Abstract:
A microfluidic system isolates blood cells of a minute volume of a blood sample by using a passive-settlement approach or an active-flow approach, rather than an approach that requires centrifugation. In the passive-settlement approach, the microfluidic system yields a hematocrit measurement by detecting the volume of red blood cells that settle under the influence of gravitational force. According to the active-flow approach, blood cells are isolated at one or more porous regions having pores dimensioned which preferentially pass constituents of the blood sample.

Description:
TECHNICAL FIELD  
         [0001]    The invention relates generally to isolating blood cells of a blood sample and more particularly to determining measurements for a small volume of blood.  
         BACKGROUND ART  
         [0002]    In clinical chemistry, the identification of the composition of a person&#39;s blood is used as an important diagnostic tool. Blood is primarily serum, but includes three major types of cells. Serum comprises approximately sixty percent to seventy percent of a human blood sample, while approximately thirty to forty percent of the sample is cellular. Plasma within the sample is more than ninety percent water, with the remainder consisting of proteins, lipids, salts and the like. The three major blood cell types are red blood cells (RBCs), white blood cells (WBCs) and platelets.  
           [0003]    The category “hematology” encompasses a wide variety of different measurements, including hemoglobin, hematocrit, total RBC count, total WBC count, total platelet count, differential WBC count, platelet function and calculated RBC indices. Additionally, newer tests are becoming standard additions to this list, including RBC morphology, reticulocyte counts, and neutrophil maturation. Routinely, a large quantity of blood is drawn from a vein in the arm of a person when clinical lab tests are requested. Typically, more than ten milliliters (mis) are drawn. This sample volume is significantly more than is needed for one test, but it is common to run multiple clinical tests.  
           [0004]    For tests that do not require cell-related counts, small scale filtration devices are available for at least significantly reducing the hematocrit content. That is, such devices may be used to yield plasma having little or no hematocrit content. U.S. Pat. No. 6,319,719 to Bhullar et al. describes a capillary hematocrit separation structure. A capillary pathway from an inlet port is dimensioned so that the driving force for the movement of a blood sample through the pathway is capillary pressure. Obstacles are fixed within the capillary pathway. Each obstacle has a concave portion facing a reaction region at the downstream side of the obstacles. The obstacles can take on a variety of shapes, but should be dimensioned so that the hematocrit accumulates within the concave portions.  
           [0005]    In the case of blood sample analysis that requires cellular considerations, available analyzers range from small benchtop instruments with limited menus to large automated analyzers with significant versatility. The minimum required volume of blood also varies considerably. For example, a large state-of-the-art analyzer may require 150 microliters (μl) of whole blood for a full five-part differential, while a more limited analyzer may require a volume of 12 μl whole blood for a menu of five measured parameters.  
           [0006]    As previously noted, one procedure for assessing the health of a patient is to determine the patient&#39;s hematocrit. The hematocrit is the percentage of the volume of a blood sample that is occupied by red blood cells. This measurement may be acquired using different techniques. Most comprehensive hematology analyzers derive the hematocrit value from several other direct and indirect measurements. First, the RBC count and a quantitative hemoglobin measurement are obtained. Then, the RBC index, “mean cell hemoglobin content” (MCHC), is calculated for the sum of the RBC count and the hemoglobin measurement. This parameter is then used to calculate hematocrit. Typically, at least one of the measurements is acquired using centrifugal separation of cell populations.  
           [0007]    Microhematocrit techniques commonly employ centrifugal separation. One such technique is to use a small capillary tube which is filled with anticoagulated blood and centrifuged under selected conditions, e.g., for five minutes at 14,500 revolutions per minute. Because of the higher density of the red blood cells, these cells collect at the bottom of the tube. Thus, the volume of packed RBCs relative to the total volume of whole blood can be expressed as the percent hematocrit. This is often a relatively inexact measurement, giving values with +/−five percent variation. However, the technique is inexpensive and is used widely. U.S. Pat. No. 6,204,066 to Wardlaw describes a method of determining the sedimentation rate of erythrocyte. An anticoagulated sample of whole blood is placed in a transparent capillary tube, which is then subjected to centrifugation. The position of the erythrocyte/plasma interface in the blood sample is determined at selected time intervals during the centrifugation of the blood sample. The interface positions can be mapped. A value which reflects the sedimentation rate of the sample can be derived from a calculated slope and a Y intercept of the calculated slope, thereby arriving at a conventional gravity sedimentation rate value.  
