Abstract:
A fluidic medical diagnostic device permits measurement of analyte concentration or a property of a biological fluid, particularly the coagulation time of blood. The device has at one end a sample port for introducing a sample and at the other end a bladder for drawing the sample to a measurement area. A first channel carries the sample from the sample port to the measurement area, and a stop junction, between the measurement area and bladder, halts the sample flow. A second channel, which runs from the first channel to an edge of the device, determines whether the sample volume is sufficient to permit an accurate measurement. The desired measurement can be made by placing the device into a meter, which measures a physical property of the sample, typically optical transmittance, after it has interacted with one or more reagents on the device.

Description:
CROSS-REFERENCE TO PRIOR PROVISIONAL APPLICATION 
     This application claims the benefit of U.S. Provisional Application No. 60/093,421, filed Jul. 20, 1998. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a fluidic medical diagnostic device for measuring the concentration of an analyte in or a property of a biological fluid. 
     2. Description of the Related Art 
     A variety of medical diagnostic procedures involve tests on biological fluids, such as blood, urine, or saliva, and are based on a change in a physical characteristic of such a fluid or an element of the fluid, such as blood serum. The characteristic can be an electrical, magnetic, fluidic, or optical property. When an optical property is monitored, these procedures may make use of a transparent or translucent device to contain the biological fluid and a reagent. A change in light absorption of the fluid can be related to an analyte concentration in, or property of, the fluid. Typically, a light source is located adjacent to one surface of the device and a detector is adjacent to the opposite surface. The detector measures light transmitted through a fluid sample. Alternatively, the light source and detector can be on the same side of the device, in which case the detector measures light scattered and/or reflected by the sample. Finally, a reflector may be located adjacent to the opposite surface. A device of this latter type, in which light is first transmitted through the sample area, then reflected through a second time, is called a “transflectance” device. References to “light” throughout this specification and the appended claims should be understood to include the infrared and ultraviolet spectra, as well as the visible. References to “absorption” are meant to refer to the reduction in intensity as a light beam passes through a medium; thus, it encompasses both “true” absorption and scattering. 
     An example of a transparent test device is described in Wells et al. WO94/02850, published on Feb. 3, 1994. Their device comprises a sealed housing, which is transparent or translucent, impervious, and rigid or semi-rigid. An assay material is contained within the housing, together with one or more assay reagents at predetermined sites. The housing is opened and the sample introduced just before conducting the assay. The combination of assay reagents and analyte in the sample results in a change in optical properties, such as color, of selected reagents at the end of the assay. The results can be read visually or with an optical instrument. 
     U.S. Pat. No. 3,620,676, issued on Nov. 16, 1971 to Davis, discloses a calorimetric indicator for liquids. The indicator includes a “half-bulb cavity”, which is compressible. The bulb is compressed and released to form a suction that draws fluid from a source, through a half-tubular cavity that has an indicator imprinted on its wall. The only controls on fluid flow into the indicator are how much the bulb is compressed and how long the indicator inlet is immersed in the source, while the bulb is released. 
     U.S. Pat. No. 3,640,267, issued on Feb. 8, 1972 to Hurtig et al., discloses a container for collecting samples of body fluid that includes a chamber that has resilient, collapsible walls. The walls are squeezed before the container inlet is placed into the fluid being collected. When released, the walls are restored to their uncollapsed condition, drawing fluid into and through the inlet. As with the Davis device, discussed above, control of fluid flow into the indicator is very limited. 
     U.S. Pat. No. 4,088,448, issued on May 9, 1978 to Lilja et al., discloses a cuvette, which permits optical analysis of a sample mixed with a reagent. The reagent is coated on the walls of a cavity, which is then filled with a liquid sample. The sample mixes with the reagent to cause an optically-detectable change 
     U.S. Pat. Nos. 4,426,451; 4,868,129; 5,104,813; and 5,230,866 disclose various devices for diluting and/or analyzing biological fluid samples. The devices include a “stop flow junction” to control the flow of the sample. The junction consists of an abrupt change in the cross-sectional area of a flow channel. Typically, the junction is formed when a small-diameter capillary channel enters a larger channel. The stop junction creates a back pressure that stops the normal blood flow until some additional pressure, such as hydrostatic pressure, acts to cause the sample to break through the junction into the larger channel. 
