced from the sample, is captured using the image dissector, and a signal representative of the captured light is sent to the programmable analyzer, where it is processed into an image. The image is produced in a manner that permits the light transmittance or fluorescence intensity captured within the image to be determined on a per unit basis; e.g., “per unit basis” being an incremental unit of which the image of the sample can be dissected, such as a pixel. 
     An example of an acceptable image dissector  38  is a charge couple device (CCD) type image sensor that converts light passing through (or from) the sample into an electronic data format image. Complementary metal oxide semiconductor (“CMOS”) type image sensors are another example of an image sensor that can be used. The signals from the image dissector  38  provide information for each pixel of the image, which information includes, or can be derived to include, intensity, wavelength, and optical density. Intensity values are assigned an arbitrary scale of, for example, 0 units to 4095 units (“IVUs”). Optical density (“OD”) is a measure of the amount of light absorbed relative to the amount of light transmitted through a medium; e.g., the higher the “OD” value, the greater the amount of light absorbed during transmission. OD can be quantitatively described in optical density units (“ODU”) or fractions thereof; e.g., a MilliODU is a 1/1000 th  of an ODU. One “ODU” decreases light intensity by 90%. “ODU” or “MilliODU” as a quantitative value can be used for images acquired or derived by transmission light. 
     The programmable analyzer  40  includes a central processing unit (CPU) and is in communication with the cartridge positioner  34 , sample illuminator  36 , and image dissector  38 . The programmable analyzer  40  is adapted (e.g., programmed) to send and receive signals from one or more of the cartridge positioner  34 , the sample illuminator  36 , and an image dissector  38 . For example, the analyzer  40  is adapted to: 1) send and receive signals from the cartridge positioner  34  to position the cartridge  30  and chamber  10  relative to one or more of the optics, illuminator, and image dissector; 2) send signals to the sample illuminator  36  to produce light at defined wavelengths (or alternatively at multiple wavelengths); and 3) send and receive signals from the image dissector  38  to capture light for defined periods of time. It should be noted that the functionality of the programmable analyzer may be implemented using hardware, software, firmware, or a combination thereof. A person skilled in the art would be able to program the processing unit to perform the functionality described herein without undue experimentation. 
     Now referring to  FIGS. 6-8 , an automated image analysis of biological fluid sample may include using a variety of imaging techniques, each designed to gather image data that permits identification and analysis of specific constituents within the sample. Using a whole blood sample as an example, an automated analysis device will preferably be operable to produce information relating to each of the constituents within the sample; e.g., RBC indices, WBC count, WBC differential, platelet enumeration, reticulocyte enumeration, etc. To acquire the desired image information, the analysis device may image the sample using several different imaging techniques; e.g., imaging the sample at multiple different wavelengths of light (e.g., using an epi-fluorescence light source and/or a transmission light source at different wavelengths); imaging the sample at different resolutions; imaging at different focal positions, etc. 
     The following patents and patent applications describe analysis devices operable to use different imaging techniques for acquiring data that permits specific identification and analysis of constituents within the sample: U.S. patent application Ser. No. 13/204,415, entitled “Method and Apparatus for Automated Whole Blood Sample Analyses from Microscopy Images” (which is hereby incorporated by reference in its entirety) discloses methods for performing a WBC differential on a whole blood sample that include imaging a blood sample at a variety of different wavelengths, using an epi-fluorescence light source and a transmission light source; U.S. Pat. No. 8,472,693, entitled “Method and Apparatus for Determining at Least One Hemoglobin Related Parameter of a Whole Blood Sample” (which is hereby incorporated by reference in its entirety) discloses methods for determining RBC indices including RBC cell volume (CV), mean cell volume (MCV), cell hemoglobin concentration (CHC), mean cell hemoglobin concentration (MCHC), and mean cell hemoglobin content (MCH), as well as their population statistics, using transmission light sources; U.S. patent application Ser. No. 13/730,095, entitled “Method and Apparatus for Automated Platelet Identification Within a Whole Blood Sample From Microscopy Images” (which is hereby incorporated by reference in its entirety) discloses methods for identifying and enumerating platelets within a sample that utilizes epi-fluorescent light sources; and U.S. patent application Ser. No. 13/729,887, entitled “Method for Rapid Imaging of Biologic Fluid Samples” (which is hereby incorporated by reference in its entirety) discloses methods for analyzing a sample at high and low image resolutions, which resolutions may facilitate data acquisition. 
