WO2004063722A1

WO2004063722A1 - Method and apparatus of hemorheometer

Info

Publication number: WO2004063722A1
Application number: PCT/KR2003/002862
Authority: WO
Inventors: Sehyun Shin
Original assignee: Kyungpook National University Industry-Academy Cooperation Foundation
Priority date: 2003-01-08
Filing date: 2003-12-27
Publication date: 2004-07-29
Also published as: AU2003288762A1

Abstract

An apparatus and method for determining the aggregation of red blood cells and the viscosity of blood over plural shear rates by measuring the light intensity and pressure differential over time. The apparatus and method utilize a slit, a sample reservoir, a waste sample collector, a vacuum generator, a light source, a light sensor and a pressure sensor, such as a precision differential pressure transducer. In the apparatus and method, sample liquid filled in the sample reservoir is flowing through the slit due to the pressure difference generated by the vacuum generator. Then, the light from the light source is irradiated on the blood sample and the amount of transmitted light is detected by the light sensor to determine blood cell aggregation. Simultaneously, the blood viscosity can be determined by measuring the decreasing pressure differential with the pressure sensor.

Description

METHOD AND APPARATUS OF HE ORHEO ETER

Technical Field

The present invention relates to the measurement of he orheological characteristics, and more particularly to a method and apparatus for simultaneously measuring the viscosity and cell aggregation of blood in a quasi-equilibrium state at each moment while measuring a reduction in driving pressure with time at each time.

Background Art

As blood cell aggregation is known as a factor having a direct effect on the viscosity and rheological characteristics of blood, the development of an apparatus for measuring the blood cell aggregation has been attempted. Particularly, a laser-assisted optical rotational cell analyzer (LORCA) disclosed in Clinical Hemorheology and Microcirculation, Vol. 21, pp. 1-11, 2001 is a technology for measuring the blood cell aggregation, wherein laser beam is irradiated on a blood sample_, at a rotational Couette flow condition in a double concentric circular tube structure, the backscattered light is collected by a photodiode sensor, and the intensity of the collected light is computerized. In this case, to minimize the blood cell aggregation before the measurement, a shear rate of at least 500 (1/s) is maintained for 5 seconds, and then the rotary motion is suddenly stopped. Also, in this measurement, an increase in blood cell aggregation with time is measured by the intensity of the backscattered light. This measurement is meaningful as a syllectogra for determining a change in blood cell aggregation with time, but it has a shortcoming in that the blood cell aggregation at an actual shear rate for blood flow cannot be directly measured.

Furthermore, another method for measuring the blood cell aggregation was disclosed in Oguz K. Baskurta, H.J. Meiselmanb, Ercument Kayara et al . , "Measurement of red blood cell aggregation in a plate-plate shearing system by analysis of light transmission", Clinical Hemorheology and Microcirculation, Vol. 19, pp. 307-314, 1998. In this method, a blood sample is put between two circular plates, and the lower circular plate is rotated at a suitable speed to minimize the blood cell aggregation, and then the rotary motion is suddenly stopped. The light from a light source is irradiated on the blood sample to transmit through or backscatter from the sample, the transmitted light is collected by a light sensor, and an increase in blood cell aggregation with time is measured by the intensity of the collected light. This measurement of blood cell aggregation using the light transmission or backscattering technique was already known in the art .

Meanwhile, Korean Patent Application No. 10-2003-0000939 discloses a vacuum viscometer using a precise pressure sensor, wherein fluid is transported from a sample reservoir via a capillary tube to a waste sample reservoir with the application of vacuum, while slow release of the applied vacuum pressure is measured with a pressure sensor and converted into viscosity. Moreover, Korean Patent Application No. 10-2003-0041026 entitled "a hemorheometer" discloses an apparatus for measuring blood cell aggregation, wherein the operational principle of the vacuum viscosity disclosed in Korean Patent Application No. 10- 2003-0000939 is applied to a slit-shaped flow restrictor, while the light from a light source is irradiated on a blood sample, and the amount of the backscattered light is detected by a sensor to determine blood cell aggregation. However, the above-mentioned LORCA and the hemorheometer disclosed in Korean Patent Application No. 10-2003-0041026 have shortcomings in that at least two separate light sensors are required to detect the backscattered light, and noise significantly affects the measurement since very small amounts of light signals backscattered from blood cells should be detected. Also, in the LORCA, a technique for detecting the backscattered light is unavoidably applied because the outer circular tube of the double concentric circular tube structure rotates, such that it is difficult to attach a sensor on the rotating location. Moreover, in the existing measurement apparatuses, a portion which was in contact with a blood sample should be washed after the measurement, such that it is difficult to use the apparatuses in the diagnostic field.

