RU2513999C1 - Early diagnostic technique for severity and method for prediction of clinical outcome of acute radiation injury - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: peripheral blood erythrocytes are analysed. The assessment of erythrocyte analysis is ensured by introducing an integral value that is a relative shift of a mean radius of peripheral blood erythrocytes (RSMR PBE) as compared to a normal value, and if 72 hours after the total radiation RSMR PBE increases as compared to the normal value by more than 10%, the moderate acute radiation injury is diagnosed, and the animal is expected to survive; if 72 hours after the total radiation RSMR PBE increases as compared to the normal value by 10% or more, a lethal dose is diagnosed, and the animal is expected to die for 30 days.

EFFECT: early diagnosis of the severity and prediction of the clinical outcome of the radiation injury.

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Description

The invention relates to medicine, in particular to radiation biology, and can be used for early diagnosis of severity and prediction of the outcome of acute radiation injury due to the introduction of an integral indicator - the relative shift in the average radius of peripheral blood red blood cells.

At the moment, a very urgent problem of practical medicine is the creation of an express method for early diagnosis of the severity of radiation damage, which, along with predicting the outcome of acute radiation damage, would also contribute to the urgent conduct of therapeutic and preventive radiation protection measures after mass lesions.

A known method for the diagnosis of radiation damage to people by detecting autoantibodies in blood serum by indirect immunofluorescence (NRIF) on cryostatic thymus media and a 14-day culture of thymic epithelial cells in newborn mice (Abstract "Abstract: Human Immune Status and Radiation." - M., 1991, - p.31).

However, cryopreservation of cells and tissues, the use of laboratory animals, immunofluorescence and immunohistochemical techniques are very expensive, complex and lengthy methods, the assessment of the results of which is also subjective.

Relatively close in terms of the goal is a method for diagnosing the severity of acute radiation injury (patent RU No. 2099709, G01N 33/483, publ. 12/20/1997) by examining peripheral blood, which consists in simultaneously determining the strength of red blood cells, white blood cell count and erythrocyte aggregation half-life with the subsequent establishment of a correlation between the severity of acute radiation injury and the maximum deviation of the severity index of radiation injury, which is calculated by the formula J = Ud / τ (L + n), where J is the severity index and radiation injury (relative units); Ud is the maximum strength of red blood cell aggregates (B); τ is the half-life of red blood cell aggregation (s); L is the content of leukocytes (thousand / μl); n is a correction factor, n is 0 when L is not equal to zero, n is 0.1 when L is zero, and with a maximum deviation J recorded in the range from 0.31 to 3.0, light radiation damage is diagnosed, from 3.1 to 9.0 - average, from 9.1 and above - severe radiation damage, and in the range of values of J from 0.05 to 0.30, the norm is diagnosed.

The main disadvantages of this method are, firstly, the complexity and high cost of the method; secondly, the limited use of the method due to a sharp decrease in the level of leukocytes and lymphocytes (up to a complete absence) at high and even medium doses, which does not allow differentiating the severity of acute radiation injury and predicting its outcome, and also leads to low sensitivity of these methods .

The closest technical solution, taken as a prototype, is a method for diagnosing the severity of acute radiation injury [Mkoyan F.A. The diffractometric analysis of the erythrocyte population of white rats in radiation injury / F.A. Mikoyan. - Yerevan, 1994. - 125 p.] Based on the study of characteristic changes in the size composition of peripheral blood erythrocytes in the RPP according to the modal size of peripheral blood erythrocytes (MPEC) by laser diffractometry of a dry blood smear. The severity of acute radiation injury is judged by the magnitude of the relative shift of the MPEC compared to the norm, which is obtained using a diffractometric assessment of this parameter in the first hours-days of RPP. Prediction of the outcome of acute radiation injury is made by comparing the relative shifts of MREPK compared with the norm for different stages of RPP, followed by finding the corresponding point on the dose-effect curve three days after radiation injury.

