Initiation of an analytical measurement

Initial method of analysis and measurement in blood

Patent No. W094/02850 [owned by Wells et al.]. The device includes a sealed casing, which is transparent or translucent, airtight, and rigid or semi-rigid. An analyte and the same or multiple analysis reagents located in predetermined positions are packed in the housing. Only open the housing to import the sample before proceeding with the analysis. The combination of the analytical reagent and the test substance in the sample causes the optical characteristics of the selected reagent, such as color, to change at the end of the analysis. The result can be read visually or with an optical instrument.

US Patent No. 3620676 issued to Davis on November 16, 1971 discloses a liquid color indicator. The indicator includes a compressible "hemispherical cavity". The spherical cavity is compressed and released to form a suction force to draw fluid from a fluid source through the half-tubular cavity, and an indicator is imprinted on the wall of the half-tubular cavity. The flow control unit of the fluid inflow indicator only has the compression degree of the spherical cavity and the length of the indicator inlet immersed in the fluid source when the spherical cavity is released.

U.S. Patent No. 3,640,267 issued to Hurtig et al. on February 8, 1972 discloses a container for collecting body fluid samples, which includes a compartment with a plurality of flexible and collapsible walls. The walls of the compartments are squeezed before the inlet of the container is placed in the fluid to be collected. The compartment walls return to their uncollapsed state when released and draw fluid through the inlet. This device, like the David device mentioned above, has quite limited control of the fluid flow entering the indicator.

US Patent No. 4088448 issued to Lilja et al. on May 9, 1978 discloses a cuvette that can perform optical analysis on a sample mixed with a reagent. The reagent is coated on the wall of a cavity, and then the cavity is filled with liquid samples. The sample is mixed with the reagent to produce a detectable optical change.

Several patents described below disclose devices for diluting and/or analyzing biological fluid samples. These devices include valve-like designs to control the flow of samples.

U.S. Patent No. 4426451 issued to Columbus on January 17, 1984 discloses a multi-zone fluid device, which has a pressure-actuable device to control fluid flow between zones. The device utilizes pressure balance on the meniscus of the interface between the first zone and the second zone with different cross-sections. When both the first zone and the second zone are at atmospheric pressure, the surface tension generates a back pressure to prevent the meniscus from proceeding from the first zone to the second zone. The structure of this interface or "stop junction" allows the liquid to flow into the second region only when sufficient pressure is applied to the liquid in the first region to push the meniscus out of the second region.

US Patent No. 4,868,129 issued to Gibbon et al. on September 19, 1989 discloses that the back pressure in a stop joint can be overcome by the hydrostatic pressure on the liquid in the first zone, for example, in the first zone There is a fluid column.

U.S. Patent No. 5230866 issued to Shartle et al. on July 27, 1993 discloses a fluid device with a plurality of stop joints, in which the back pressure caused by surface tension at the stop joints is for example due to The gas in the second zone is blocked and compressed and strengthened. The compressed gas can then be unclogged before applying additional hydrostatic pressure to the first zone to allow the fluid to exit before flowing into the second zone. By changing the back pressure of a plurality of stop joints in parallel, it is possible to form "rupture junctions" with a lower maximum back pressure

U.S. Patent No. 5,472,603 (see also U.S. Patent No. 5,627,041) issued to Schembri on December 5, 1995 discloses the use of centrifugal force to overcome the back pressure in a stop joint. When the flow stops, the first zone is at atmospheric pressure plus a pressure less than the centrifugal pressure required to overcome the back pressure. The second zone is at atmospheric pressure. To continue the flow, additional centrifugal pressure must be applied to the first zone to overcome the meniscus back pressure. The second zone is maintained at atmospheric pressure.

Announced on October 29, 1997, European Patent Application No. 0803288 filed by Naka et al. discloses a device and method for analyzing samples, which includes: sucking the sample into the device, and then The sample and a reagent are allowed to act in an analysis section. The analysis is done by optical or electrochemical devices. Other embodiments have multiple analysis sections and/or a bypass pipeline. The flow between these sections is balanced without the use of stop joints.