Disclosure of Invention

Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and an object of the present invention is to provide a hemorheometer which can simultaneously measure blood viscosity and blood cell aggregation for a very small amount of a blood sample over a range of interest shear stresses and shear rates by one measurement in a very short time, as well as a measuring method using the same.

Another object of the present invention is to provide a hemorheometer which has a simple structure and production costs and is easily operated such that it can be used as a disposable product, as well as a measuring method using the same. The above objects can be accomplished by providing the following method for measuring blood cell aggregation. Brief Description of Drawings

FIG. 1 is a block diagram schematically showing the structure of a hemorheometer according to the present invention; FIG. 2 is a schematic diagram of a hemorheometer according to the present invention;

FIG. 3 is a graphic diagram showing a change in pressure with time;

FIG. 4 is a graphic diagram showing the measured results of blood viscosity, obtained from the measurement shown in FIG. 3, as a function of shear rate;

FIG. 5 is a graphic diagram showing the structure of a hemorheometer for measuring blood cell aggregation according to the embodiment of FIG. 1; FIG. 6 is a graphic diagram showing the measured results of blood cell aggregation, obtained by the embodiment of FIG. 5, as a function of shear rate;

FIG. 7 is a schematic diagram showing disposable parts of the apparatus of FIG. 1, in a disassembled state; FIG. 8 is a schematic diagram showing a disposable sample reservoir in the apparatus of FIG. 1; and

FIG. 9 is a schematic diagram of a vacuum generator in the apparatus of FIG. 5.

Best Mode for Carrying Out the Invention

The present invention is directed to a hemorheometer using variable driving pressure and a slit-shaped flow restrictor. FIG. 1 is a block diagram showing the schematic structure of the hemorheometer according to the present invention, and FIG. 2 is a schematic diagram showing the structure of the hemorheometer according to the embodiment of FIG. 1. Referring to FIGS. 1 and 2, the hemorheometer of the present invention comprises: a sample reservoir 10 in which one blood sample 15 is injected and stored; a slit-shaped flow restrictor 21 whose one end is connected in flow communication with the sample reservoir such that the liquid sample is introduced into and passed through the flow restrictor 21 while generating high flow resistance; a waste sample collector 23 which is connected in flow communication with the other end of the flow restrictor such that the liquid sample flowing out of the flow restrictor is stored in the waste sample collector 23; a vacuum generator 40 for applying a lower pressure than atmospheric pressure to the waste sample collector 23 via a connecting tube 24 and a valve unit 25; a pressure sensor 27 whose one end is connected with the vacuum generator 40 and which is adapted to continuously measure a change in pressure with time; a light source unit 61 which is attached to one side of the flow restrictor; a light sensor unit 64 for detecting the light transmitted through the liquid sample; a processor 51 where values measured in the pressure sensor 27 and the light sensor unit 64 are stored, calculated and processed; a display 52 for displaying the data from the processor on a screen; a storage unit 53 for storing the data; and an output unit 54 for outputting the data.

Hereinafter, the operational principle of the hemorheometer according to the present invention, and a measuring method using the same, will be described in detail with FIGS. 1 and 2.

