Erythrocyte aggregation and erythrocyte sedimentation rate measuring device

Abstract

The invention relates to a device that simultaneously measures both erythrocyte aggregation and erythrocyte sedimentation rate from values obtained by measuring the reactances of erythrocyte suspensions (blood samples).

Description

[0001] ERYTHROCYTE AGGREGATION AND ERYTHROCYTE SEDIMENTATION RATE MEASURING DEVICE

[0002] TECHNICAL FIELD

[0003] The invention relates to a device that simultaneously measures both erythrocyte aggregation and erythrocyte sedimentation rate from values obtained by measuring the reactances of erythrocyte suspensions (blood samples).

[0004] BACKGROUND

[0005] In the absence of blood flow, erythrocytes cluster as biconcave disks forming parallel surfaces in the plasma. This is called aggregation (Figure 1B). These clusters disperse when blood flow begins due to the current forces acting on the erythrocytes. This event is a reversible aggregation. This aggregation does not occur when the same cells are suspended in simple salt solutions (Figure 1A). Erythrocyte aggregation is affected by changes in the cellular properties of both the plasma and the erythrocytes. Fibrinogen, acute phase reactants, changes in plasma globulin fractions, osmolarity, pH changes and increases in hematocrit value affect erythrocyte aggregation.

[0006] Various approaches and methods are used to determine erythrocyte aggregation in vitro. The most frequently used of these methods are; measurement of light transmittance or light reflection from erythrocyte suspensions (photometric measurements), microscopic indices of erythrocyte aggregation, erythrocyte sedimentation rate, low shear viscosity and electrical measurements of erythrocyte suspensions (impedance, capacitance).

[0007] All internal and external factors affecting erythrocyte aggregation also affect the erythrocyte sedimentation rate, i.e. there is a linear relationship between them. Photometric measurements are widely used to investigate aggregation in erythrocyte suspensions. Other measurement methods are used only rarely.

[0008] Photometric measurement devices are produced for commercial purposes and are quite expensive. In these devices that make photometric measurements, various flow geometries (such as nested cylinders, cone-plate, parallel surfaces, rectangular flow channel) are used, which are especially important in the disaggregation processof aggregates. These measurement systems have systems that provide blood flow (disaggregation).

[0009] First described 120 years ago (Westergren method), erythrocyte sedimentation rate (ESR) is a widely used, non-specific screening test. It is the best-known method for measuring erythrocyte sedimentation rate and the method that best distinguishes high and low values. In this method, four volumes of blood are mixed with one volume of 3.8% sodium citrate. The standard Westergren glass tube is filled to the zero point at the top, adjusted to the vertical position and left for one hour. The results are expressed as x / mm. Sedimentation consists of three phases; the first phase is the formation of rolls of erythrocytes (aggregation), the second phase is the formation of aggregates of erythrocytes, and the third phase is the slow phase of sedimentation (due to the accumulation of erythrocyte aggregates at the bottom of the tube). As alternative methods of erythrocyte sedimentation rate, different principles such as centrifugation or especially photometric aggregometry are used. In these devices, the measurement of the first phase of erythrocyte sedimentation rate, which is roll formation, is taken as the basic principle.

[0010] AIM OF THE INVENTION

[0011] Our invention simultaneously measures both erythrocyte aggregation and erythrocyte sedimentation rate from the values obtained by measuring the electrical properties (X, Reactance) of erythrocyte suspensions (blood samples).

[0012] With our invention, it takes approximately 2.5 minutes between the start of the measurement and the calculation of the values, and it gives results much faster than many commercial devices.

[0013] The measurement of erythrocyte aggregation is a parameter that is not used in the clinic but is used in research laboratories. However, erythrocyte sedimentation rate is used in almost all large or small health institutions. With our invention, two devices that allow these two measurements and are sold separately in the state of the art have been brought together under one roof. Especially due to the limited resources of research laboratories, it is quite costly to access devices that measure erythrocyte aggregation. Even today, in many hospitals, the measurement of erythrocyte sedimentation rate is performed with various versions of the classical method (Westergren method) and takes quite a long time (30-60 minutes) depending on the device we invented. In addition, most devices are not fully automated. Thetechnical effects of our invention are that both measurement parameters are in a single device, it gives results in a very short time and its cost is very low compared to its peers. It is also thought that it will contribute to the development of our country by reducing the external dependency of health professionals and institutions.

