JPH0252220B2 - - Google Patents

Description

Claim 1: comprising an absorbent strip and a membrane, the membrane having a 3.0
having capillary-like pores having an average pore size in the range of 8.0 to 8.0 microns, the membrane and the strip being in at least partial surface contact at one end of the strip; 4.0×10 ⁵ ±15% pore number/
A red blood cell deformability measuring device characterized by having a pore density of cm ² , a tensile strength of at least 3000 psi, and a specific gravity of 0.94 to 0.97 gm/cc. 2. A device according to claim 1, characterized in that the average pore size of the pores is 47±0.2 microns. 3 one side of the membrane is in surface contact with the absorbent strip;
3. The device according to claim 2, wherein the other surface of the membrane is in partial surface contact with a transparent membrane. 4. Device according to claim 3, characterized in that the absorbent strip and the membrane are adhered to an adhesive support layer. 5. Device according to claim 4, characterized in that the transparent membrane is adhered to an adhesive support layer. 6. Place the blood sample on a membrane with capillary-like pores having an average pore size of 3.0 to 8.0 microns so that the blood passes through the membrane into at least partial surface contact with the membrane by capillary action. blood permeates the absorbent strip and flows laterally along the absorbent strip for a predetermined distance, the time required for the blood to travel the predetermined distance laterally is measured, and the membrane is nearly flat on the surface of the membrane.ゞPerpendicular linear through pores and a pore density of 4.0×10 ⁵ ±15% pore number/cm ² ,
Tensile strength of at least 3000psi and 0.94 to 0.97g
A method for measuring deformability of red blood cells characterized by having a specific gravity of m/cc. 7. Process according to claim 6, characterized in that the average pore size is 4.7±0.2 microns. Description The present invention relates to a method and apparatus for rapidly determining the level of deformability of human red blood cells. The invention further relates to a method and apparatus for rapidly measuring the relative viscosity of fluids. The deformability of red blood cells is an important parameter in determining whether red blood cells are properly performing their functions, particularly regarding oxygen supply to tissues and removal of metabolites. The functions of these red blood cells occur primarily in the capillaries of the peripheral circulatory system. The average diameter of a healthy red blood cell is approximately 7.5 microns, but it is known that when passing through capillaries, it deforms to approximately 3 microns without hemolysis. Because red blood cell deformability coincides with the onset of many diseases, such as sickle cell anemia, diabetes, and various heart diseases, the ability of red blood cells to pass through capillaries is used as a measure of red blood cell deformability. At the same time, it can be used as an indicator of abnormal red blood cells. It is known that in order for healthy red blood cells to pass through the body's microcirculatory capillary system, these red blood cells need to undergo significant deformation. Lessin et al. reported that normal human red blood cells have greater deformability than those of people with sickle cell disease, hereditary spherocytosis, and sickle cell anemia (Blood Cells 3 , 241-
262, 1977). The method for measuring deformability of red blood cells published by Lessin et al. uses a positive pressure cell filtration system to filter a suspension of red blood cells through a microfilter.
Subsequently, the resistance of the suspended substance as it passes through the microfilter is measured by changing the flow rate to cause a pressure change. However, Lessin
This system by others is complex and expensive, and
It has the disadvantage that measurement takes time. Reid et al. have published a method for measuring the deformation rate of red blood cells by the amount of red blood cells that pass through a membrane filter per minute (J. Clin. Pharm. 29 , 855-858, 1976).
This system by Reid et al. requires the use of whole blood, and filtration must be performed with negative pressure to draw the blood through a membrane filter. This system is expensive because it requires a vacuum device, takes some time to measure, and has the disadvantage that the pressure drop tends to change during filtration, making it difficult to make accurate measurements. . Therefore, when using the system of Reid et al., care must be taken at all times to maintain a relatively constant negative pressure on the suction device. Other methods for measuring the deformability of red blood cells include:
Iclopipette method by Rand (Biophysics
Journal, 4 , 155, 1964), and Bessis and
A measurement method using a laser deflection instrument called an ectasytometer by Mohandas (Blood Cells
1 , 307-314, 1975). Instruments for measuring the deformability of red blood cells include a red blood cell rigidometer and an optical microscope by Kiesewetter (Scand.J.Clin.Lab.Invest.41, 229-232,
1981) has been published. Co-pending U.S. application filed August 31, 1981