The proposed diffractometric method for assessing MREPK has a number of significant drawbacks: firstly, the results of the analysis substantially depend on the choice of the site on a dry blood smear (subjective factors); secondly, the smear drying process leads to a change in the size of red blood cells, thirdly, the semi-automated version of the technical implementation of the method creates additional difficulties, which leads to a 1-2% loss in accuracy.

The technical result of the invention is to improve the accuracy of early diagnosis of severity and predict the outcome of acute radiation injury in the early post-radiation period (RPP).

The technical result is achieved by the fact that in the method of early diagnosis of severity and prediction of the outcome of acute radiation injury by examining peripheral blood erythrocytes, the new one is that an integral indicator is introduced for the assessment - the value of the relative shift of the average radius of peripheral blood erythrocytes (BOS SREPK) compared to the norm and carry out an automated assessment of this indicator by computer processing of digital information of the red blood cell microimage in a thin layer of peripheral with the help of an optical microscope and a CCD camera at different stages of the early post-radiation period, and if 72 hours after total irradiation, the BOS of the SREPK increases by more than 10% compared to the norm, then they diagnose acute radiation injury of moderate severity and predict that the animal will survive if 72 hours after total exposure to BOC, SREPK decreases by 10% or more compared to the norm, then a lethal dose is diagnosed and the animal is predicted to die within 30 days.

The invention is illustrated by drawings. Figure 1 presents a block diagram of a device for digital processing of microimages, with the help of which the average radius of the population of red blood cells of a certain area of a thin layer of peripheral blood is estimated. Figure 2 presents a graph of the time dependence of the relative shifts (in percent compared with the norm; norm (N) -0%) of the average radii of erythrocytes in rats subjected to total single exposure (data are normal and 24, 48 and 72 hours after irradiation doses of 300 P, 600 P and 1000 P). Figure 3 presents a graph of the dose dependence of the relative shifts (in percent compared with the norm; norm (N) -0%) of the average radii of rat erythrocytes three days after total single exposure (doses 300P, 600P and 1000 P).

The essence of the method lies in the fact that they estimate the average radius of peripheral blood red blood cells (SREC) for a sufficiently large sample using the technique for estimating the average radius of a set of objects that are close in shape to a circle by computer processing digital data of a micro image of a thin layer of the studied drug. The severity of acute radiation injury is judged by the magnitude of the relative shift of the SREK compared with the norm - after the first, second and third days of the early post-radiation period (RPP), based on the results of the assessment of this parameter. Prediction of the outcome of acute radiation injury is made by comparing the relative shift (BOC) of the SRECC compared with the norm for different stages of the RPP, with the subsequent finding of a point on the correlation curve, the dose - effect three days after radiation injury.

It is generally accepted that in the process of radiation sickness caused by various doses of radiation, the shift dynamics of the studied parameter reflects the change in the severity of radiation damage to the body. The severity of radiation damage to the body depends on two main factors: the dose of radiation and the initial functional state of the body. Consequently, the need for developing a new method for early diagnosis of the severity of acute radiation injury is obvious, which will allow for an individual approach to achieving the goal without a lot of time and money. It is also obvious that it is necessary to choose a research parameter that can be measured throughout the entire RPP. This is especially important in emergency situations, when there is a need for examination of victims of mass destruction.

The implementation of the method of early diagnosis of severity and prediction of the outcome of acute radiation injury will become possible using the well-known methodology for assessing the average radius of a population of objects by performing a two-dimensional Fourier transform from an image of objects and finding a two-dimensional periodogram of this image [Kveglis L.I. Dissipative structures in thin nanocrystalline films / L.I. Kveglis, V.B. Kashkin. - Krasnoyarsk: Siberian Federal University, 2011, - p.88-101].

These differences allow us to conclude that the proposed technical solution meets the criterion of "novelty." Signs that distinguish the claimed device from the prototype are not identified in other technical solutions when studying this and related areas of technology and, therefore, provide the claimed solution with the criterion of "inventive step".