U.S. Patent No. 5,700,695 issued to Yassinzad (Yassinzadeh) et al. on December 23, 1997 discloses a device for collecting and manipulating biological fluids, which uses a "thermal pressure chamber" to provide a sample transfer Through the driving force of the device.

US Patent No. 5736404 issued to Asinzad et al. on April 7, 1998 discloses a method for determining the clotting time of a blood sample. The method involves oscillating one end of the sample in a channel. The oscillating motion is produced by alternately increasing and decreasing the pressure on the sample.

European patent EP 0 922 954 A2 discloses a method by monitoring the first and second derivatives of a parameter (such as the reflectance of a mixture of biological fluid and a reagent) to confirm whether there is a sample fluid on a test paper.

Summary of the invention

The present invention provides an initial method for measuring the concentration or characteristics of a analyte in a biological fluid, the biological fluid presents a "rouleaux" reorganization. "Rouleaux formation" refers to a stack of red blood cells, which gives this fluid (usually whole blood) a special optical sign. The method includes:

a) Provide a meter that measures the concentration of the test substance or a physical characteristic of a blood sample on a medical diagnostic fluid device,

b) Insert the device into the meter, the device contains

i) The same port, which is used to introduce a biological fluid sample into the device,

ii) A measurement area in which the concentration or physical characteristics of the analyte is measured,

iii) A pipe with a first end and a second end for providing a fluid passage from the sample port at the first end to the measurement area,

c) applying the biological fluid sample to the sample port,

d) illuminate the sample port and monitor the light scattered by the sample for a predetermined period of time, and

e) Only during the predetermined period of time that the scattered light has increased rapidly and then decreased before measuring the concentration or physical characteristics of the analyte, so that the meter only measures when the biological fluid is whole blood.

In another embodiment, the method of the present invention makes the measurement of the concentration or characteristic of a analyte in a biological fluid effective only when the biological fluid contains whole blood. The method includes:

a) Provide a meter that measures the concentration or physical characteristics of a blood sample on a medical diagnostic fluid device,

b) Insert the device into the meter, the device contains

i) The same port, which is used to introduce a biological fluid sample into the device,

ii) A measurement area in which the concentration or physical characteristics of the analyte is measured,

iii) A pipe with a first end and a second end for providing a fluid passage from the sample port at the first end to the measurement area,

c) applying the biological fluid sample to the sample port,

d) illuminate the sample port and monitor the light scattered by the sample for a predetermined period of time,

e) Measure the concentration or physical characteristics of the test substance, and

f) Only during the predetermined period of time that the scattered light has increased rapidly and then decreased to make the measurement effective, so that the meter only makes the measurement effective when the biological fluid is whole blood.

In another embodiment, the present invention includes an initial method for measuring the concentration or physical characteristics of a analyte in a biological fluid, the method comprising:

a) Provide a meter that measures the concentration or physical characteristics of a blood sample on a medical diagnostic fluid device,

b) Insert the device into the meter, the device contains

i) A transparent sample port for introducing a biological fluid sample into the device,

ii) A measurement area in which the concentration or physical characteristics of the analyte is measured,

iii) A pipe with a first end and a second end for providing a fluid passage from the sample port at the first end to the measurement area,

c) applying the biological fluid sample to the sample port,

d) illuminate the sample port and monitor the light transmitted through the sample for a predetermined period of time, and

e) Only during the predetermined period of time that the transmitted light has first sharply decreased and then increased before measuring the concentration or physical characteristics of the analyte, so that the meter only measures when the biological fluid is whole blood.

The method of the present invention can be widely used in various devices for measuring the concentration or physical characteristics of the analyte in the blood, but it is particularly suitable for measuring the prothrombin time (PT time) of whole blood. At this time, the measurement area has a component that catalyzes the stepped coagulation of blood.