[0014] LIST OF FIGURES

[0015] Figure 1A. Microscope image showing absence of erythrocyte aggregation. Figure 1B. Microscope image showing erythrocyte aggregation in roll form. Figure 2. Schematic representation of the invention

[0016] Figure 3. Curves derived from reactance values derived from measurements conducted with the invention.

[0017] Correspondences of the numbers given in the figures:

[0018] 1. Electrodes

[0019] 2. Glass Capillary

[0020] 3. Peristaltic Pump (Servo Control Motor)

[0021] 4. Processor-Control Unit

[0022] 5. LCR Meter Analysis-Calculation

[0023] 6. Printer

[0024] 7. Peristaltic Pump (Servo Control Motor)

[0025] 8. Washing and Calibration Solution

[0026] 9. Waste Bin

[0027] 10. Power supply connection

[0028] 11. Control Panel-LCD Screen

[0029] 12. Aluminum Block

[0030] 13. Plastic-Silicone Pipes

[0031] 14. Interconnection Cables

[0032] 15. Three-Way Tap

[0033] 16. Sample

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The invention pertains to a device that simultaneously measures the aggregation and sedimentation rates of erythrocytes from the values derived by measuring the reactances of erythrocyte suspensions (blood samples). The device inquestion comprises the following components: two electrodes (1), a glass capillary (2) that serves as the transport path for the sample (17) and solution (9), a peristaltic pump (servo control motor) (3), a processor-control unit (4) with analysis-calculation (6) functions, an LCR meter (5), a printer (7), a peristaltic pump (servo control motor) (8), a washing and calibration solution (9), a waste bin (10), a power supply connection (11) and a control panel-LCD screen (12), an aluminum block (13) which is the housing for the electrodes (1) and the glass capillary (2), allowing for measurements to be taken at a temperature that is comparable to that of the human body (36.5 - 37.5°C). The device further comprises interconnection cables (15) and a three-way tap (16). The three-way tap (16) is a valve used to manually control the flow of liquids and gases. It has a valve that can be turned in three directions. Thus, the flow of the closed areas is completely stopped and thus the flow to the desired area is provided. It provides the flow of liquids between the two peristaltic pumps (3, 8) and the control of air entry into the system. The silicone plastic pipes (14) are used in the connections of this tap made of plastic to the system. While it provides the connection between the sample (17) and the washing and calibration solution (9) with its two ends connected to the system, it provides air entry when desired from the empty part.

[0036] Within the measurement device described above, the two stainless steel electrodes (1) are placed at the beginning and end of the glass capillaries (2). The connections in all other parts of the device, which is the subject of the invention, are made of silicone plastic pipes (14).

[0037] By connecting the device to the power supply (11) and operating it with the operating buttons on the front panel (12), the peristaltic pump (3) is activated. In this way, the blood sample (17) to be measured is transported to the system for measurement by the peristaltic pump (3). Ten seconds after the peristaltic pump (3) starts operating, the mini LCR meter (5) to which the electrodes (1) are connected is operated by the processor-control unit (4). The LCR meter (5) is a test device used to measure basic electrical properties such as inductance (L), capacitance (C) and resistance (R) in electronic circuits. The LCR meter measures the impedance (resistance to alternating current) of the components under test by sending alternating current (AC) signals to them. Based on these measurements, the device calculates the L, C or R values of the component under test. Within the scope of our invention, after the LCR meter (5) starts working, it takes measurements for 5seconds while the peristaltic pump (3) is running. The measurement process is as follows: the sample (17) included in the system by the peristaltic pump (3) is passed through the electrodes (1) and electric current is applied to the sample (17) during this passage and the LCR meter (5) monitors the changes that occur during the current through the sample. Following this, the processor-control unit (4) receives a signal from the LCR meter (5), which signifies that the initial measurement has been completed. The peristaltic pump (3) is halted by the processor-control unit (4) in response to this signal. Without interruption, the LCR meter (5) measures for 120 seconds. In other words, the LCR meter (5) measures Reactance (X, ohm) at a frequency of 100 kHz, with 1 second intervals, for a total of 125 seconds, including the initial measurement. The values obtained at the conclusion of the measurement (6) are used to calculate the "Erythrocyte Aggregation" and "Erythrocyte Sedimentation Rate." The LCD screen (12) displays the outcomes. Optionally, the results can be printed using a printer (7). The memory of the device records all measurements. The software that is configured to execute these operations within the processor-control unit (4) is responsible for the control and implementation of the aforementioned process, as well as the generation of signals for the start, measurement, and stop of the aforementioned equipment.