           [0008]    A modification of this technique is to add a plastic “float” to the blood sample to enhance the separation of cell types. Additionally, chemical and fluorescent coatings on the microtube may be used to stain the individual cell types as they migrate past a position, so as to label discrete layers with different fluorescence or absorption. Again, the red blood cells migrate to the bottom of the tube. The hematocrit is obtained directly from the measurement of the volume of red blood cells relative to the total volume of the blood sample.  
           [0009]    U.S. Pat. No. 6,350,613 to Wardlaw describes another approach. In this approach, a whole blood sample is placed within a sample chamber having convergent opposing walls. At least one of the convergent walls of the chamber is transparent, so that the blood sample can be observed. By mixing fluorescent dyes with a blood sample, target cells in the sample can be enumerated and differentiated by using a scanning instrument which is able to measure different wavelength color signals emitted from target cells. The varying thicknesses provided by the convergent walls produce a first thickness region in which red blood cells and quiescent monolayers of red blood layers of the sample will reside after the sample fills the chamber. Larger constituents, such as white blood cells and nucleated red blood cells, will reside in greater thickness regions of the chamber. The non-nucleated red blood cells which reside in the greater thickness regions will agglomerate to form rouleaux. A related invention is described in U.S. Pat. No. 6,235,536 to Wardlaw.  
           [0010]    While the prior art techniques operate well for their intended purposes, further improvements are desired. For example, in the determination of a hematocrit measurement, centrifugation is typically required, so that the patient or the blood sample must enter a laboratory setting. Moreover, a relatively large sample volume is often required. What is needed is an approach that yields sufficiently accurate cellular measures from a minute quantity of blood. What is also needed is a more convenient approach to acquiring hematocrit measurements and other measurements that require blood cells to be isolated, thereby enabling Point-of-Care testing.  
         SUMMARY OF THE INVENTION  
         [0011]    A microfluidic system is used to isolate blood cells from other constituents of a minute volume of a blood sample, without requiring centrifugal separation. According to one approach, the separation is carried out by allowing gravitational force to provide separation on the basis of differences in densities. A density gradient medium having a density less than that of at least one blood cell type (RBC), but greater than that of plasma, may be introduced into a microfluidic separation region to allow the cells present in the minute volume of blood to settle through the density medium. In a second approach, the microfluidic system includes a microfluidic path having at least one porous region with pores that are dimensioned to inhibit passage of a specific blood cell type. In one contemplated embodiment, a first porous region includes pores dimensioned to block passage of white blood cells, which are typically the largest constituents of a blood sample, while a second porous region has pores dimensioned to block passage of red blood cells.  
           [0012]    The term “microfluidic” feature, as applied to flow regions, such as inlets, outlets and flow channels, shall be defined herein as a feature having a cross sectional dimension (e.g., width, height or diameter) of no more than 500 μm and preferably no more than 200 μm. As applied to non-flow regions, the term “microfluidic” is used herein to define a region in which fluids are introduced and removed via microfluidic features.  
           [0013]    In the gravity-separation approach, the microfluidic system yields a hematocrit measurement by detecting the volume of red blood cells within a known volume of blood. The separation region may be a microfluidic channel or may be a chamber. The density medium ensures at least one well defined gradation that is based upon relative densities of blood constituents. Due to the laminar flow properties inherent in microfluidics, the blood fluid will tend to remain on top of the density medium, resisting any mixing of layers. However, the red blood cells will fall through the density medium and will settle on the lower surface of the microfluidic channel or chamber. The layer of red blood cells can then be detected using any of a number of means, such as optical, electrical, and/or thermal. The volume of red blood cells is compared to the total volume of the blood sample to provide the hematocrit measurement. A density gradient medium may be selected to also allow passage of white blood cells. The portion of white blood cells is small compared to the portion of red cells, so that the white cell “contamination” can be readily factored into the hematocrit measurement.  
