RU2276788C2 - Spectropolarimetric express method of estimation of degree of heaviness of condition of patients based on usage of blood serum - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: spectropolarimetric express method of estimation of degree of heaviness of condition of patients is based upon testing blood serum, of patients in isotonic solution by passing polarized and non-polarized light through it. Intensity of radiation is measured within values of fluorescent polarizer among 0° and 90° in any 10° at values of polarizer's angle at the side of excitement among 0° and 90°.

EFFECT: improved precision of method.

1 tbl, 6 dwg

Description

The invention relates to measuring equipment in medicine, namely clinical and laboratory diagnosis of the severity of the condition of patients.

A known method for assessing the functional state of patients, based on chemiluminescence - spontaneous emission of biological material, which in its sensitivity is close to radio-immune, while remaining quite simple and express (Chernitsky EA, Slobozhanina EI "Spectral luminescent analysis in medicine" , Minsk, Science and Technology, 1989). It is 2-3 orders of magnitude higher in sensitivity than the method of electron paramagnetic resonance (Veselovsky V.A. “The Chemiluminescent Analysis Method in Biology.” In Sat: Spectroscopic Methods of Research in Physiology and Biochemistry. L .: Nauka, 1987, p. 34- 37).

Spontaneous serum chemiluminescence is associated with the constant occurrence of lipid peroxidation reactions in the blood plasma, accompanied by luminescence and under physiological conditions (A. Zhuravlev, "Spontaneous Biochemiluminescence of Animal Tissues". In: Biochemiluminescence. - M .: Nauka, p. 3-30), since one-electron oxygen reduction is a small but relatively constant fraction of the total oxygen reduction in various oxygen-consuming cells (Vladimirov Yu.A., Sharov A.P., Malyugin E.F. "Hemilyum nestsentsiya plasma in the presence of the ions Fe 2+. "Biophysics 1973, t.18, s.148-152). However, it is not accurate enough.

The express method is known for assessing the severity of the condition of patients by determining the degree of destabilization of erythrocyte membranes by studying their biophysical properties (Yakubova P.P., Murin A.V. AC 1636731 A1, G 01 N 21/00, 1990). The method consists in examining a sample of a suspension of erythrocyte membranes in an isotonic solution, passing unpolarized light through it and determining the degree of polarization, passing polarized light through the same sample, determining the degree of background depolarization of the sample at an excitation wavelength of 493 nm, determining the ratio of the first parameter to the second and an increase in this ratio compared with the physiological norm for determining the degree of destabilization of erythrocyte membranes, which correlates with the severity of the condition sick. The method is modern, with a high degree of accuracy reflects changes in the biophysical properties of red blood cells, however, it cannot serve as an integrated assessment of changes in the physicochemical characteristics of vital systems of the body, reflecting only the properties of one of the links of homeostasis.

Closest to the proposed one is the spectropolarimetric express method for assessing the severity of the condition of patients by examining a sample of biological material in an isotonic solution, passing unpolarized light through it and determining the radiation intensity, then passing through the same sample polarized light and determining the radiation intensity, while blood serum is used as biological material, the maximum excitation wavelength is set to 360 nm, and the maximum fluorescence wavelength ntsii - 450 nm. By coefficient

where I 90/0 is the radiation intensity of the selected wavelengths when the polarizer of the exciting radiation is set to 90 °, and the polarizer of the emission radiation to 0 °;

I 0/0 - the radiation intensity of the selected wavelengths when both polarizers are set to 0 °,

determine the degree of metabolic changes in the body, allowing to judge the severity of the condition of patients. In addition, a clinical blood test is performed and the concentration of medium molecular peptides in the blood serum is determined (RF Patent N 2168165 C2, Bull. N 15, 05/27/2001). However, the method is not accurate enough.

The purpose of the invention is to increase the accuracy of determining the severity of the condition of patients.

This goal is achieved by studying a blood serum sample in an isotonic solution by passing exciting radiation through the same sample and recording fluorescence radiation at a maximum excitation wavelength of 360 nm, a maximum fluorescence wavelength of 450 nm, and then determining the radiation intensity of the selected wavelengths, where I (90 / 0) is the radiation intensity when the exciting radiation polarizer is set to 90 °, and the fluorescent radiation polarizer is set to 0 °, I (0/0) is the radiation intensity when and polarizer are set to 0 °. The study is characterized in that in addition, after determining I (90/0), the position of the polarizer on the excitation side is not changed, the radiation is measured every 10 ° on the fluorescence side and a polarized radiation curve is obtained: after determining I (0/0), the position of the polarizer is not changed to side of the excitation, measure radiation every 10 ° on the side of fluorescence and get a curve of non-polarized radiation, then assess the severity of the condition of the patients according to the position of the two curves obtained compared with the norm in the system coordinates: radiation intensity - the value of the angle of the polarizer on the side of fluorescence.

Spectropolarimetric express method for assessing the severity of the condition of patients is as follows. Examine the patient's blood serum at a dilution of 1:10 0.85% NaCl solution (sample).

The studies are carried out on an MPF-4 Hitachi spectrofluorimeter (Japan) in quartz cuvettes with a diameter of 10 mm using polarizing prisms on the side of the exciting and fluorescence radiation of the given wavelengths. The operating mode of the Hitachi MPF-4 spectrofluorimeter: excitation radiation wavelength - 360 nm, fluorescence wavelength - 450 nm, diode voltage 700V, slit widths of monochromators on the side of excitation and fluorescence, respectively, 10 nm each, signal sensitivity of the sample - 30.

