RU2379687C2 - Method for determining blood cell sedimentation dynamics - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: offered invention concerns medicine, namely clinical laboratory diagnostics. Substance of the invention consists as follows: analysed blood sample is mixed with an anticoagulant, applied on a transparent horizontal hydrophobic surface (a cell) in as a sessile drop with using an autosampler, illuminated from below with a light flux of the diametre 1-1.5 mm falling perpendicularly to the drop base. Then cell sedimentation dynamics is recorded by measuring variation of the light flux intensities through the blood drop within its central axial area during a time frame.

EFFECT: reduced amount of blood required for the analysis, reduced time of the analysis, improved information value of the analysis, reduced number of random factors affecting the cell sedimentation process.

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Description

The present invention relates to medicine, namely to laboratory clinical diagnostics. The invention can be used for laboratory tests in pediatric practice, as well as for research purposes.

The value of the erythrocyte sedimentation rate (ESR) is a non-specific indicator widely used in clinical practice to assess the presence of inflammatory processes in the human body in various diseases and allows monitoring the course of the disease and its treatment.

Known in Russia (classical) method for assessing the erythrocyte sedimentation rate, performed according to the Panchenkov method. A glass graduated tube to a specified level is filled with a mixture of blood with 3.8% sodium citrate (anticoagulant) in a ratio of 4: 1 and placed vertically in a tripod under the clamp (to prevent leakage of blood). One hour after the start of the measurement, the distance (in mm) by which the column of red blood cells has dropped from the initial level [Laboratory methods of research in the clinic] is determined by the divisions on the tube. / Ed. Menshikova V.V. - M .: Medicine, 1987. - 368 p.].

The disadvantage of this method is the long analysis time (more than 1 hour), as well as the difficulties that arise when sampling the volume of capillary blood necessary for the study (at least 0.3 ml) and associated with this fact, violations of the rules for taking and preparing blood for research. In addition, subjective factors related to the professional training of medical personnel, the cleanliness of the tubes, the accuracy of mixing blood with an anticoagulant, and the temperature stabilization of the sample during the study have a significant influence on the research process. In general, all these facts lead to an analysis error. It is also recognized that a single measurement, taken 1 hour after the tubes were placed in a tripod, gives only the average ESR value and is insufficient. It is important to record the dynamics of the erythrocyte sedimentation process, which increases the information content of the analysis.

A known method for determining the dynamics of sedimentation of blood cells, including mixing whole blood with an anticoagulant, taking the mixture into a standard pipette for measurements, placing it vertically in a tripod and registering the position of the erythrocyte-plasma boundary at regular intervals (EN 2103672, publ. 27.01 .1998).

This method of studying ESR takes at least 1 hour and requires about 0.3 ml of blood to conduct, as in the classical Panchenkov method.

There is a method of determining the dynamics of changes in the erythrocyte sedimentation rate, including standard blood sample preparation, its collection in capillaries for measuring hematocrit and their placement in a vertical position around the perimeter in the centrifuge rotor. In the process of rotation of the centrifuge, the height of the column of plasma free from red blood cells is measured at specified intervals in capillaries (RU 2256917, publ. 20.07.2005).

The use of capillaries of smaller sizes than standard tubes for measuring ESR allows one to reduce the amount of blood needed, and centrifugation - to reduce the time during which the cells settle, and, accordingly, the time of the study. However, reducing the diameter of the tube significantly changes the erythrocyte sedimentation conditions, since the contact of the sedimenting aggregates with the vessel wall has a significant effect, which leads to erroneous results and insufficient reproducibility of the research results. The use of centrifugation of the tubes violates the "physiology" of the settling process, which is also undesirable.

The closest analogue of the proposed method is a method for determining the dynamics of sedimentation of blood cells, the essence of which is that they use a standard disposable immunological tablet, into the well of which, with the help of an automatic dispenser, test blood with an anticoagulant is placed and the dynamics of sedimentation of blood cells is recorded by measuring over a specified time changes in the intensity of luminescence of blood plasma over the upper boundary of the layer of sedimenting blood cells (RU 2313091, publ. 20.12.2007).

The disadvantage of this method is the need to add to the sample substances that increase the plasma luminescence, which can ambiguously affect the cell sedimentation rate.

The objective of the present invention is to reduce the amount of blood required for the study, reducing the analysis time, increasing the information content of the analysis, as well as reducing the number of random factors affecting the cell sedimentation process.

The problem is solved due to the fact that in the method for determining the dynamics of sedimentation of blood cells, as well as in the prototype, using the automatic dispenser, the test amount of blood with an anticoagulant is placed on a transparent hydrophobic surface and illuminated with a light flux.