[0038] During the measurement of the sample (17) with the device in question, blood may remain in the equipment where the measurement is made. In order to clean this, there is a cleaning mode within the processor-control unit (4) that can be selected by using the keys on the control panel (12) after the measurement. When this mode is activated, both peristaltic pumps (3, 8) in the system are operated by the processorcontrol unit (4) and the blood remaining in the system can be cleaned with a washing solution (9) and transferred to the waste bin (10). Then, both pumps (3, 8) are stopped. Then, only one peristaltic pump (3) is operated empty without the sample (17) and thus air is allowed into the system via the three-way tap (16). Thus, all the solution in the system is cleaned and the next measurement can be started. If the measurement will be interrupted for a long time after all the measurements are completed, distilled water should be loaded into the system and it should be kept waiting in this way.

[0039] The cleansing solution is also employed as a calibration solution (9). Calibration should be conducted every 10 days for each new calibration solution (9), or if the solution (9 is incomplete). Solution (9) that is not utilized within a monthshould be discarded. Within the processor-control unit (4), there is a calibration function. The Reactance (X, ohm) value is calculated as in the sample (17) and the calibration mode is initiated by pressing the calibration start button on the control panel (12). The device's memory stores the results of this calculation as "fixed values." The content of the calibration solution is a sample with a hematocrit adjusted to 40%, consisting of 4 units of erythrocyte packs and 6 units of BPS.

[0040] The processor-control unit (4) calculates the parameters of the device in question using the formulas provided below, which are based on the 125 Reactance values derived from the measurement. The software in the processor-control unit (4) is prepared to execute the procedures and formulas specified below.

[0041] Erythrocyte Aggregation (EA, unitless):

[0042] A: The average of five values obtained from the 49th and 53rd seconds of the blood sample (Whole Blood).

[0043] B: The average of five values obtained with the calibration solution (PBS) between the 49th and 53rd seconds.

[0044] A EA = - B

[0045] Erythrocyte Sedimentation Rate (ESR, mm / hour):

[0046] X: The difference between the 62nd and 125th second values, as measured with the whole blood sample.

[0047] Y: The difference between the 62nd and 125th second values, as determined by the calibration solution (PBS).

[0048] X ESH = - ¥

[0049] In order to illustrate the efficacy of our invention, measurements were implemented. Two different samples were used in the measurement. The initial of these is a suspension of erythrocytes and plasma, referred to as "Whole Blood." The hematocrit value of the blood obtained from the vein was regulated to 40% using a syringe. The second sample is a suspension of erythrocytes and PBS, which isreferred to as "PBS." Additionally, the hematocrit value of this suspension was established at 40%. The difference of the second suspension from the first suspension is that it does not contain plasma. Because the cellular properties of erythrocytes (shape, deformability and membrane surface properties), the properties of the suspension medium (the concentration of proteins such as plasma fibrinogen and macroglobins, hematocrit, pH and osmolarity) and the shear forces in the medium effect erythrocyte aggregation. Based on this, the only difference between the two samples prepared is the presence and absence of the suspension medium plasma. Apart from this, the shear forces in the system are the same. These two suspensions were measured within the device that is the subject of our invention. As a result of these measurements, the curves shown in Figure 3 emerge from the obtained reactance values. In the curve of the suspension called "Whole Blood", the first five seconds are the disaggregation section where blood flow occurs. At the end of this period, when the blood flow is suddenly stopped, a rapid decrease in the curve is observed, followed by a rapid increase. Erythrocytes can change shape reversibly under the effect of shear forces acting on them, and this is defined as erythrocyte deformability. In other words, erythrocytes elongate in the direction of the flow and behave like a liquid drop and become oriented. When the flow is eliminated, they return to their original state, the biconcave disk shape. The changes recorded in the obtained curve after the peristaltic pump (3, 8) is stopped, i.e., the time it takes for the erythrocytes to change shape and return to their original state. At the end of this period, the exit velocity in the curve slows down and forms a plane after a while. In short, the curve reaches a peak and starts to decrease again. The section in the curve where the ascent rate slows down (Phase 1) is considered the initial phase of erythrocyte aggregation. Since small one-dimensional erythrocyte aggregates are formed, the curve still continues to rise, albeit slightly. At the end of this period, the ascent rate of the curve decreases to almost a standstill and a plateau forms. This section (Phase 2) is considered the roll formation phase of erythrocyte aggregation. The section where two- and three-dimensional large erythrocyte aggregates form allows the curve to reach its peak. The average of the first five seconds of the Phase 2 section, when it is the fastest, constitutes the "A" measurement in the formula used to calculate erythrocyte aggregation. As an alternative to the calculation method used, the average of all measurement read in the Phase 2 section can also be taken. However, taking the first section of this section, when it is the fastest, makes itpossible to reach the same result. The section where the curve decreases from the peak to the end of the measurement (Phase 3) was evaluated as the sedimentation phase of erythrocyte aggregates; in other words, the erythrocyte sedimentation phase. Because the erythrocyte aggregates acted like a cluster and began to settle towards the bottom of the glass capillary with a 1 mm internal diameter that was horizontally positioned, both with their own weight and the effect of gravity. As a result, the measured reactance value decreased over time. The difference between the section where the obtained curve became almost flat and almost reached the middle of Phase 2, where it was thought that erythrocyte aggregates were the most dense, and the last value measured, reflects the erythrocyte sedimentation rate. This change is represented by “X” in the erythrocyte sedimentation rate formula.