     U.S. Pat. No. 5,627,041, issued on May 6, 1997 to Shartle et al., discloses a diagnostic device that includes a stop junction, and the force that causes sample to break through the junction is a centrifugal force provided by rotating the device. 
     European Patent Application EP 0 803 288, of Naka et al., published on Oct. 29, 1997, discloses a device and method for analyzing a sample that includes drawing the sample into the device by suction, then reacting the sample with a reagent in an analytical section. Analysis is done by optical or electrochemical means. In alternate embodiments, there are multiple analytical sections and/or a bypass channel. The flow among these sections is balanced without using stop junctions. FIG. 9 of Naka et al. depicts a “liquid pooling portion” at the sample inlet and an air vent passage branching from the drawing channel. These two elements, in combination, permit a two-stage process for introducing the sample to the analytical section. 
     U.S. Pat. No. 5,700,695, issued on Dec. 23, 1997 to Yassinzadeh et al., discloses an apparatus for collecting and manipulating a biological fluid that uses a “thermal pressure chamber” to provide the driving force for moving the sample through the apparatus. 
     U.S. Pat. No. 5,736,404, issued on Apr. 7, 1998, to Yassinzadeh et al., discloses a method for determining the coagulation time of a blood sample that involves causing an end of the sample to oscillate within a passageway. The oscillating motion is caused by alternately increasing and decreasing the pressure of the sample. 
     U.S. Pat. No. 5,208,163, issued on May 6, 1993 to Charlton et al., discloses a sample analysis device that includes a metering chamber and capillary that allows an operator to determine that sample has been applied in excess of the amount needed for the measurement. 
     SUMMARY OF THE INVENTION 
     The present invention provides a fluidic diagnostic device for measuring an analyte concentration or property of a biological fluid. The device comprises 
     a first layer and second layer at least one of which has a resilient region over at least part of its area, separated by an intermediate layer, in which cutouts in the intermediate layer form, with the first and second layers, 
     a) a sample port for introducing a sample of the biological fluid into the device; 
     b) a measurement area, in which a physical parameter of the sample is measured and related to the analyte concentration or property of the fluid; 
     c) a first channel, having a first end and a second end, to provide a fluidic path from the sample port at the first end through the measurement area; 
     d) a bladder at the second end of the first channel, comprising at least a part of the resilient region in at least the first or second layer and having a volume that is at least about equal to the combined volume of the measurement area and first channel; 
     e) a stop junction in the first channel between the measurement area and bladder that comprises a co-aligned through-hole in at least the first or second layer, the through-hole being overlaid with a third layer; and 
     f) a second channel having a first end in fluid communication with the first channel at a first point between the sample port and measurement area and a second end vented, in which 
     (i) at least the first or second layer has a transparent section at a predetermined second point adjoining the second channel and 
     (ii) the volume of the part of the second channel lying between the first and second points is at least about equal to the volume of the measurement area. 
     The device is particularly well adapted for measuring prothrombin time (PT time), with the biological fluid being whole blood and the measurement area having a composition that facilitates the blood clotting cascade. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a plan view of a device of the present invention. 
     FIG. 2 is an exploded view of the device of FIG.  1 . 
     FIG. 3 is a perspective view of the device of FIG.  1 . 
     FIG. 4 is a schematic of a meter for use with a device of this invention. 
     FIG. 5 is a graph of data that is used to determine PT time. 
     FIG. 6 is a plan view of an alternative embodiment of a device of this invention. 
     FIGS. 6A-6F depict a time sequence during which a sample is admitted to the device of FIG.  6 . 