1. A sample of a blood serum in an isotonic solution is irradiated with unpolarized light. The exciting monochromator polarizer is set to zero (0 °) and its position is not changed. The polarizer of the fluorescent monochromator is set to the zero position (0 °), the radiation intensity is determined (I 0/0), then the polarizer on the fluorescence side is changed by 10 degrees and the intensity of the radiation transmitted through the sample is measured (I 0/10), and so on , increasing the angle of the polarizer on the fluorescence side by 10 degrees to the level of 90 degrees (I 0/90). Thus, the polarizer on the excitation side is constantly set to zero (0 degrees), the polarizer on the fluorescence side is set from 0 to 90 degrees with a “step” of 10 degrees. In each position, the value of the radiation intensity is recorded. A curve of unpolarized radiation transmitted through a blood serum sample is obtained.

2. Repeat the experiment with the same sample, but set the polarizer of the exciting monochromator 90 degrees and do not change the position of the polarizer on the side of the excitation, and the polarizer on the fluorescence side - 0 degrees, again measure the radiation intensity at given wavelengths (I 90/0) , and so on until the measurement of radiation intensity (I 90/90). Thus, the polarizer on the excitation side is constantly set at 90 degrees, the polarizer on the fluorescence side is set from 0 to 90 degrees with a “step” of 10 degrees. In each position, the value of the radiation intensity is recorded. A curve of polarized radiation transmitted through a blood serum sample is obtained.

3. Next, combining both polarization curves in the same coordinate system "radiation intensity is the value of the angle of the polarizer on the fluorescence side", evaluate the nature of the intersection of the curves and determine the angle of their intersection.

The study is economical in time (takes 10 minutes) and the use of reagents (only physiological saline is required), is highly sensitive and reliable.

Studies were conducted in 129 resuscitation patients of varying severity. 2 groups of patients were allocated: 1) moderate severity (patients were transferred from the intensive care unit and discharged home with a successful outcome) - 86 patients; 2) a heavy contingent of patients with a fatal outcome - 43 patients. The control group consisted of donors of both sexes aged 18 to 36 years (N = 39). A total of 168 patients were examined.

Figure 1 shows the nature of changes in the intensities of polarized radiation passing through a sample of blood serum in the control group (donors). In patients with moderate severity, the nature of the curve changes slightly, assuming a more asymmetric shape with a clear decrease in the "weight" fraction of the left shoulder (figure 2). Moreover, the intersection angle of the polarization curves shifts from the level of 38 degrees (in the control group) to the direction of decrease (Table 1), reaching the average values in the group of patients with moderate severity of the order of 34 degrees.

Table 1. The angle of intersection of the polarization curves in patients of varying severity: Control (donors) Moderate patients Severe contingent of patients (dying patients at various times until death) Dying sick Day to death: 5 3 2 one N (amount) 39 86 43 5 6 8 22 The average value of the angle of intersection of the curves (degrees) 38 34.5 fifteen 16 eighteen fifteen 12 Standard deviation four 5.3 13 fifteen eighteen fifteen fourteen Differences: Significant Significant Significant

The degree of this change in the nature of overlapping polarization curves increases in the heavy patient population (Fig. 3), acquiring a "qualitative" jump in the modification of the shape of polarization curves in patients with a high degree of risk (Figs. 4-6).

For this group of patients, the absence of “intersection” of the polarization radiation curves is significant, respectively, the angle of intersection of the polarization curves is 0 degrees, which is determined by the fundamentally different nature of the interaction of light with a sample of biological material. Moreover, a similar trend in the biophysical properties of the sample can be traced already 3 days before death (Fig.6).

Thus, the studies allow us to draw the following conclusion.

The proposed spectropolarimetric express method for assessing the severity of the condition of patients using blood serum in comparison with the prototype is more accurate, since 20 definitions of radiation intensity are carried out to construct polarization curves, and not 2 definitions. Accordingly, the degree of severity is estimated not by the calculated coefficient, but by the nature of the intersection of the polarization curves, which gives a detailed, more visual picture of the nature of the change in the intensity of light emission passing through the blood serum sample. This, in turn, makes it possible to conduct studies with a lower probability of systematic error.

The proposed spectropolarimetric method for assessing the severity of the condition of patients allows taking into account the number of patients with a low probability of developing complications and death and, conversely, patients with an inevitable unfavorable prognosis. This methodology allows you to calculate and argue the real need of material resources for the intensive care unit.

Claims (1)

Spectropolarimetric express method for assessing the severity of the condition of patients, which consists in examining a blood serum sample in an isotonic solution by passing exciting radiation through the same sample and recording fluorescence radiation with a maximum excitation wavelength of 360 nm, a maximum fluorescence wavelength of 450 nm, and then determine the intensity radiation of selected wavelengths, where I (90/0) is the radiation intensity when the polarizer of the exciting radiation is set to 90 °, and the polarizer flu of rescent radiation - by 0 °, I (0/0) - radiation intensity when both polarizers are set to 0 °, characterized in that, additionally after determining I (90/0), the position of the polarizer on the side of the excitation is not changed, the radiation is measured every 10 ° on the fluorescence side and get a polarized radiation curve; after determining I (0/0), the position of the polarizer on the side of the excitation is not changed, the radiation is measured every 10 ° on the side of the fluorescence and a curve of unpolarized radiation is obtained, then the severity of the patient’s condition is estimated by the position of the two obtained curves compared to the norm in the coordinate system: radiation intensity - the value of the angle of the polarizer on the side of fluorescence.

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