Detailed description of the invention

The present invention relates to a measurement starting method of a device for analyzing certain biological fluids (especially whole blood). The device is combined with an appropriate meter and is generally of a type that correlates a physical parameter or a component of the blood with the concentration of a test substance in the blood or fluid characteristics. Although there are a variety of physical parameters such as electrical, magnetic, fluid or optical parameters that can constitute the benchmark for measurement, the change of optical parameters is a preferred benchmark, and the following detailed description will refer to this device as an optical device. Similarly, the method can be adapted to a variety of device designs, including devices involving capillary action filling; however, we here provide details of a particularly suitable device, which includes an additional sample area; a pouch for generating a The suction pulls the sample into the device; a measurement area where the sample can experience a change in optical parameters such as light scattering; and a stop joint for accurately stopping the flow of fluid after the measurement area is filled. (Applying the modification of the method of the present invention to other devices and performing other measurements only involve routine experiments.)

Preferably, the measurement area of the device is substantially transparent, so that the area can be illuminated by a light source located on one side and the transmitted light can be measured on the opposite side. The measurement of the sample may be an unchanged parameter, but usually the sample undergoes a change in the measurement area, and the change in the transmitted light is a measure of the characteristics of the object or fluid of interest. Another option is to use a detector on the same side of the light source to detect light scattered from a fluid sample or pass through the sample and then reflect (reflected by a reflector on the opposite side) and pass through the sample twice. Light.

This type of device is suitable for various blood analysis tests, such as determining biochemical or hematological characteristics, or measuring the concentration of proteins, hormones, carbohydrates, lipids, drugs, poisons, gases, electrolytes, etc. The procedures for conducting these tests are described in the literature. The tests mentioned in the literature are as follows:

(1) Analysis of chromogenic factor XIIa (and other clotting factors): Rand, MD, et al., Blood, 88, 3432 (1996).

(2) Factor X analysis: Bick, RL embolism and hemostatic disorder: clinical and actual room operation. Chicago, ASCP Press, 1992.

(3) Dilution Lessel's Snake Venom Test (DRVVT): Exner, T. et al., Blood Coagulation, Fibrin, 1, 259 (1990).

(4) Immunoturbidimetric and immunoturbidimetric analysis of protein: Whicher, JT, CRC Crit. Rev. Clin LabSci. 18: 213 (1983).

(5) TPA analysis: Mann, KG et al., Blood, 76,755, (1990); and Hartshorn, JN et al., Blood, 78,833, (1991).

(6) APTT (analysis of activated local thrombosis time): Proctor, RR and Rapaport, SIAmer. J. Clin. Path, 36, 212 (1961); Brandt, JT and Triplett, DAAmer. J. Clin. Path, 76, 530 (1981) ); and Ke1sey, PR Thromb. Haemost. 52, 172 (1984).

(7) HbAlc analysis (glycosylated heme analysis); Nicol, DJ et al., clinical chemistry,<u>29,</u>1694(1983)。

(8) Total hemoglobin: Schneck et al., Clinical Chemistry,<u>32/33,</u>526 (1986); and U.S. Patent No. 4088448.

(9) Factor Xa: Vinazzer, H., Proc. Symp. Dtsch. Ges. Klin. Chem., 203 (1977), edited by Witt, I.

(10) Colorimetric analysis of nitrogen oxides: Schmidt, HH et al., Biochemistry,<u>2,</u>22(1995)。

This method is particularly suitable for a device to measure prothrombin time (PT time). The details of this device are disclosed below. The modifications required to make the method and the device suitable for, for example, the preceding applications only require routine experimentation.

Fig. 1 is a plan view of a device 10 suitable for the method of the present invention. Figure 2 is an exploded view of the device and Figure 3 is a perspective view of the device. The sample is applied to the sample port 12 after the bladder 14 has been compressed. Obviously, the area of the layer 26 and/or the layer 28 close to the cutout portion of the capsular bag 14 must be elastic to allow the capsular bag 14 to be compressed. About 0.1 mm thick polyester has appropriate elasticity and elasticity. Preferably, the thickness of the top layer 26 is about 0.125 mm, and the thickness of the bottom layer 28 is about 0.100 mm. When the pouch is released, suction draws the sample through the tube 16 to the measurement area 18, which preferably contains a reagent 20. To ensure that the measurement area 18 can be filled with samples, the volume of the bladder 14 is preferably at least approximately equal to the sum of the volume of the pipe 16 and the measurement area 18. If the measurement area 18 is illuminated from below, the layer 28 must be transparent in the portion close to the measurement area 18. For coagulation tests, reagent 20 contains thrombogenic substances, which do not have swelling reagents normally found in freeze-dried reagents.