First, the liquid blood sample 15 is injected into the sample reservoir 10. At this time, a portion of the liquid sample injected into the sample reservoir also flows into the flow restrictor 21 connected to the sample reservoir 10, due to a capillary effect. Then, the vacuum generator 40 operates to generate a lower vacuum pressure than atmospheric pressure while the generation of the vacuum pressure is measured by the pressure sensor 27 via the connecting tube. When the waste sample collector 23 is opened by the valve unit 25, driving pressure occurs due to the difference between the atmospheric pressure of the sample reservoir and the vacuum pressure of the waste sample collector, so that the liquid sample is pulled up from the sample reservoir into the waste sample collector through the flow restrictor. In this case, the waste sample collector is a closed container. Thus, as the liquid sample flowing out of the flow restrictor gradually flows into the waste sample collector, the internal vacuum of the waste sample collector is released and finally the pressure of the waste sample collector equilibrates with the sum of atmospheric pressure and the water head of the flow restrictor 21 so as to stop the flow of the liquid sample. During this process, the light from a light source 61 (e.g., laser diode or light-emitting diode (LED) ) attached to one side of the flow restrictor is irradiated on the blood sample, and the light transmitted through the blood sample is detected by the light sensor 64 (e.g., photodiode). The detected light is converted into an electrical signal and stored in the processor 51. At this time, the pressure value with time measured by the pressure sensor 27 is also stored in the processor and converted into shear rate, shear stress and viscosity. The electrical signal transmitted from the light sensor 64 is analyzed in the processor and converted into blood cell aggregation, and the converted value is provided on a display such as a screen.

FIG. 3 is a graphic diagram showing a change in pressure with time, measured according to the present invention. In this case, the difference between atmospheric pressure and the pressure of the waste sample collector connected with the vacuum generator is measured with time. The pressure difference is gradually reduced from high initial pressure difference, and upon the end of the measurement, the pressure of the waste sample collector equilibrates with atmospheric pressure.

Meanwhile, the pressure difference between the sample reservoir and the waste sample collector is measured and converted into volume by the ideal gas equation for air, and a change in volume with time can be converted into the flow rate at any moment. Thus, the pressure difference corresponding to the driving pressure, and the flow rate calculated therefrom, can be converted into shear rate and shear stress by the known equation.

Meanwhile, in order to remain the liquid level of the sample reservoir 10 substantially unchanged until the end of the measurement, a reservoir having large bottom area as shown in FIG. 2 is preferably used as the sample reservoir 10. Moreover, as shown in FIG. 2, one end of the flow restrictor is preferably maintained higher than the bottom surface of the waste sample collector, such that there is no change in the water head of the waste sample collector even when the waste sample is filled in the waste sample collector. Namely, a difference in water head until the end of the measurement can be preferably calculated based on only the length of the flow restrictor. In other words, the water head is expressed as pgL wherein p is the density of the liquid sample, g is the acceleration of gravity, and L is the length of the flow restrictor. Hereinafter, the principle for converting the pressure measured in the present invention into shear rate and shear stress will be described with reference to the time-pressure graph shown in FIG. 5. If the measured pressure is the pressure difference (ΔP) between the atmospheric pressure of the sample reservoir and the vacuum pressure of the waste sample collector, it will be reduced with time and then the final pressure of the waste sample collector will equilibrate with atmospheric pressure .

If the ideal gas equation is applied to the air pressure and volume of the waste sample reservoir using the pressure difference measured as described above, the internal volume (V) at each time can be calculated.

As the liquid sample is transported into the waste sample reservoir through the flow restrictor, the pressure P_w(t) of the waste sample reservoir is gradually increased with time while the internal air volume V_w(t) of the waste sample collector is reduced. In this case, since the pressure P (t) is a value either measured by the pressure sensor or converted, the air volume V_w(t) of the waste sample collector at each time can be calculated by the above equation. A reduction in the internal air volume of the waste sample collector as calculated above equals to an increase in the volume of the introduced liquid sample .

Meanwhile, linear differentiation of a change in volume of the liquid sample with time makes the volume flow rate (Q) of the test fluid passed through the flow restrictor per unit time.

Q = [ΔVliq/Δt ]

Using the driving pressure and flow rate at both ends of the flow restrictor, the shear rate (γ) can be calculated by the following equation on the assumption that the flow restrictor is a rectangular channel whose height, width and length are H, W and L, respectively: γ = (1/3) [6Q/(WH²)] [2+ {d(ln Q)/d(ln τ) }]

The shear stress (τ) is calculated by the following equation: τ = [ΔP(t)H/L]/[ (1+2H/W)]

Although the equations for calculating the shear rate and the shear stress were derived only for the flow restrictor 21, such as a rectangular channel or slit-shaped tube, engineering equations for calculating the shear rate and the shear stress for tubes, such as a circular tube, were also known, and can be used to calculate the shear rate and stress by the same principle.