No. 298195 describes a red blood cell deformability monitor consisting of a series of specialized filters with different pore sizes. In this case, the red blood cells pass through the filter and are absorbed into the absorbent material at the end of the filter. With the exception of the method described in co-pending application no. 298195, all of the methods described above involve various specialized equipment and accessories. All of these devices are complex and expensive. The red blood cell filtration device of the present invention is advantageous because it does not require specialized electronic equipment or its accessories. Although the method of co-pending application No. 298195 does not use special and expensive equipment, the method of measurement is more complicated than that of the present invention. The co-pending application has a series of filters with different pore sizes, allowing for quantification of deformability. In contrast, the present invention is advantageous because it allows quantitative measurement of the flow rate of fluid through the filter membrane, in particular fluid containing red blood cells. An object of the present invention is to provide a simple, rapid and convenient method for quantitatively evaluating the deformability of healthy and abnormal red blood cells. A further object of the present invention is to provide a completely self-contained and disposable red blood cell deformability quantitative measuring device. A further object of the present invention is to provide a red blood cell deformability quantitative measuring device that can quantitatively measure the flow rate of a fluid, particularly a fluid containing red blood cells, using a single filter. According to the invention, the flow rate of a fluid, in particular the deformability of red blood cells, can be determined by placing a sample of fluid, in particular blood, on a membrane characterized by pores with an average diameter of 3.0 to 8.0 microns, and by capillary action the fluid (blood ) permeates through the membrane and into an absorbent strip that is in at least partial surface contact with the membrane and flows along the absorbent strip for a predetermined distance, such that the fluid (blood) It is measured by measuring the time taken. FIG. 1 is an exploded view of one embodiment of a test strip according to the present invention. FIG. 2 is an assembled view of the test strip of FIG. FIG. 3 is a plan view of an embodiment of the present invention in which a test strip is combined with a plurality of absorbent strips. The device of the invention is preferably utilized for measuring the filtration rate of red blood cells, but can be generally applied to flow measurement of any fluid to determine fluid viscosity. The amount of fluid (blood) sample required for the device of the invention is 10 microliters. Another advantage of the present invention is that the amount of sample required is small, as conventional blood filtration measurement devices require a sample of about 100 microliters. The red blood cell filtration device according to the invention comprises an absorbent strip and a membrane, both of which are at least partially in surface contact with each other. Membranes are thin films with strictly defined pore sizes. The membrane is preferably made of a special polycarbonate material having tightly sized capillary pores and having a thickness of approximately 10 microns manufactured according to the method disclosed in U.S. Pat. No. 3,303,085. It is a thin film. The membrane was manufactured by Hema-
It is commercially available under the name fil ^M. The average pore size of the membrane may be in the range of 3.0 to 8.0 microns, with a preferred average pore size of about 5 microns. The pores of the Hema-fil ^M membrane run straight through and perpendicular to the surface within ±34° and are evenly and randomly distributed over the surface. This precise placement of the pores ensures even deposition of the sample over the surface of the membrane, allowing continuous filtration to accurately size the particles. The membrane is patented
Since it is produced in a two-step process as described in No. 3303085, it is possible to obtain membranes with specific average pore sizes and densities. Hema-fil ^M
The actual maximum pore size of the membrane is 4.7±0.2 microns. The thickness variation of the Hema-fil ^M membrane is less than 5% from the nominal thickness of 10 microns. The pore density (number of pores/cm ² ) and pore size of the Hema-fil ^M membrane are tightly and specifically controlled to provide the best combination of strength and flow rate. The range of pore density is 4.0×10 ⁵ (±15
%) Number of pores/ ^cm2 . This membrane is flexible;
Does not tear easily and has a tensile strength of at least 3000 psi. The empty weight of the membrane is very low and constant. For a given pore size, the weight change is ±5
Do not exceed %. The nominal weight is 1 mg/cm ² for pore sizes between 3.0 and 8.0 microns. This membrane is water,
It is easily wetted by many other fluids and has no bactericidal or bactericidal properties. The specific gravity of Hema-fil ^M membrane is 0.94 to
0.97gm/cc, approximately