As shown in Figures 1, 2 and 3, the stop joint 22 is adjacent to the bladder 14 and the measurement area 18; Zone 18 and/or pocket 14 are separated. When the sample reaches the stop joint 22, the flow stops. For blood coagulation measurement, it is very important to stop the flow of the sample at this point to allow the renewable money string-like red blood cell tandem structure. This step is an important step for monitoring blood coagulation using the method of the present invention. Here, it is indicated that the formation of the string-shaped red blood cell structure is reversible, and the string-shaped red blood cell series generated in the sample port earlier is eliminated as the blood passes through the pipe 16. The operating principle of the stop joint is disclosed in U.S. Patent No. 5,230,866, which is incorporated herein by reference.

As shown in FIG. 2, all the aforementioned elements are formed by the cutouts in the middle layer 24 sandwiched between the top layer 26 and the bottom layer 28. The layer 24 is preferably a double-sided adhesive tape. The stop joint 22 is formed by an additional cut-out portion in the layer 26 and/or 28, which is aligned with the cut-out portion in the layer 24 and sealed with the sealing layer 30 and/or 32. As shown in the figure, the stop joint preferably includes cutouts in both layers 26 and 28, and has sealing layers 30 and 32. Each cut-out portion used by the stop joint 22 is at least as wide as the pipe 16. FIG. 2 also shows an optional filter membrane 12A covering the sample port 12. The filter membrane can separate red blood cells from the whole blood sample and/or can contain a reagent that interacts with the blood to provide additional information. For the reasons explained below, red blood cells must be visible from "below", so if the filter membrane filters out red blood cells, it must be transparent. The optional reflector 18A may be located on or near one of the surfaces of the layer 26 and positioned above the measurement area 18. If there is the reflector, the device becomes a penetrating reflector.

The method of using the strips shown in Figures 1, 2 and 3 can be understood with reference to the schematic diagram of the meter element shown in Figure 4. The first step performed by the user is to activate the meter, thereby supplying energy to the strip detector 40, the sample detector 42, the measurement system 44, and the optional heater 46. The second step is to insert the strip. The strip is preferably not transparent at least in its partial area. The strip inserted in this way can block the illumination of the light-emitting diode 40a of the detector 40b (a better way is that the intermediate layer is made of opaque material, so that the background light Will not enter the measurement system 44.). The detector 40b thereby senses that a strap has been inserted and triggers the capsular bag actuator 48 to compress the capsular bag 14. Then, the measurement display 50 instructs the user to apply the sample to the sample port 12, which is the third and final step that the user must perform to start the measurement procedure.

It is important to operate the device appropriately to sense that a "correct (appropriate)" sample (ie whole blood) has been applied. Therefore, the meter must not report the measurement result when a non-whole blood sample causes the detector 42b to detect a change in light. These changes may be caused by the movement of the strip, an object (such as a finger) running near the sample port, or even the application of serum to the sample port 12. Everything can cause wrong results. To avoid this type of error, a preferred method of the present invention involves illuminating the sample port 12 with a light-emitting diode 42a and measuring diffuse reflection (ie, "scattered") light with a detector 42b positioned orthogonal to the plane of the strip 10 . If a whole blood sample has been applied to the sample port 12, the signal detected by the detector 42b will increase sharply due to the scattering in the blood sample, and then will be weakened as red blood cells begin to pile up into a string of money (a string of red blood cells in series) .