FIG. 4 is a graphic diagram showing blood viscosity and shear rate which were converted from the pressure shown in FIG. 3 by the above principle. The graph in FIG. 4 directly shows the characteristic of the inventive hemorheometer in that blood viscosities over a wide range of shear rates can be obtained by only one measurement in a short time of 1-2 minutes.

FIG. 5 shows the structure of the hemorheometer for measuring blood cell aggregation according to the embodiment shown in FIG. 1. The light from a point light source 61, such as a laser diode, is irradiated on the flow of the liquid sample through one optically transparent side of the flow restrictor 21. A portion of the irradiated light is backscattered by the blood cells and aggregated blood cells contained in the fluid, and the remaining portion is transmitted through the fluid, and collected by the light sensor 64 attached to the opposite side to the light source 61.

In this case, the aggregation of blood cells varies depending on the shear stress of the flow within the shear flow fields, while the light irradiated from the light source is transmitted at an amount varying depending on the aggregation of blood cells so that the intensity of the light detected by the light sensor is measured to be blood cell aggregation. Here, as the shear rate of the flow is reduced, the aggregation of the blood cells is increased and the light irradiated on blood is mostly transmitted so that the intensity of the light collected by the light sensor shows a high value. On the other hand, as the shear rate is increased, the aggregation of blood cells is reduced and the blood cells are separately present, in which case the light irradiated on blood is mostly backscattered and the transmitted light is reduced so that the intensity of the light collected by the light sensor has a relatively low value.

Particularly, in the inventive apparatus, the flow rate and shear rate have high values at an initial stage and then reduce and approach a value of zero over time. Thus, the shear rate is reduced with time so that the blood cell aggregation and also the intensity of the transmitted light are increased.

FIG. 6 is a graphic diagram which shows a light intensity- shear rate graph measured according to an embodiment of the apparatus for measuring blood cell aggregation shown in FIG. 5, as a function of shear rate. In this case, an index of light intensity is the amount of transmitted light, and can be used as an index of blood cell aggregation. As known in the art, the measured blood cell aggregation can also be converted into erythrocyte sedimentation rate (ESR) .

As shown in FIG. 7, parts of the hemorheometer which are in contact with blood are all made of a disposable product. In this case, in order to maintain the connection between the flow restrictor 21 and the waste sample collector in an airtight state, a silicon tube 22 is inserted around the flow restrictor 21 and then inserted into the waste sample collector. The sample reservoir 10, the flow restrictor 21 and the waste sample collector 23, which are parts of the inventive hemorheometer coming in direct contact with the liquid sample as shown in FIGS. 1 and 2, can be made of a disposable product in an integral or assembled form, using a material selected from the group consisting of silicon, quartz, silica, glass, laser- processable polymer, injection-molded polymer and ceramic, by a suitable process. For example, using a plastic material as a substrate, a structure as shown in FIG. 7 can be easily made in an integral form by a microinjection technique. This plastic substrate is very suitable for disposable use in terms of economic efficiency, such that if it is used for the measurement of a sample, such as blood, which can be contaminated with a virus, it can be discarded after the measurement.

As shown in FIG. 8, the sample reservoir 10 is designed in such a manner that one end of the flow restrictor can be tightly inserted into and supported by a hole 14 formed in the cover 12 of the sample reservoir. Also, for exposure to atmospheric pressure, another hole 13 is formed in another portion of the cover 12. This cover 12 is securely assembled with a container 11 having large bottom area. The liquid sample, such as blood, is injected into the reservoir through the hole 13 using a device, such as a syringe. Furthermore, as shown in FIG. 2, the flow restrictor 21 inserted and fixed in the sample reservoir 10 is inserted and fixed in a body 30 in a simple one-touch operation, and maintained at a sealed state after the fixing. Also, to make assembling and disassembling easy, the flow restrictor is designed in such a manner that it is spaced apart from the bottom surface of the body at a given interval.