     FIG. 7 is a schematic of a device that includes multiple measurement areas, a single bladder, and a single bypass channel. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention relates to a fluidic device for analyzing biological fluid. The device is of the type that relates a physical parameter of the fluid, or an element of the fluid, to an analyte concentration in the fluid or to a property of the fluid. Although a variety of physical parameters—e.g., electrical, magnetic, fluidic, or optical—can form the basis for the measurement, a change in optical parameters is a preferred basis, and the details that follow refer to an optical device. The device includes a sample application area; a capillary-fill channel to accumulate at least enough sample to permit a valid measurement to be made; a measurement area, in which the sample may undergo a-change in an optical parameter, such as light scattering; a bladder, to create a suction force to draw the sample from the capillary channel to the measurement area; and a stop junction to stop flow after the measurement area has been filled. 
     Preferably, the device is substantially transparent over the measurement area, so that the area can be illuminated from one side and the transmitted light measured on the opposite side. The change in transmitted light is a measure of the analyte or fluid property of interest. Alternatively, light that is scattered from a fluid sample or light that passes through the sample and is reflected back through a second time (by a reflector on that opposite side) can be detected by a detector on the same side as the light source. 
     This type of device is suitable for a variety of analytical tests of biological fluids, such as determining biochemical or hematological characteristics, or measuring the concentration in such fluids of proteins, hormones, carbohydrates, lipids, drugs, toxins, gases, electrolytes, etc. The procedures for performing these tests have been described in the literature. Among the tests, and where they are described, are the following: 
     (1) Chromogenic Factor XIIa Assay (and other clotting factors as well): Rand, M. D. et al., Blood, 88, 3432 (1996). 
     (2) Factor X Assay: Bick, R. L. Disorders of Thrombosis and Hemostasis: Clinical and Laboratory Practice. Chicago, ASCP Press, 1992. 
     (3) DRVVT (Dilute Russells Viper Venom Test): Exner, T. et al., Blood Coag. Fibrinol., 1, 259 (1990). 
     (4) Immunonephelometric and Immunoturbidimetric Assays for Proteins: Whicher, J. T., CRC Crit. Rev. Clin Lab Sci. 18:213 (1983). 
     (5) TPA Assay: Mann, K. G., et al., Blood, 76, 755, (1990).; and Hartshorn, J. N. et al., Blood, 78, 833 (1991). 
     (6) APTT (Activated Partial Thromboplastin Time Assay): Proctor, R. R. and Rapaport, S. I. Amer. J. Clin. Path, 36, 212 (1961); Brandt, J. T. and Triplett, D. A. Amer. J. Clin. Path., 76, 530 (1981); and Kelsey, P. R. Thromb. Haemost. 52, 172 (1984). 
     (7) HbA1c Assay (Glycosylated Hemoglobin Assay): Nicol, D. J. et al., Clin. Chem. 29, 1694 (1983). 
     (8) Total Hemoglobin: Schneck et al., Clinical Chem., 32/33, 526 (1986); and U.S. Pat. No. 4,088,448. 
     (9) Factor Xa: Vinazzer, H., Proc. Symp. Dtsch. Ges. Klin. Chem., 203 (1977), ed. By Witt, I. 
     (10) Colorimetric Assay for Nitric Oxide: Schmidt, H. H., et al. , Biochemica, 2, 22 (1995). 
     The present device is particularly well suited for measuring blood clotting time—“prothrombin time” or “PT”—and details regarding such a device appear below. The modifications needed to adapt the device for applications such as those listed above require no more than routine experimentation. 