Withstands temperatures of 140℃. The absorbent strip may be any material that provides uniform absorption with reproducible absorbance and preferably facilitates lateral flow of red blood cells. Preferably, the absorbent strip is filter paper, but may also be a synthetic material such as absorbent plastic. The flow rate along the absorption strip varies depending on the width of the strip. When used to analyze the deformability of red blood cells, the width of the absorption surface of the absorption strip is preferably about 3 millimeters or less. The device is used to measure the time required for a fluid sample, such as a blood sample, to be absorbed along the length of an absorbent strip. The timer is started when the sample is placed on the membrane where there is surface contact between the membrane and the absorption strip. Stop the timer when blood reaches the opposite end of the strip. The same procedure is repeated with multiple strips using blood samples, after which the average absorption time is calculated for multiple values. An embodiment of the present invention is shown in FIGS. 1 and 2. FIG. As shown in FIG. 1, the device has a flat rectangular shape and includes a base layer 10, an absorbent strip 1
1. It has a filter membrane 12, a transparent tape layer 13, and an opaque cover tape layer 14. base layer 10
supports the device and is preferably opaque and coated with a pressure sensitive adhesive on one side. It is also desirable to color the base layer 10 to provide a contrasting background color so that the absorbing strips 11 can be distinguished. Absorbent strip 11 and filter membrane 12 are applied to the adhesive surface of base layer 10. Filtration membrane 1
2 is located above the absorbent strip 11. The transparent tape layer 13 has a filter membrane 12 and an absorbent strip 11.
, creating an exposed area of the filtration membrane at one end of the absorbent strip. Opaque tape cover 1
4 is located on the transparent tape layer 13 and forms the upper end of the absorbent strip 11 (the end of the absorbent strip). FIG. 3 is another embodiment of the invention. This example has a base layer 20, a plurality of absorbent strips 21, a common filtration membrane 22, a tape layer 23, and a cover tape layer 24. Common filtration membrane 22, tape layer 2
3 and the cover tape layer 24 are each fixed as in FIGS. 1 and 2. First, a small amount (approximately 10 microliters) of sample fluid, usually blood, is placed on top of the filtration membrane in partial surface contact with one end of the absorbent strip. After a short time, the forefront of the fluid (blood) can be seen moving along the absorption strip 11. As the fluid progresses along the strip, the absorbent front is visible even in clear fluids. The time taken for the fluid to be absorbed to the end of the absorption strip is the readout from the device and is timed by the user. The fluid flow rate is calculated from the amount of fluid absorbed and the absorption time. The device according to the invention differs from conventional filtration systems in several respects in its basic operating principle. first,
First, the driving force is due to the capillary force of the absorbent strip. Second, the length of the fluid flow path along the absorbent strip increases over time. As a result, as flow progresses, the effective pressure gradient acting in the system decreases monotonically. The reduction in pressure gradient therefore results in a certain fundamental difference in fluid flow motion in this device compared to conventional systems. In the device according to the invention, the fluid flow rate varies inversely proportional to the amount of fluid absorbed, which is directly proportional to the square root of the absorption time. The time required to absorb a given amount of fluid is proportional to the square of the amount absorbed. Therefore, due to these relationships, the flow rate is initially high, but rapidly decreases over time. The performance of the red blood cell filter device according to the invention can be evaluated on blood samples from healthy and diseased individuals. The apparatus used for these evaluations has a plurality of absorption strips designed as shown in FIG. This device has five absorbent strips, the length and width of each strip being 3.38±0.16 mm and 9.76±0.04 mm, respectively. At the ends of these absorbent strips
Covered with Nuclepore's Hema-fil ^M membrane.
The membrane is a polycarbonate capillary pore membrane with a pore size of 4.7±0.2 microns;
×10 ⁵ number of pores/cm ² ; film thickness of 10.3±0.3 microns. The average length and average area of the membraned ends of the strips are 3.34±0.09mm and 3.34±0.09mm, respectively.
It is 11.3± ^0.6mm2 . The overall dimensions of the device in Figure 3 are:
It is 38mm x 76mm. The average absorption volume of each strip is 5.3 microliters. Table 1 shows the results of the test on normal blood. From the same table, it can be seen that the average absorption time Ta varies greatly depending on the hematocrit value H. Hematocrit is the concentration of red blood cells in a suspension medium, expressed as a percent by volume. The average absorption time depends exponentially on the hematocrit value H when the hematocrit value H is greater than or equal to 5%. If you draw a graph of L _o T _a vs. H based on the values in Table 1, it will be a straight line if H is greater than or equal to 8.8%. The average absorption time in Table 1 can be calculated using the following formula: T _a =K exp (0.055H) (1) where H is expressed in % and T _a is expressed in seconds. The values of K are listed in the fourth row of Table 1. K is determined by the following formula: K=T _a exp (-0.055H) (2) Here, the unit of K is seconds. In Table 1, H is 5
% or more or equal to 5%, the average K value is
It is 4.4±0.4 seconds. It can be seen that the parameter K is a very important parameter for blood samples. In each case, a 10 microliter blood sample was applied to the apparatus of FIG. 1, which employed a Hema-fil ^M membrane. The absorbent strip is Whatman filter paper No.
It was 41.