Fig. 5 shows that the intensity of the scattered light (I) rapidly increases and then decreases as a function of time (t). This is a characteristic curve A of a blood sample. The figure also shows curve B, which is a characteristic curve of a non-whole blood sample.

In another embodiment, as shown in FIG. 4A, the transmitted light is measured instead of scattered light. In this case, the string-like red blood cell structure phenomenon causes the detected signal to rapidly weaken and then increase (that is, the inversion of curve A).

The detector system 42 is programmed to first request a whole blood signal of the type shown in FIG. 5 (curve A or its inversion, depending on the situation), and then cause the actuator 48 to release the bladder 14 to allow the sample to enter the tube 16. This of course requires a delay time (preferably at least 5 seconds) than if the sample is passed without first determining whether the sample is whole blood. However, the delayed-release pouch 14 generally does not affect the readings described below. The release of the bladder 14 causes suction in the pipe 16 to draw the sample through the measurement area 18 to the stop joint 22. The light from the light-emitting diode 44a passes through the measurement area 18, and the detector 44b monitors the light transmitted through the sample in the coagulated blood. When there are a plurality of measurement areas, the measurement system 44 includes a light-emitting diode/detector pair (like 44a and 44b) for each measurement area. The analysis of the transmitted light versus time function (described below) can calculate the clotting time, which is displayed on the metering display 50. The temperature of the sample is preferably maintained at about 37°C by the heater 46.

In another embodiment, the capsular bag 14 is released under any circumstances, but the measurement of the concentration/physical characteristics of the analyte becomes valid only when the detector 42 detects the sign of the sample. If the sign is not detected, the user will see an error signal on the display 50.

Figure 6 depicts a typical "clot signature" curve in which the current from the detector 44b is plotted as a function of time. The blood is first detected in the measurement area by the detector 44b at time 1. In the time interval A between points 1 and 2, blood fills the measurement area. The decrease in current in this time interval is due to the scattering of light by red blood cells, so it is an approximate measurement of blood cell volume. At point 2, the sample has filled the measurement area and stopped flowing, and its movement has been blocked by the stop joint. Then the string-like red blood cell structure in series allows more and more light to pass through the sample (and less scatter) in the time interval between points 2 and 3. At point 3, blood clot formation terminates the string-like red blood cell tandem structure and the amount of transmission through the sample reaches its maximum. The clotting time can be calculated from the interval B between points 1 and 3 or the interval between points 2 and 3. After that, the blood changes from liquid to semi-solid gel, and the amount of light transmission is correspondingly reduced. The decrease in current C between the maximum value 3 and the end point 4 is related to the fibrinogen in the sample.

The device depicted in FIG. 2 and described above is preferably composed of thermoplastic plastic sheets 26 and 28 laminated on both sides of the thermoplastic plastic intermediate layer 24 with adhesives. The cut-out portion forming the element shown in FIG. 1 can be formed by laser cutting or die cutting of the layers 24, 26, and 28, for example. Another option is that the device can be constructed of molded plastic. The surface of the plastic sheet 28 is preferably hydrophilic. (Plastic film 9962 produced by 3M Company in St. Paul, Minnesota.) However, the surface does not have to be hydrophilic because the sample fluid can fill the device without capillary force. Therefore, the plastic sheets 26 and 28 may be untreated polyester or other conventional thermoplastic plastic sheets. Similarly, since there is no gravity during filling, the device has to be used in any direction. Unlike the capillary action filling device with vents that may leak the sample, the device of the present invention vents through the sample port before the sample is applied, that is, the strip portion first inserted into the meter does not have any openings, thus reducing the risk of contamination.

FIG. 7 is a plan view of another embodiment of a device suitable for the method of the present invention, wherein the device includes a bypass pipe 52 connecting the pipe 16 and the bag 14. 7A, 7B, and 7C can know the function and operation of the bypass pipe. The three figures show the time sequence of drawing samples into the device 10 for measurement.

FIG. 7A illustrates the situation after the user applies the sample to the strap when the bag 14 is compressed. This can be done by applying one or more drops of blood. The sample remains there while the meter determines whether the sample contains whole blood. If the sample contains whole blood, release the compression of the capsular bag.