     For an automated analysis device  12 , it is desirable to produce the desired information in a minimum amount of time. It is also desirable to produce the desired information using a minimal amount of image data, thereby reducing the image data handling and storage requirements of the device. 
     For at least these reasons, aspects of the present invention coordinate the performance of those imaging techniques used to identify and/or analyze specific constituents within the sample with the position of the constituents within the sample. In other words, imaging techniques used to identify and/or analyze a constituent within a sample are implemented only in areas of the sample where that specific constituent is likely to be present (e.g., where a statistically significant population of the constituent is likely to be present), and not in areas where that specific constituent is not likely to be present. This selective implementation of imaging techniques can be utilized in instances where multiple types of analyses of the sample are to be performed or in instances when only one or more select type of analysis is to be performed. The analysis device  12  is adapted perform the imaging techniques described below. 
     According to aspects of the present invention, the sample chamber  10  is mapped to provide locatable sub-regions within the sample chamber. The mapping is described herein after as an orthogonal map (e.g., an X-Y orthogonal) having tiles. The term “tiles” as used herein refers to sub-regions defined by the rows and columns of the map. The tiles are not limited to any particular geometry or size, and are not required to have four equal length sides. The chamber mapping is not limited to an orthogonal mapping. The tiles may represent individual image fields, and the collective image fields may capture all or substantially of the sample residing within the chamber  10 . The tiles may be collectively assembly to form a single image of all, or substantially all, of the sample residing within the chamber  10 . 
       FIG. 6  diagrammatically illustrates an orthogonal mapping  50  applied to an analysis chamber  10  that includes tiles numbered T 1 -T 86 . Tiles T 1  and T 2  are reference tiles separated from the chamber, and are used to produce a reference image for a glue line  22 /air interface. Tiles T 83 -T 86  are aligned with chamber regions located at the filling edge  23  of the chamber where the chamber inlet  21  resides. In the chamber embodiment shown in  FIG. 6 , tile T 83  is aligned with a chamber region predominantly filled with a glue line  22 , and tile T 86  is aligned with a chamber partially filled with a glue line  22 . Consequently, the chamber inlet  21  is disposed there between, aligned with tiles T 84 , T 85 , and a portion of T 86 . Tiles T 3 , T 18 , T 19 , T 34 , T 35 , T 50 , T 51 , T 66 , T 67 , and T 82  are aligned with the right lateral edge  52  of the chamber  10 , and the chamber region aligned with each tile is at least partially filled with a glue line that forms the right lateral boundary of the analysis chamber  10 . Tiles T 10 , T 11 , T 26 , T 27 , T 42 , T 43 , T 58 , T 59 , T 74 , and T 75  are aligned with the left lateral edge  54  of the chamber  10 , and the chamber region aligned with each tile is at least partially filled with a glue line  22  that forms the left lateral boundary of the analysis chamber  10 . Tiles T 3 -T 10  are disposed along the edge  56  of the chamber  10  opposite the filling edge of the chamber  10 . 
     In some applications, constituents within a sample residing within an analysis chamber  10  will assume a repeatable non-uniform distribution within the analysis chamber. The term “repeatable non-uniform distribution” is used to mean that when the same type of sample (e.g., undiluted whole blood) is disposed in a particular type of analysis chamber (e.g., the chamber described above as being defined by planar members; or a slide), certain constituents within the sample repeatably occupy particular regions within the chamber in a non-uniform distribution; i.e., not every chamber region occupied by the sample has the same type and/or number of constituents as the other chamber regions.  FIG. 8  below provides an example of a repeatable non-uniform distribution of whole blood within an analysis chamber. Hence, a statistically significant number (e.g., a number adequate to perform an analysis) of certain constituents will be repeatably present in certain regions of the analysis chamber and not in other regions. A predetermined representative version of the repeatable non-uniform distribution can, for example, be determined by evaluating a meaningful number of samples of the same type disposed within the same chamber. It has been determined that in these instances, constituents within the sample will repeatably occupy certain regions within the chamber. The predetermined distribution may, therefore, be empirically based on the statistical positions of the constituents within a meaningful number of filled chambers. In these applications, the positions of the statistically significant numbers of constituents within the sample are known, and can be accessed without the need to perform an imaging step for the purpose of finding the locations of the aforesaid constituents within the image of the sample chamber. 