           [0014]    Typically, when blood is drawn and allowed to stand undisturbed, red blood cells naturally settle by gravity. This is because the red cells are denser than the white blood cells and platelets. The age-dependent buoyant density of the red cells is in the range of 1.085 to 1.12, as compared to 1.077 for white blood cells. Thus, the less dense cell types remain at the interface of the aqueous plasma and the settled red cells. In a 10 milliliter blood collection tube, the settling may take hours. However, using the present invention, the hematocrit measurement may be acquired in a time frame of 1 to 2 minutes. An advantage of the use of density gradient medium over passively allowing the red blood cells to settle from a volume of blood is that the medium provides a “sharper” demarcation of blood constituents, thereby providing a more easily detected interface from which to base hematocrit measurements.  
           [0015]    Alternatively, porous structures can be used to provide blood cell isolation. Red blood cells tend to have a mean cross sectional dimension of 7 μm, while white blood cells have a mean cross sectional dimension of approximately 20 μm. The differences in sizes can be used to broadly separate these two types of blood cells from other blood constituents.  
           [0016]    In one embodiment of this approach in which blood constituents are separated by size, red blood cells can be concentrated into a discrete volume by using a first porous region to separate white blood cells and a subsequent porous region to separate the red blood cells from the plasma and the platelets. The volume of red blood cells can be compared to the volume of red blood cells plus plasma, giving a hematocrit measurement as a percentage. A correction can be made for the presence of low percentages of white cells in the plasma volume. Either a pressurized flow or gravitational force may be used to direct a blood sample through the porous region. Each porous region may be formed by a single layer having holes or may be formed of a number of different structures that are spaced apart by a minute distance to selectively block types of blood constituents.  
           [0017]    One advantage of the invention is that cell separation and hematocrit measurements can be obtained in a microfluidic platform without the requirement of centrifugation. Thus, a point-of-care diagnostic tool can be fabricated. Another advantage of the invention is that smaller blood volumes are required. In the current centrifugation devices, required volumes as low as 10 μl have been used, but the present invention allows hematocrit measurements to be performed using a blood sample of 1 μl. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0018]    [0018]FIG. 1 is a perspective view of one embodiment of a microfluidic system for providing blood sample separation in accordance with the invention.  
         [0019]    [0019]FIG. 2 is a block diagram of components of the microfluidic system of FIG. 1 connected to a source of a blood sample.  
         [0020]    [0020]FIG. 3 is a representation of constituents of a blood sample separated using a passive-settlement approach in accordance with the invention.  
         [0021]    [0021]FIGS. 4 and 5 are side views of a microfluidic channel of FIG. 1 having a single porous region for isolating constituents of a blood sample.  
         [0022]    [0022]FIG. 6 is a side view of a microfluidic channel of FIG. 1 having two porous regions for isolating constituents of a blood sample.  
         [0023]    [0023]FIG. 7 is a side view of a microfluidic channel of FIG. 1 having a porous region that is formed in a manner different from the porous region of FIG. 5.  
         [0024]    [0024]FIG. 8 is a side view of a microfluidic channel of FIG. 1 having two porous regions formed in a manner consistent with that of FIG. 7.  
         [0025]    [0025]FIG. 9 is a side view of a microfluidic channel of FIG. 1 in which a porous region is positioned along a vertical segment of the microfluidic channel. 
     
    
     DETAILED DESCRIPTION  
       [0026]    With reference to FIGS. 1 and 2, a microfluidic system  10  may be connected to a source  12  of a blood sample. Two different configurations  14  and  16  of microfluidic features are shown in FIG. 1 for isolating selected constituents of a blood sample, but the system is more likely to use a single configuration. The first configuration utilizes a passive-settlement approach to induce blood cell separation, while the second configuration  16  utilizes an active-flow approach. Both of these approaches will be described in greater detail as follows.  
         [0027]    The microfluidic system  10  includes a sampling body  18 . The sampling body may be a substrate made of a material such as a polymer, glass, silicon, or ceramic. Polymers are often used in fabricating microfluidic devices. Polymer materials that are well suited for this application include materials selected from the following classes: polyimide, polycarbonate, polystyrene, polyester, polyamide, polyether, polyolefin, and mixtures of such materials.  
         [0028]    In the embodiment of FIGS. 1 and 2, the sampling body  18  includes electronic features, mechanical features, and fluidic features. The electrical features include a detector  20  and a processor  22 . The electrical features may be integrated into the substrate or may be separately attached. For example, the processor may be an integrated circuit chip that is separately fabricated and then connected to the substrate material. The mechanical features that are represented in FIG. 1 are six valves  24 ,  26 ,  28 ,  30 ,  32  and  34 . However, the valves are not included in embodiments in which blood samples are manipulated through passageways by capillary action. FIG. 2 represents a simplification of the fluidic features, since the drawing only shows an inlet  36 , a separation chamber  38 , and an outlet  40 .  