According to the invention, the analyzed blood is placed on a transparent horizontal hydrophobic surface in the form of a lying droplet, which is illuminated from below by a light flux falling perpendicular to the base of the droplet and the dynamics of cell sedimentation is recorded by measuring over a specified time interval the changes in the intensity of the light flux passing through the droplet of blood in its central axial area.

Using for studying the dynamics of sedimentation of sample cells in the form of a lying drop significantly (up to 20-30 μl) reduces the amount of blood needed for the study.

The process of sedimentation of cells in a droplet sample (with a drop height of about 2 mm) ends depending on the sedimentation rate after about 10-40 minutes, and therefore less time is required for the study compared to the classical method.

Obtaining in the course of the study curves reflecting the dynamics of cell sedimentation can increase the information content of the analysis, since these graphs are characteristic for each subject and vary depending on the physiological state and, accordingly, can provide additional information about the state of the subject. In addition, the initial portion of the photometry curve of a dropping blood sample carries information about the aggregation processes occurring in the sample, and subsequently leading to cell sedimentation.

Due to the absence of contact between the cells and the vessel wall during subsidence, the action of random factors associated with the purity of the capillary and the influence of wall effects on the subsidence process is eliminated.

The essence of the proposed method is illustrated by the description and drawings.

Figure 1 shows a diagram of a method for assessing the dynamics of sedimentation of blood cells.

Figure 2 schematically shows a number of processes that occur during the sedimentation of cells in a drop sample of blood.

Figure 3 is a structural diagram of a device that was used to implement the proposed method for assessing the dynamics of sedimentation of blood cells.

Figure 4 shows the experimental dependence of the change in the value of the output voltage (U _o ) of the photocurrent amplifier (US) on the height of the plasma layer (h) above the surface of the sedimenting layer of cells in a droplet sample (in accordance with the data of Fig. 5) and a linear approximation of experimental data ( dashed line).

Figure 5 presents a graph 5.1, showing the change in time of the output voltage of the photocurrent amplifier (US) with a change in the height of the plasma layer (h) above the surface of the settling layer of cells in the droplet sample shown in graph 5.2. Point "a" indicates the moment of appearance of a clearly visible interface between the phases of the blood cell - plasma. Point "b" denotes the characteristic portion of the inflection curve of the photometry.

Figure 6 shows photographs of the lateral projection of a blood drop in which the cells are sedimented, taken at the initial moment of sedimentation - photo 6.1, after 3 minutes - photo 6.2, after 5 minutes - photo 6.3, after 7 minutes - photo 6.4, after 9 minutes - photo 6.5, after 13 minutes - photo 6.6.

Figure 7 shows the experimental graphs reflecting the dynamics of sedimentation of blood cells in a drop for blood samples with different ESR values (measured by Panchenkov), where curve 7.1 for 2 mm / h, curve 7.2 for 8 mm / h, curve 7.3 for 17 mm / h, curve 7.4 for 23 mm / h, curve 7.5 for 42 mm / h, curve 7.6 for 70 mm / h.

On Fig presents the relationship between the parameter U _t10 -U _t0 , characterizing the sedimentation rate of cells in the drop, and the ESR value of the test blood sample, measured by the Panchenkov method.

Figure 9 presents experimental graphs that reflect the dynamics of changes in the sedimentation of blood cells in the drop for patients before (curve "a") and after treatment after 13 days (curve "b").

Chart 9.1. Patient B., female 16 years old, diagnosed with pneumonia, ESR (according to Panchenkov) before treatment 25 mm / h, after treatment 12 mm / h.

Schedule. 9.2. Patient X., husband. 33 years old, diagnosis - pneumonia, ESR before treatment 46 mm / h, after treatment 16 mm / h.

Schedule. 9.3. Patient I., female 35 years old, diagnosis - pneumonia, ESR before treatment 70 mm / h, after treatment 60 mm / h.

Chart 9.4. Patient S., female 62 years old, diagnosis - pneumonia, ESR before treatment 14 mm / h, after treatment 15 mm / h.

Schedule. 9.5. Patient T., female 69 years old, diagnosis - pneumonia, ESR before treatment 36 mm / h, after treatment 42 mm / h.

Chart 9.6. Patient I., husband. 76 years old, diagnosis - gangrene of the lower extremity, ESR before treatment 60 mm / h, after treatment 60 mm / h. A deterioration was observed.