[0050] If erythrocytes are resuspended in buffers that do not contain protein or polymer, erythrocyte aggregation disappears. Similarly, erythrocyte sedimentation does not occur. Measurements made with these buffers, which are usually made with PBS, are considered zero (0). In the measurement made with the erythrocyte suspension prepared with PBS using the device in question, after stopping the peristaltic pump (3), a slight increase that can be neglected in the curve and then a structure that almost does not change until the end of the measurement appears (Figure 3). There are very large differences between the curves that can be seen with the eye. The only difference between the two suspensions represented by the curves is whether or not there is plasma in the suspension medium. The reactance value obtained as a result of this measurement is considered as zero (0), that is, there is no aggregation - sedimentation. The values calculated for “Whole Blood” above are calculated in the same way for “PBS”. In other words, when the calculation method for “A” in the formula is calculated for “PBS”, “B” in the formula is found, and when the value calculated for “X” is calculated for “PBS”, “Y” in the formula is found. In order to find the coefficient of variation of the whole blood value according to the PBS value, the “A” value is divided by “B” to calculate erythrocyte aggregation, and the “X” value is divided by “Y” to calculate erythrocyte sedimentation rate parameters.

Claims

CLAIMS1. A device for measuring erythrocyte aggregation and erythrocyte sedimentation rate by measuring reactance (X, ohm) characterized by comprising; two electrodes (1), a glass capillary (2) which is a transportation path of a sample (17) and a solution (9) in the system, a peristaltic pump (servo control motor) (3), a processor-control unit (4) with analysis-calculation (6) functions, an LCR meter (5), a peristaltic pump (servo control motor) (8), a waste bin (10), a power supply connection (11), a control panel-LCD display (12), an aluminum block (13) which the houses electrodes (1) and the glass capillary (2) and thus enables measurements to be made at a value close to normal body temperature (36.5 - 37.5°C), a three-way tap (16).

2. The device according to claim 1 characterized by comprising a printer (7) that facilitates the printing of the results obtained.

3. The device according to claim 1 characterized by comprising silicone plastic pipes (14) and interconnection cables (15) that enable the parts to be connected to each other.

4. The electrodes (1) mentioned in claim 1 characterized by being placed at the beginning and end parts of the glass capillaries (2).

5. The device according to claim 1 characterized by comprising the processorcontrol unit (4) that further comprises a software configured to perform operating the LCR meter (5) to which the electrodes (1) are connected 10 seconds after the peristaltic pump (3) starts working; generating the signal that will allow the LCR meter (5) to take measurements while the peristaltic pump (3) is running for 5 seconds after it starts working; generating the signal necessary to stop the peristaltic pump (3) after a signal from the LCR meter (5) indicating that the first measurement has been made; generating the signal for the LCR meter (5) to take measurements for 120 seconds without interruption at this stage; measuring the Reactance (X, ohm) at 1 second intervals during this period and the calculating the erythrocyte aggregation and erythrocyte sedimentation rate from the values obtained at the end of the measurement.

6. The device according to claim 5, characterized in that comprising the software Aconfigured to use formula EA= - for calculating the erythrocyteBxaggregation rate and formula ESH = — _ for calculating the erythrocyte ¥sedimentation rate.

7. The device according to claim 1, characterized by comprising the processorcontrol unit (4) further comprises a cleaning mode.

8. The device according to claim 1, characterized by comprising the processorcontrol unit (4) further comprises a calibration mode.

9. The device according to claim 1, characterized by comprising a memory unit.

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