【table】

[Table] The red blood cell suspension used to obtain the results in Table 1 was prepared with normal (AA) blood from a healthy person. A sample of blood is taken from a vein to prevent clotting.
EDTA (ethylenediaminetetraacetic acid) was added.
Red blood cells were separated by centrifugation and washed with salt diluted in phosphate buffer. Red blood cell suspensions of various hematocrit values were prepared by diluting this phosphate buffered salt. Average absorption time Ta determined by the above equation (1)
From the relationship between the hematocrit value H and the hematocrit value H, normal red blood cells and abnormal red blood cells can be determined. The value 0.055 in the exponent of equation (1) is the slope of the straight line in the graph of L _o T _a versus H. This value, 0.055, is found in both normal and abnormal red blood cells. What is important is that the parameter K given by equation (2) alone specifies the filtration ability of red blood cells and, therefore, the deformability of red blood cells. In practicing the present invention, the average absorption time of a given blood sample is determined using a red blood cell filtration device.
Measure Ta. Sample hematocrit value H
shall be measured separately using well-known techniques.
Next, the parameter K is determined by substituting T _a and H into equation (2). The value of K is unique for a blood sample and determines the filtration or deformability of red blood cells. Table 2 shows the test results obtained with the present red blood cell filter device on normal and abnormal blood samples. The first and second columns show the sample number and identification name, the third column shows the measured hematocrit value, the fourth column shows the average absorption time, and the fifth column shows the calculated K. Footnotes in Table 2 indicate blood sample details.

[Table] 1. Healthy, fresh blood, whole blood, male, oriental. 2. Healthy, fresh blood, whole blood, female, white. 3. Separate (2), wash, and suspend in salt solution diluted with phosphate buffer. 4 Normal, 1 day after collection, whole blood. 5 Normal, whole blood 30 days after collection, expired warranty period from blood bank. 6 Dilute (5) with a salt solution diluted with phosphate buffer saline (PBS). 7 Sickle red blood cell constitution, 1 day after collection, whole blood. 8 Sickle red blood cell constitution, 7 days after collection, whole blood. 9 Diabetes, 2 days after collection, whole blood. 10 Diabetes, 7 days after collection, whole blood, patient A. 11 Diabetes, 7 days after collection, whole blood, patient B. 12 Sickle cell disease, fresh blood, whole blood. Dilute 13 (12) with PBS. 14 (12) was separated by centrifugation. 15 Sickle cell disease, 1 day after collection, anti-sickle drug administration (-14 days, DMA). 16 Sickle cell disease, fresh blood, anti-sickle drug DMA administration (-42 days). 17 Sickle cell disease, 1 day after collection, female, pregnant. From the results shown in Table 2, normal blood (AA)
It can be seen that the K value of is in the range of 1.5 to 4 seconds.
Blood K values for sickle cell disease range from 65 to 140 seconds. Therefore, in general, K in the blood of a sick person
The K value is high, and it can be said that the K value of normal people's blood is low. The data in Table 2 shows that the conditions of the blood sample (e.g. disease name, age, presence or absence of previous medication, duration of medication, and whether it is whole blood or diluted blood) have important effects on the K value. It shows that it gives. It can be seen from equation 1 that when H=0, the relationship Ta=K is obtained. Therefore, K is simply the average absorption time at H=0, and is the point where Ta(H) intersects the Ta axis. Table 3 shows a comparison of the results obtained with the conventional filtration mode of the red blood cell filtration device according to the invention.

【table】

[Table] The pressure filtration method is the same as above, manufactured by Nuclepore.
Hema-fil ^M was used. In the pressure filtration method,
The drain time T _D is between the first and second descent,
Measured under gravity at the exit of the filter holder. A 200 ml sample was poured onto the surface of a 13 mm diameter filter. The volume of one drop is approximately 66 microliters. As expected, the results of filtration drain time showed large changes with hematocrit values. T _a /T _D in the fifth column is the ratio of average absorption time to filtration drain time. This ratio is almost constant, with an average value of 15±0.2. From the results in Table 3,
The results of the present absorption filtration device can be interpreted as being exactly the same as those obtained with conventional filtration methods.