Figure 7B depicts the situation after the pouch has been uncompressed. The resulting pressure drop in the inlet pipe 16 starts to draw the sample into the measurement area 18. When the sample reaches the stop joint 22, the sample encounters a back pressure that stops the sample and causes additional samples to be drawn into the bypass pipe.

Figure 7C depicts the situation when a reading is taken. The sample stops in the measurement area 18. The sample also fills part or all of the pipe 16 (as shown in the figure).

Figure 8 depicts a preferred embodiment of a device suitable for the method of the present invention. The device is a multi-pipe device including a bypass pipe 152. The function of the bypass pipe 152 in the device is similar to the function of the bypass pipe 52 of the device in FIG. 7 described above. The measurement area 118 is filled with thrombogenic substances. Preferably, the measurement areas 218 and 318 preferably contain a control substance, preferably the control substance described below. The measurement area 218 contains thrombogenic material, bovine eluate, and recombinant factor VIIa. The selected component is to normalize the clotting time of the blood sample by counteracting the effect of an anticoagulant such as warfarin. The measurement area 318 contains thrombogenic substances and bovine eluate to locally overcome the effects of anticoagulants. Therefore three measurements are made on this strip. The main measurement, that is, the clotting time of the sample, is measured on the measurement area 118. However, the measurement value is valid only when the measurement results on the measurement areas 218 and 318 are within a predetermined range. If either or both of these control measurements are outside the range, it means that the test needs to be re-tested. The extended stop joint 122 stops the flow in all three measurement zones.

The following examples demonstrate devices suitable for the method of the present invention, but do not have any limiting meaning.

Example one

A method of tape suitable for the method of the present invention is as follows: First, make a double-sided adhesive tape sandwiched between two release liners (RX 675SLT produced by Scapa Tapes, Windsor, Connecticut) Through a stacking and rotating punching processing system. Cut the top release liner and tape through the pattern shown in Figure 7 (except for the stop joint), but do not cut through the bottom release liner. The bottom release liner is then removed as waste along with the cut from the tape. The hydrophilic polyester film (plastic film 3M9962 produced by 3M Company in Sao Paulo, Minwei State) was laminated on the exposed bottom side of the tape. Then the reagent (thrombogenic substance produced by Ortho Clinical Diagnostics in Narita, New York) was printed by inkjet printing (using the product produced by Hewlett Packard, Oregon) Printing head 51612A] is printed on the reagent area (18) of the polyester film. Untreated polyester film (Adhesives Research Company, Canyon Rock City, Pennsylvania) AR1235 produced by Research) Cut out the sample port, then align and superimpose on the top of the double-sided adhesive tape (after the release liner has been removed from the tape). Then a punch punches the three stacked layers to cut out the stop joint. Finally, a single-sided adhesive tape (MSX4841 produced by 3M Company in St. Paul, Minnesota) was applied to the outside of the polyester layers to seal the stop joint.

Example two

Here, follow the procedure similar to the previous example 1 to make a stripe of the type depicted in FIG. 8. Among them, the reagents sprayed on zones 118, 218 and 318 are; thrombogenic substance; thrombogenic substance, bovine eluate and recombinant factor VIIa; thrombogenic substance and bovine eluate. The cattle eluate (plasma barium citrate cattle eluate) can be obtained from Haemotologic Technologies in Burlington, Vermont; and the recombinant factor Vlla can be obtained from the American Medical Diagnostic Center in Greenwich, Connecticut Obtained.

The measurement of a whole blood sample using this example strip produces a curve of the type shown in FIG. 6 for each measurement area. The data from the curve of the control area (measurement areas 218 and 318) is used for the data of the curve of the nuclear eyelid measurement area 118. Therefore, it is possible to obtain a more reliable clotting time than a band with a single measurement area.

The above describes the details of the present invention. It is obvious to those skilled in the art that many modifications and changes can be made without departing from the spirit and scope of the present invention.