The principle of the method for assessing the dynamics of sedimentation of blood cells is illustrated in figure 1. The test blood in the form of a lying drop 1 is placed on a transparent hydrophobic cell 2 between the coaxially located optical radiation source 3 and the optical radiation receiver 4. Moreover, the optical radiation source is located under the drop 1, and the optical radiation receiver 4 is above it. The transparent hydrophobic cell 2 is structurally designed so that the drop 1 located on it does not spread and retains its shape. The luminous flux from the optical radiation source 3 falls perpendicular to the base of the droplet 1 and passes through its central axial part. The optical radiation receiver 4 registers the radiation transmitted through the drop 1 in its axial region. The intensity of the light flux incident on the optical radiation receiver 4 is determined by the optical properties of the translucent sample and changes over time depending on the processes occurring in the droplet sample 1.

In a drip sample of whole blood with an anticoagulant, as well as in a glass capillary, the processes of aggregation of red blood cells and their sedimentation by gravity occur.

The process of formation of cell aggregates leads to an increase in the transparency of the sample, and this fact is widely used in modern optical aggregometers.

A number of processes are specific for the sedimentation of cells in a drip sample, which were the basis of the proposed method.

Figure 2 schematically shows a number of processes that occur during the sedimentation of cells in a drop sample of blood, affecting its optical properties. Despite the fact that the surface of the droplet at the blood-air interface has a shape close to spherical, the upper boundary of the settling layer of cells in the droplet is almost flat. That is, there is a redistribution of cells settling in the central axial region of the droplet over the entire volume of the sedimenting layer (figa). This leads to a decrease in the number of cells in the axial region and, accordingly, an increase in the transparency of this region of the drop.

The formation of a transparent plasma layer in the form of a lens 5 above the settling layer of cells 6 (Fig.2.b) leads to a change in the scattering properties of the droplet sample - "focusing" of the light flux - and, accordingly, an increase in the density of the light flux above the droplet.

As a result of these basic processes, the intensity of the light flux passing through the axial region of the studied blood drop 1 and reaching the photodetector 4 changes over time and reflects the processes of cell aggregation and sedimentation in the droplet sample.

To implement the method, a device was used (patent for PM of the Russian Federation No. 47526, 2005), the structural diagram of which is presented in figure 3. The device consists of a thermostabilized chamber of the primary transducer 7 (PP), including a vertically coaxially located optical radiation source 3 (AI) and an optical radiation receiver 4 (AI), between which a cuvette with an investigated liquid droplet sample 1 (PR) is placed. The composition of the chamber of the primary transducer 7 (PP) also includes a temperature sensor 8 (TD) and a heating element 9 (NE). In the side wall of the camera of the primary transducer 7 (PP), a video camera lens 10 (VK) is integrated, focused on the drip sample 1 (PR). The camcorder 10 (VK) is electrically connected to the video input board of a personal computer 11 (PC). In addition, the device includes: a stable current source 12 (IST) connected to an optical radiation source 3 (AI); thermoregulation unit 13 (TP), electrically connected to a temperature sensor 8 (TD) and a heating element 9 (NE); a photocurrent amplifier 14 (US) electrically connected to an optical radiation receiver 4 (PI) and a recording device 15 (RU). The power source 16 (IP) is electrically connected to the thermoregulation unit 13 (TP), a stable current source 12 (IST) and a photocurrent amplifier 14 (US). To reduce the evaporation of liquid samples in the chamber there is a system for maintaining high humidity.

The photocurrent amplifier 14 (US) is a current (photodiode) -voltage converter and has a linear dependence of the output voltage (U _o ) on the intensity of the light radiation incident on the light-emitting radiation receiver 4 (PI) associated with it. The luminous flux from the optical radiation source is collimated and has a diameter of 1-1.5 mm. The optical radiation receiver 4 (PI) has an angular aperture of 5-10 ° and is located above the surface of the droplet sample at a distance of 2-3 mm.

As a recording device 15 (RU), a digital voltmeter V7-38 was used.

The method is as follows.

The device (figure 3) for research should be turned on 30 minutes before the start of the study to establish the necessary mode in the primary converter chamber: temperature 37 ° C ± 0.1 ° C, relative humidity 100%.

Blood is taken from the finger (vein) of the subject according to standard methods into a test tube of the required size. The taken blood is immediately diluted with a solution of 3.8% sodium citrate in a ratio of 4: 1. Tubes (or other devices) used to draw blood should also be rinsed with a solution of 3.8% sodium citrate to eliminate blood coagulation. The study of the dynamics of sedimentation of blood cells by the proposed method is preferably carried out immediately after the above procedures for the preparation of blood. Other blood preparation options are possible depending on the objectives of the study.