     FIG. 1 is a plan view of a device  10  of the present invention. FIG. 2 is an exploded view and FIG. 3 a perspective view of the device. Blood is applied to sample port  12  after bladder  14  has been compressed. Clearly, the region of layer  26  and/or layer  28  that adjoins the cutout for bladder  14  must be resilient, to permit bladder  14  to be compressed. Polyester of about 0.1 mm thickness has suitable resilience and springiness. Preferably, top layer  26  has a thickness of about 0.125 mm, bottom layer  28  about 0.100 mm. Blood is drawn from port  12  by capillary action into channel  13 . It doesn&#39;t flow into channel  16 , because that path is not vented. Blood continues to flow into channel  13  until it reaches a point at which the volume of blood in channel  13  is at least about equal to the combined volume of measurement area  18  and the volume of channel  16  lying between the measurement area and channel  13 . When blood reaches that point—“E” (for “enough-sample”) in channel  13 —a sensor (described below) senses that enough blood has been drawn into the device and bladder  14  is released. When the bladder is released, suction draws blood from port  12  and channel  13  through channel  16  to measurement area  18 . In order to ensure that measurement area  18  can be filled with blood, the volume of bladder  14  is preferably at least about equal to the combined volume of channel  16  and measurement area  18 , which preferably contains a reagent  20 . If measurement area  18  is to be illuminated from below, layer  28  must be transparent where it adjoins measurement area  18 . For a PT test, reagent  20  contains thromboplastin that is free of bulking reagents normally found in lyophilized reagents. 
     As shown in FIGS. 1,  2 , and  3 , stop junction  22  adjoins bladder  14  and measurement area  18 ; however, a continuation (i.e., “neck”) of channel  16  may be on either or both sides of stop junction  22 , separating the stop junction from measurement area  18  and/or bladder  14 . When the blood reaches stop junction  22 , blood flow stops. For PT measurements, it is important to stop the flow of blood as it reaches that point to permit reproducible “rouleaux formation”—the stacking of red blood cells—which is an important step in monitoring blood clotting using the present invention. The principle of operation of stop junctions is described in U.S. Pat. No. 5,230,866, incorporated herein by reference. 
     As shown in FIG. 2, all the above elements are formed by cutouts in intermediate layer  24 , sandwiched between top layer  26  and bottom layer  28 . Preferably, layer  24  is double-sided adhesive tape. Stop junction  22  is formed by an additional cutout in layer  26  and/or  28 , aligned with the cutout in layer  24  and sealed with sealing layer  30  and/or  32 . Preferably, as shown, the stop junction comprises cutouts in both layers  26  and  28 , with sealing layers  30  and  32 . Each cutout for stop junction  22  is at least as wide as channel  16 . Also shown in FIG. 2 is an optional filter  12 A to cover sample port  12 . The filter may separate out red blood cells from a sample and/or may contain a reagent to interact with the blood to provide additional information. Of course, the filter will offer resistance to passage of the blood sample into sample port  12  and from there into the channels. Thus, a filter must be selected with that in mind. A suitable filter comprises an anisotropic membrane, preferably a polysulfone membrane of the type available from Spectral Diagnostics, Inc., Toronto, Canada. If optional reflector  18 A, which may be on or adjacent to a surface of layer  26  and positioned over measurement area  18 , is present, the device becomes a transflectance device. 
     The method of using the strip of FIGS. 1,  2 , and  3  can be understood with reference to a schematic of the elements of a meter shown in FIG. 4, which contemplates an automated meter. Alternatively, manual operation is also possible. (in that case, bladder  14  is manually depressed before blood is applied to sample port  12 , then released when blood reaches point E on channel  13 .) The first step the user performs is to turn on the meter, thereby energizing strip detector  40 , enough sample detector  42 , measurement system  44 , and optional heater  46 . The second step is to insert the strip. Preferably, the strip is not transparent over at least a part of its area, so that an inserted strip will block the illumination by LED  40   a  of detector  40   b . (More preferably, the intermediate layer is formed of a non-transparent material, so that background light does not enter measurement system  44 .) Detector  40   b  thereby senses that a strip has been inserted and triggers bladder actuator  48  to compress bladder  14 . A meter display  50  then directs the user to apply a blood sample to sample-port  12  as the third and last step the user must perform to initiate the measurement sequence. 