         [0029]    The first configuration  14  of microfluidic features will be described with reference to FIGS. 1 and 3. In this embodiment, flow from the inlet  36  is controlled by capillary action or by the valve  26  to allow a blood sample to enter a separation chamber  42 . In a passive device, the microfluidic structure should be designed to enhance blood flow to the bottom of the separation chamber. The inlet flow may be pressurized to ensure that the entire sample volume reaches the chamber. A precisely metered volume may be required in some applications, such as a hematocrit measurement device in which the measure of total volume is based upon an input measurement. A second input  44  to the separation chamber may be used to introduce a density medium, which is sometimes referred to as a density gradient medium. During manufacture, the second input may be used to “load” the device with the density medium. Thus, blood samples would be introduced only when the density medium is present. Alternatively, the density medium may be inserted during the fabrication process and the second input may be deleted. However, the density medium is not critical to all applications.  
         [0030]    Red blood cells (erythrocytes) are denser than white blood cells and platelets. The age-dependent buoyant density of red blood cells is in the range of 1.085 to 1.125, while the density of the white blood cells may be 1.077. When whole blood is introduced into the separation chamber  42 , layers will be formed, as represented in FIG. 3. Red blood cells  46  will rapidly fall through the density medium  48 . On the other hand, white blood cells and platelets  50  will rest within the density medium atop the red blood cells. The uppermost layer is the serum portion  52  of the blood sample.  
         [0031]    Due to the laminar flow properties inherent in microfluidics, the blood fluid will tend to remain layered on top of the density gradient fluid. At the lowest level of the separation chamber is the red blood cell layer  46 . In this position, the red cells can be detected using the detector  20 . As one example, the detector may be an optical device which is able to determine the height of the red blood cell layer. The volume of red blood cells can then be compared to the volume of the whole blood that was introduced to the microfluidic system  10 , so as to calculate a hematocrit measurement, as is well known by persons skilled in the art. Since the chamber has a known maximum volume, the volume of the red blood cell layer  46  can be compared to the volume of the chamber to yield a micro-hematocrit calculation. In one application, the starting volume of whole blood is 1 μl.  
         [0032]    As an alternative to blood analysis on-board the sampling body  18 , the layer  46  of red blood cells may be caused to flow through a first outlet  54  to a station (not shown) for analysis. Similarly, the flow of red blood cells may be directed to an off-board analytical station following a hematocrit determination by the detector  20  and processor  22  of FIG. 2. An outlet valve  28  regulates flow of the red blood cell layer  46  through the first outlet  54 . A second outlet  56  resides above the first outlet and may be used to control the flow of other separated constituents, such as the white blood cells within the layer  50 . The second outlet valve  30  controls flow through the second outlet  56 .  
         [0033]    In some applications, the plasma may be of interest, in addition to blood cells. Thus, following separation of a blood sample into constituents, the same or different outlets may be used to independently remove the constituents. As other alternatives, the separated blood cells can be independently analyzed on-board the sampling body  18  or the blood cells can be removed for off-board analysis in order to leave the plasma for on-board measurement or more sophisticated analysis.  
         [0034]    As a substitute for the separation chamber, a separation capillary may be used. The separation capillary should be oriented to accommodate gravitational separation of blood constituents. Thus, the capillary may be oriented to be vertical when the sampling body  18  is rested on a table in its intended position.  
         [0035]    Referring now to the second configuration  16  of microfluidic features in FIG. 1, one or more porous regions may be formed to separate a whole blood sample into constituents. Microfluidic structures may be used to effect separation of cells from the serum. By inserting structures of a given size and/or given orientation, cells can be preferentially blocked. This technique operates best for separating cells having significantly different sizes. For example, red blood cells having a mean diameter of 7 μm can be relatively easily separated from white blood cells, which have a mean diameter of approximately 20 μm. More precisely fabricated structures are necessary to separate different classes of the same cell type, such as separating populations of white blood cells that differ in size by 2 μm to 3 μm.  