     For example, using the chamber  10  described above as an example, a whole blood sample may be drawn into the chamber inlet  21  along the filling edge  23  by capillary forces. As the sample is drawn into the chamber  10 , the sample travels toward the opposite end  56  of the chamber  10 , and laterally outward toward the glue lines  22  that form the lateral boundaries of the chamber  10 . As the whole blood sample distributes within the chamber  10 , constituents (e.g., WBCs, RBCs, platelets, plasma) distribute in a repeatable non-uniform pattern. Specifically in the aforesaid non-uniform distribution, a statistically significant number of the WBCs within the sample will travel a limited distance into the chamber  10  and will populate the chamber region  58  proximate the chamber inlet  21 . In  FIG. 7 , the chamber region  58  that will repeatably contain a statistically significant number of the WBCs within the sample is aligned with tiles T 53 -T 56 , T 60 -T 64 , T 69 -T 72 , and T 78 -T 79 , and portions of T 68 , T 65 , T 52 , T 48 -T 45 , T 57 , and T 73 . 
     A statistically significant portion of the RBCs within the sample will also travel a distance into the chamber  10 , but will travel further into the chamber  10  than the WBCs. The RBCs will thereby populate a chamber region  60  contiguous with, but a distance further away from the chamber inlet  21  than the chamber region  58  populated by the statistically significant number of WBCs. In  FIG. 7 , the chamber region  60  that will repeatably contain a statistically significant number of the RBCs within the sample is aligned with tiles T 22 , T 23 , T 27 -T 33 , T 42 -T 36 , T 43 , T 58 , and T 49 , and portions of T 65 , T 52 , T 59 , T 25 , T 24 , T 21 , and T 20 . The disparity in travel distance is at least in part attributable to the height of the analysis chamber (e.g., a 4 micron separation between the interior surfaces of the planar members  14 ,  16  that form the chamber  10 ), and the relative sizes of WBCs vs. RBCs. WBCs are on average substantially larger in size than RBCs, and their travel within the chamber may therefore be impeded by frictional contact with the interior surfaces of the planar members  14 ,  16 . RBCs in their normal state will typically not contact both interior surfaces of the planar members  14 ,  16 . Plasma and platelets are distributed throughout the chamber by capillary flow. 
     Analyses directed specifically to plasma may be performed in the chamber regions beyond where the statistically significant portions of RBCs are located, which regions are relatively free of WBCs and RBCs and consequently predominated by plasma. In  FIG. 7 , the chamber regions that will repeatably be predominated by plasma within the sample are shown as region  62 , which region is aligned with portions of tiles T 13 - 16 , T 19 -T 21 , and T 25 -T 27 . Note that region  62  is arbitrarily shown here for description purposes, and regions predominated by plasma may also be found elsewhere. As a result of their substantially smaller relative size, platelets can be imaged throughout the chamber. 
     The above described repeatable alignment of chamber regions containing certain constituents within the sample and particular tiles is provided as a non-limiting example, and alternative tile/constituent alignments may be used; e.g., the aforesaid alignment may be influenced by the height of the chamber and/or the volume of the sample introduced into the chamber. 