     Capillary action draws the blood sample into enough sample channel  13 , which optionally has a reactant to react with the blood. LED  42   a  illuminates the edge of channel  13 , designated “E” in FIG.  1 . When channel  13  is empty; i.e., when no blood extends to point E, background light (e.g., from LED  42   a ) falls on detector  42   b . As blood is drawn into the strip and enters channel  13 , the blood at the “enough sample” point, E, changes the amount of light from LED  42   a  that is reflected to detector  42   b , which, in turn, signals actuator  48  to release bladder  14 . Depending on the materials of the strip and sample and the geometry of the optics, the light can be either increased or decreased to signal actuator  48 . The resultant suction in channel  16  draws blood through measurement area  18  to stop junction  22 . Light from LED  44   a  passes through measurement area  18 , and detector  44   b  monitors the light transmitted through the blood as it is clotting. Analysis of the transmitted light as a function of time (as described below) permits a calculation of the PT time, which is displayed on the meter display  50 . Preferably, blood temperature is maintained at about 37° C. by heater  46 . 
     FIG. 5 depicts a typical “clot signature” curve in which the current from detector  44   b  is plotted as a function of time. Blood is first detected in the measurement area by  44   b  at time  1 . In the time interval A, between points  1  and  2 , the blood fills the measurement area. The reduction in current during that time interval is due to Light scattered by red cells and thus an approximate measure of the hematocrit. At point  2 , blood has filled the measurement area and is at rest, its movement having been stopped by the stop junction. The red cells begin to stack up like coins (rouleaux formation). The rouleaux effect allows increasing light transmission through the sample (less scattering) in the time interval between points  2  and  3 . At point  3 , clot formation ends rouleaux formation and transmission through the sample reaches a maximum. The PT time can be calculated from the interval B between points  1  and  3  or the interval between points  2  and  3 . Thereafter, blood changes state from liquid to a semi-solid gel, with a corresponding reduction in light transmission. The reduction in current C between the maximum  3  and endpoint  4  correlates with fibrinogen in the sample. 
     The device pictured in FIG.  2  and described above is preferably formed by laminating thermoplastic sheets  26  and  28  to a thermoplastic intermediate layer  24  that has adhesive on both of its surfaces. The cutouts that form the elements shown in FIG. 1 may be formed, for example, by laser- or die cutting of layers  24 ,  26 , and  28 . Preferably, the surface of sheet  28  is hydrophilic (Film 9962, available from 3M, St. Paul, Minn.). 
     FIG. 6 is a plan view of another embodiment of the device of the present invention, in which the device includes a bypass channel  52  that connects channel  16  with bladder  14 . The function and operation of the bypass and “enough sample” channels can be understood by referring to FIGS. 6A-6F, which depict a time sequence during which a blood sample is drawn into device  10  for the measurement. 
     FIG. 6A depicts the situation after a user has applied a blood sample to the strip, while bladder  14  is compressed. This can be accomplished by applying one or more drops of blood to sample port  12 . 
     FIG. 6B depicts the situation after blood has been drawn by capillary action into enough-sample channel  13  of the strip and reached point E, thereby triggering release of the bladder and causing reduced pressure in channel  16 . 
     FIG. 6C depicts the situation as blood is drawn into channel  16  from channel  13  and sample port  12 . The materials and dimensions of channels  16  and  13  are selected to ensure that blood is preferentially drawn from channel  13 , before the blood in port  12  has been depleted. 
     FIG. 6D depicts the situation when the blood sample has been drawn into the measurement area  18 . When the blood reaches stop junction  22 , it encounters a back pressure that causes it to stop and causes additional blood to be drawn into the bypass channel. 
     FIG. 6E depicts the “endpoint”; i.e., the situation when a reading is taken. Blood is at rest in measurement area  18 . Excess blood has been drawn into bypass channel  52 . 