FIG. 9 is a schematic diagram showing an embodiment of the vacuum generator shown in FIG. 1. A step motor 42 is connected to a device 43, such as a linear motion (LM) guide, such that when the device 43 is moved backward by a given amount under the control by the processor 51, vacuum pressure is formed in a piston-cylinder device or syringe 41. The formed vacuum pressure is transferred into the waste sample collector 23 and the pressure sensor 27 through the connecting tube 26. Meanwhile, since the rheological characteristic of liquid greatly varies depending on temperature, the measurement temperature of liquid needs to be controlled. Thus, in the present invention, the substrate may be inserted into a heat exchanger having a water jacket such that it can be heated or cooled. Alternatively, a thermoelectric component may be attached to the substrate, or a halogen lamp can be included such that the substrate can be preheated to a predetermined temperature .

Industrial Applicability

As described above, the present invention has the effect of simultaneously measuring viscosity and blood cell aggregation for a blood sample in a very short time, and also the effect of collectively measuring viscosity and blood cell aggregation at a wide range of shear flow fields for a very small amount of a blood sample in a very short time. Particularly, in the inventive apparatus, the sensor does not come in direct contact with the liquid sample, and portions coming in contact with the liquid sample can be all made of a disposable product, such as a disposable kit, so that the inventive apparatus is very suitable for real-time clinical application in the diagnostic fields.

Although a preferred embodiment of the present invention has been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims .

Claims

What Is Claimed Is:

1. A hemorheometer for measuring the blood cell aggregation of the circulating blood of a living body at plural shear rates while measuring a reduction in pressure with time, the hemorheometer comprising: a flow restrictor; a sample reservoir which is connected in flow communication with one end of the flow restrictor and in which a liquid sample is injected and stored, the water head of the sample reservoir being maintained at a constant level for measurement time; a waste sample collector which is connected in flow communication with the other end of the flow restrictor and in which the liquid sample flowing out of the flow restrictor is received and stored, the waste sample collector being designed in such a manner that the end of the flow restrictor inserted therein is always maintained above the level of the waste liquid sample; a differential pressure sensor, coupled to the waste sample collector, for measuring the difference between the pressure of the waste sample collector and atmospheric pressure; a vacuum generator for generating a lower pressure than atmospheric pressure; a valve unit for opening/closing the vacuum generator, the waste sample collector and the pressure sensor by an external controller; a light source placed at one side of the flow restrictor tube; a light sensor for detecting the light transmitted through the blood sample from the light source; a processor, coupled to the pressure and light sensors, for calculating the shear stress of the sample fluid over plural shear rates from the relation between given geometric parameter and pressure, using a pressure signal at each time, and for calculating the blood cell aggregation by analyzing the detected transmitted light at each time; and a display for providing the viscosity and blood cell aggregation calculated by the processor.

2. The hemorheometer of Claim 1, wherein the flow restrictor is a rectangular channel.

3. The hemorheometer of Claim 1, wherein a differential pressure generator for driving the sample fluid in the fluid restrictor consists of a vacuum generator for producing a lower pressure than atmospheric pressure.

4. The hemorheometer of Claim 1, wherein the light source is selected from the group consisting of a laser diode and a light-emitting diode (LED) .

5. The hemorheometer of Claim 1 or 2, wherein a portion of the hemorheometer coming in contact with blood, including the flow restrictor, is made of a material selected from the group consisting of silicon, quartz, silica, glass, laser-processable polymer, injection-molded polymer and ceramic.

6. The hemorheometer of Claim 1, wherein a portion of the hemorheometer coming in direct contact with the liquid sample is made of a disposable material.

7. The hemorheometer of Claim 1, wherein the light sensor is selected from the group consisting of a photodiode, a CCD sensor, a digital camera and a high-speed CCD video.

8. The hemorheometer of Claim 1, which is attached with a heater capable of preheating the hemorheometer to a predetermined temperature for receiving the liquid sample, and a temperature sensor.

9. The hemorheometer of Claim 1, wherein the flow restrictor is a circular tube.