Before the test, the blood tube is gently shaken for 5-10 seconds. Using an automatic pipette dispenser, a sample of prepared blood is applied to the surface of a transparent hydrophobic cell in the form of a lying drop with a volume of 20-30 μl and a base diameter of 4-5 mm. It is not allowed to spread a drop of blood beyond the border of the cell and the presence of air bubbles in it.

A cell with a droplet sample for 10 s is placed in the chamber of the primary transducer 8 (PP) of the device between the source 3 (AI) and the optical radiation receiver 4 (PI) and is irradiated with the light flux from the optical radiation source 3 (AI). The luminous flux passed through the drip sample 1 (PR) enters the optical radiation receiver 4 (PI), the signal from which is amplified by the photocurrent amplifier 14 (US) and is supplied to the recording device 15 (RU).

During a given time interval, starting from the moment of placing the cell with a drop in the chamber of the primary transducer and then every 30 s (or more often) using the recording device 13 (RU), measure the output voltage U _{output of the} photocurrent amplifier 14 (US) of the device. It has been experimentally shown that the most informative range of study time for sedimentation of cells in a droplet sample is 15-20 minutes.

The change in the magnitude of the output voltage U _{output of the} photocurrent amplifier recorded by the described method is linearly associated with an increase in the height of the plasma layer (h) above the surface of the settling cell layer (Fig. 4). Therefore, the graph constructed from the results of measurements of the output voltage U _{output of the} photocurrent amplifier versus time (graph 5.1 in FIG. 5) reflects the change in the position of the upper boundary of the layer of sedimenting cells in the droplet sample (graph 5.2 in FIG. 5), i.e., the settling dynamics blood cells, and can be used to analyze the sedimentation process.

The value of the height of the plasma layer (h) above the surface of the settling cell layer in order to prove a linear dependence of the change in the output voltage U _{output of the} photocurrent amplifier on this parameter was measured from the enlarged images of the lateral projection of the droplet (Fig.6) obtained in the process of sedimentation using a video camera 9 (VK) devices.

During clinical trials of the proposed method for assessing the dynamics of sedimentation of blood cells, 74 people aged 16 to 79 years (30 women and 44 men) were examined. The studied group included both subjectively healthy people and patients with inflammatory processes in the body (acute appendicitis, acute cholecystitis, purulent wounds, gangrene, frostbite, acute pneumonia, bronchial asthma, etc.).

For each subject, a blood sampling and blood test procedure was carried out using the proposed method. Simultaneously with the blood test by the proposed method, the ESR of the blood of the subjects was measured according to the standard Panchenkov method. 7. examples of graphs obtained during the study are given, which reflect the dynamics of cell sedimentation in a drop sample for blood samples with different ESR values measured according to Panchenkov.

When analyzing the results, the dynamics of cell sedimentation in a droplet sample were compared with the ESR values obtained by the standard Panchenkov method. The closest relationship between the ESR values obtained by the Panchenkov method is observed with an indicator reflecting a change in the output voltage U _{output of the} photocurrent amplifier 14 (US), measured at the initial moment of subsidence U ( _t0 ) and for 10 min U ( _t10 ): U _t10 - U _t0 . As can be seen from the dependence shown in Fig. 8, which is based on an analysis of the data of 74 examined, a direct proportional relationship of these indicators with a correlation coefficient of at least 0.93 is observed.

In a number of patients who underwent etiotropic and pathogenetic therapy, a repeated analysis of blood samples was performed. A number of comparative experimental graphs are presented in Figs. 9.1-9.6. It was found that when examining patients undergoing treatment and having a positive dynamics in the development of the pathogenetic process, repeated curves of the dynamics of cell sedimentation had a lower slew rate (Fig. 9.1-9.4). For patients in whom the disease progressed, the curves during repeated measurements had a high slew rate or practically did not differ much from the original curves (Fig.9.5-9.6).

The initial portion of the photometry curve of the droplet sample (1-2 min) to the characteristic inflection indicated by point “b” (Fig. 5), after which the rate of rise of the curve sharply increases, carries information about the aggregation processes in the sample and can also be taken into account when analyzing the process subsidence.

Thus, the proposed method allows to reduce the amount of blood required for the study, to reduce the analysis time, to increase the information content of the analysis, as well as to reduce the number of random factors affecting the cell sedimentation process.

Claims (1)

A method for determining the dynamics of sedimentation of blood cells, characterized in that, using an automatic dispenser, the analyzed blood with an anticoagulant is placed on a transparent horizontal hydrophobic surface in the form of a lying drop, the central axial part of which is illuminated from below by a light flux of 1-1.5 mm in diameter falling from the bottom of the drop and register the dynamics of cell sedimentation by measuring over a given time interval the changes in the values of the intensity of the light flux passing through a drop of blood in e central axial region.

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