     The repeatable non-uniform distribution of constituents within the sample can also be at least in part attributable to reagents disposed within the chamber. For example, in some analyses of whole blood samples it is desirable to subject at least a portion of the RBCs within the sample to an isovolumetric sphering agent; e.g., See U.S. Pat. No. 8,472,693 incorporated by reference above. In some applications, the isovolumetric sphering agent may be disposed within the chamber prior to sample entering the chamber in a manner that not all of the RBCs will be subjected to the sphering agent. As a result, and as shown in  FIG. 7 , the leading edge (i.e., the edge of the region furthest from the chamber inlet—depicted as region  60 A) of the chamber region  60  containing the statistically significant numbers of RBCs will contain a substantial number of sphered RBCs, and the remainder of the region  60  containing the statistically significant numbers of RBCs will not contain a substantial number of sphered RBCs. The sphered RBCs are particularly useful for certain types of RBC analyses. 
     As indicated above, the above described repeatable non-uniform distribution of sample constituents is provided as a non-limiting example of distribution within a specific chamber embodiment. In alternative embodiments (e.g., a smear on a slide), a non-uniform distribution may assume a completely different configuration. Regardless of the particular repeatable non-uniform distribution, according to aspects of the present invention a mapping may be applied to the chamber that allows coordination of the performance of those imaging techniques used to identify and/or analyze specific constituents within the sample with the position of statistically sufficient numbers of the particular constituents within the sample. In other words, imaging techniques used to identify and/or analyze a constituent within a sample are implemented only in areas of the sample where that specific constituent is likely to be present (e.g., where a statistically significant population of the constituent is likely to be present, and/or favorably imaged), and not in areas where that specific constituent is not likely to be present (or cannot be favorably imaged). 
     Using the determined, repeatable non-uniform distribution, the respective image analyses of particular constituents within the sample can be performed in an efficient manner in terms of time and data volume. For example, if the analysis device is commanded to perform a WBC analysis, only those mapping tiles aligned with the sample regions containing a statistically significant population of WBCs are imaged using the imaging techniques required to perform the requested analysis. Similarly, if the analysis device is commanded to perform a RBC analysis, only those mapping tiles aligned with the sample regions containing a statistically significant population of RBCs are imaged using the imaging techniques required to perform the requested analysis. As indicated above, certain RBC analyses are best performed on sphered RBCs. In those instances, only those mapping tiles aligned with the sample regions containing a statistically significant population of sphered RBCs are imaged using the imaging techniques required to perform the requested analysis. The same approach can be used for plasma analyses, etc. 
       FIG. 8  shows a diagrammatic depiction of a sample chamber with numeric values (e.g., N to N+7, where “N” is an integer) representative of the number of images necessary for multiple analyses of a whole blood sample; e.g., WBC analyses, platelet analyses, RBC analyses, plasma analyses, etc. In the depiction, the number of images necessary to be taken for all the analyses varies as a function of the position of the tile, and therefore the aligned region of the sample to be imaged at that position. Within the mapping, a group of tiles centrally located but also located closer to the chamber inlet indicate that a total of “N+7” images are to be taken for each of those tiles. That number of images represents the total number of images per tile necessary for the analyses of the WBCs, which tiles (per the repeatable non-uniform distribution of constituents within the sample) are aligned with a statistically sufficient number of WBCs in the sample. To illustrate further, within the mapping a group of tiles centrally located but also located further away from the chamber inlet indicate that a total of “N+3” images are to be taken for each of those tiles. That number of images represents the total number of images per tile necessary for the analyses of the sphered RBCs, which tiles (per the repeatable non-uniform distribution of constituents within the sample) are aligned with a statistically sufficient number of sphered RBCs in the sample. It should be noted that the imaged regions (i.e., tiles) for any particular constituents are often increased beyond what the known repeatable non-uniform distribution of constituents within the sample indicates to ensure that sufficient data is collected. The increased number of tiles also accounts for statistical variations and for variations in volumetric fill of the chamber. 
     It can be seen from the diagrammatic illustration that the total number of images for the eighty (80) tiles shown equals eighty times “N” (80*N), plus an additional one hundred and eighty (180) images. If “N+7” images were taken of all eighty tiles, it would equal 80*N images plus an additional five hundred and sixty (560) images, which represents a different of three hundred and eighty (380) images, each of which images adds significantly to the total analysis time and image storage and handling requirements. 
     While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment(s) disclosed herein as the best mode contemplated for carrying out this invention.