     FIG. 6F depicts an alternate endpoint. If the bladder has somewhat less volume and/or has not been completely compressed initially, then a reading is taken when the blood is distributed as shown in FIG.  6 F. After ambient pressure has been established in bypass channel  52 , excess blood may be drawn from sample port  12  into channel  13  by capillary forces. Note that channel  13  provides a reservoir in which excess sample can accumulate, without affecting the measurement (which is made in measurement area  18 ). 
     FIG. 7 depicts a preferred embodiment of the present device. It is a multi-channel device that includes bypass channel  152 . Bypass channel  152  serves a purpose in this device that is analogous to that served by bypass channel  52  in the device of FIG. 6, which was described above. Measurement area  118 P contains thromboplastin. Preferably, measurement areas  218  and  318  contain controls, more preferably, the controls described below. Area  218  contains thromboplastin, bovine eluate, and recombinant Factor VIIa. The composition is selected to normalize the clotting time of a blood sample by counteracting the effect of an anticoagulant, such as warfarin. Measurement area  318  contains thromboplastin and bovine eluate alone, to partially overcome the effect of an anticoagulent. Thus, three measurements are made on the strip. PT time of the sample, the measurement of primary interest, is measured on area  118 . However, that measurement is validated only when measurements on areas  218  and  318  yield results within a predetermined range. If either or both of these control measurements are outside the range, then a retest is indicated. Extended stop junction  122  stops flow in all three measurement areas. 
     The following examples demonstrate the present invention but are not intended to be in any way limiting. 
     EXAMPLE 1 
     Measurement of HbA1c 
     A device is prepared as described above and as shown in FIGS. 1,  2 , and  3 . Coated on bottom layer  28 , in alignment with sample port  12 , is a denaturant/oxidant reagent consisting of NH 4 SCN, K 3 Fe(CN) 6 , and a buffer. A suspension of anti-HbA1c antibody-latex (Ab-latex) is coated on channel  13  and dried. Measurement area  18  contains polyaspartic acid agglutinator reagent. A blood sample is applied to sample port  12 . The denaturant/oxidant causes the red blood cells to lyse and oxidizes the hemoglobin. The treated sample is then drawn into channel  13  by capillary action, where it incubates the Ab-latex. After reaching point E, bladder  14  is released, and sample is drawn into measurement area  18 , where the agglutinator reagent stops the reaction. A measurement of the optical transmission of the sample in area  18  yields the HbA1c concentration. More details relating to this type of measurement appear in U.S. Pat. No. 4,847,209, issued on Jul. 11, 1989 to L. A. Lewis et al., incorporated herein by reference. 
     EXAMPLE 2 
     Measurement of C-Reactive Protein (CRP) 
     A device is prepared as described above and as shown in FIGS. 1,  2 , and  3 . Filter  12 A is impregnated with a fluoresceinated liposome suspension and dried. Channel  13  is coated with a solution of complement and dried. The complement is then overcoated with a solution of rabbit anti-CRP antibody and dried. Measurement area  18  is coated with a solution of barbital buffer and EDTA and dried. To run the assay, blood is applied to sample port  12  through filter  12 A. Filter  12 A retains the erythrocytes but allows plasma to pass. The liposomes in filter  12 A resuspend in the plasma and move with it into channel  13 , where, at first, the anti-CRP antibody is rehydrated and mixes with the plasma-liposome mixture. The undercoated layer of complement is then exposed to the plasma-liposome-antibody mixture and the complement reaction takes place. Bladder  14  is released and the treated mixture is drawn into measurement area  18 , where the barbital buffer/EDTA reagent stops the reaction. Measurement of the fluorescent light output and the output of control(s) (in an area such as area  218  or  318 , in FIG. 7) yields the CRP concentration. Details of this measurement appear in  Immunology Methods Manual , Vol. 1, I. Lefkovitz ed., Basel Institute for Immunology, pp. 548-550, incorporated herein by reference (see also Umeda et al.,  J. Immunol. Methods  95:15-21). 
     The invention having been fully described, it will be apparent to one of ordinary skill in the art that many modifications and changes may be made to it without departing from the spirit and scope of the present invention.