         [0036]    In FIG. 1, capillary action or an inlet valve  34  regulates flow from an inlet  36  to a microfluidic channel  58  having at least one porous region. Similarly, capillary action or an outlet valve  32  controls flow from the microfluidic channel  58  to the outlet  40 .  
         [0037]    While the first configuration  14  that was described with reference to FIG. 3 involves passive settling of blood cells, the second configuration typically involves an active fluid flow. Referring to FIGS. 1, 4 and  5 , the porous region of the microfluidic channel  58  may be a single structure  60  having an array of pores  62  that are dimensioned to block the flow of blood cells. Thus, the microfluidic channel will have an upstream portion  64  with blood cells and a downstream portion  66  through which cell-free plasma flows. The porous structure  60  may be fabricated as part of the microfluidic channel structure using known micromachining techniques. Alternatively, the porous structure may be separately fabricated and then inserted into the channel. The pores may be uniform in size, but the susceptibility to clogging may be reduced by providing more than one pore size.  
         [0038]    In another embodiment, the microfluidic channel  58  of FIG. 6 has multiple porous structures  68  and  70  within the same porous region. The first structure  68  has an array of pores  72  dimensioned to block white blood cells. The pores  74  of the downstream structure  70  are smaller, since they are designed to block the flow of red blood cells. The pores  72  within the upstream structure  68  may have diameters of approximately 20 μm. As a result, white blood cells will be blocked by this structure. On the other hand, the downstream structure  70  may have pores  74  that prevent passage of constituents having sizes greater than 7 μm, so that red blood cells are blocked. The result is close to that obtained by the prior art techniques of centrifugation.  
         [0039]    While not shown in FIGS. 4, 5 and  6 , the microfluidic channel  58  may be adjacent to a detector, such as an optical detector, which senses the volume of blood cells that are blocked from flowing with the plasma through the porous region. Measurements may then be obtained. For example, a hematocrit measurement may be acquired when the red blood cells are isolated, since the volume of red blood cells can be compared to the total volume of the blood sample. As in the embodiment of FIG. 3, a 1 μl blood sample may be used in the microfluidic system.  
         [0040]    [0040]FIG. 7 illustrates another application of the active-flow approach as compared to the passive-settlement approach described with reference to FIG. 3. In FIG. 7, the microfluidic channel  58  includes an array of channel structures  76  which combine to form pores  78  between adjacent structures. As in FIG. 5, the size of the pores is designed to preferentially block constituents of a blood sample. The structure  76  may be layers that are separately fabricated using integrated circuit fabrication techniques. If the spacing between the layers is approximately 20 μm, white blood cells will be blocked, but plasma, platelets and red blood cells will pass through the pores  78 . Referring now to FIG. 8, a second array of structures  80  may be positioned downstream of the first array to preferentially block red blood cells. The pores  82  between adjacent structures  80  may be 7 μm. As a consequence, a layer of red blood cells will accumulate between the two arrays of structures. By detecting the volume of the red blood cells between the arrays, a hematocrit measurement may be acquired.  
         [0041]    Another embodiment of the active-flow approach is shown in FIG. 9. The microfluidic channel  58  includes a vertical section  84  that operates as the separation region for red blood cells. A porous structure  86  blocks the passage of the red blood cells, but allows the plasma to continue to flow. In the fabrication of the sampling body  18 , care is taken to enable light to propagate from a light source  88  to a photosensor array  90 . The photosensor array includes at least one column of photosensor elements that individually generate electrical signals in response to light. Thus, when a column of red blood cells is contained within the vertical section  84  of the microfluidic channel  58 , light will be blocked from reaching a lower portion of the array  90 . As the red blood cells continue to accumulate, a larger portion of the photosensor elements will be blocked. Therefore, the output of the photosensor array may be used as an indicator of the volume of red blood cells within a blood sample.  
         [0042]    The embodiments that have been described allow hematocrit measurements to be obtained using a hand-held cartridge. Consequently, point-of-care measurements can be acquired, rather than requiring a patient to travel to a laboratory setting having centrifugation capability or requiring the transportation of a blood sample to such a laboratory setting. Moreover, the minimum required volume for the blood sample is significantly less than the required amount using prior art techniques. A simplified test may be performed by healthcare professionals to evaluate such concerns as infections, anemia, and blood loss.