Evaluation of the average radius of the population of red blood cells of a portion of a thin layer of peripheral blood is carried out using the device, a block diagram of which is presented in figure 1. The device consists of an optical microscope 1, a special holder 2 for a blood sample, a charge-coupled device (CCD camera) 3 for digitally recording micro images of the studied area of a thin layer of blood and computer 4 for processing digital information.

A thin layer of peripheral blood is prepared according to the following scheme: blood is sampled from the test object and diluted with a 0.8% sodium chloride solution 200 times. Then the glass slide is wetted with distilled water and, using a micropipette, 1 μl of diluted blood is applied to the glass slide, wait 20 seconds and the layer is covered with a coverslip. After that, immediately using an optical microscope and a CCD camera, a digital image of a micro image of a set of red blood cell microobjects is digitally recorded.

As a result of this sequence of digital registration and analysis of the corresponding portion of a thin layer of peripheral blood, it is possible to overcome a number of disadvantages of the prototype. Firstly, by unifying the technique for preparing a thin layer of blood and choosing a fixed area for digital recording of red blood cell microimages, the dependence of the obtained data on the characteristics of an individual smear is minimized (the role of subjective factors is significantly reduced). Secondly, the digital registration of the area of the dried thin layer eliminates the influence of the drying process on the obtained data. Thirdly, the accuracy of the method is significantly improved due to the complete automation of data processing.

The inventive method is implemented as follows. For each organism subjected to a single total irradiation with a certain dose, a thin layer of peripheral blood is prepared normally (a day before irradiation) and at different stages of RPP - after 24; 48 and 72 hours after irradiation, after which a blood sample is fixed in a special holder 2, and after that, using an optical microscope 1 and a CCD camera 3, digital recording of the micro image of the studied area of a thin layer of blood is performed. All information received is transmitted to a computer 4 for digital processing. All further work is carried out with a digital database of microimages of the studied area of a thin layer of blood.

This technology leads to a significant simplification of the implementation of the method, increasing expressivity, labor productivity, reliability of the analysis results, as well as its unification and expansion of the scope.

Example 1. Rat, weight 195 g, male. The dose of total exposure is 300 R (sublethal dose for this animal species, when no death is detected during the first 30 days of RPP). According to the described scheme, 4 samples are prepared for this animal - the norm; 24 hours; 48 hours and 72 hours and evaluate the average radius of red blood cells of peripheral blood (figure 2). It can be seen from the figure (continuous line) that already on the second day of the RPP, the parameter under study - the BOS of the SREK increases by 6% and exceeds 10% after three days. In the literature, this dose is called the moderate dose. By analogy with this, it can be argued that when registering an increase in the BER of the SRREK compared with the norm of more than 10%, acute radiation injury of moderate severity occurs, and it is predicted that the animal will survive.

Example 2. Rat, weight 215 g, male. The dose of total exposure is 1000 R (an absolutely lethal dose for this animal species, when a 100% death is detected during the first 7 days of RPP). According to the described scheme, 4 samples are prepared for this animal - the norm; 24 hours; 48 hours and 72 hours and evaluate the average radius of red blood cells of peripheral blood (figure 2). It can be seen from the figure (dashed line with dots) that already 24 hours after irradiation, the studied parameter decreases by 7%, and after 72 hours this decrease reaches 14%. Thus, if the WOS of SREPK after 72 hours becomes 14% or more less than the norm, then in severity this corresponds to an absolutely lethal dose and it can be predicted that the animal will die within 7 days.

Example 3. Rat, weight 205 g, male. The total radiation dose is 600 R (the half-fatal dose for this animal species is LD50 / 30, when during the first 30 days of RPP one half of these animals die and the other half survives). According to the described scheme, 4 samples are prepared for this animal - the norm; 24 hours; 48 hours and 72 hours and evaluate the average radius of red blood cells of peripheral blood (figure 2). The study showed that for a dose of 600P there is a significant qualitative and quantitative difference in the OSI values of the SREPK between dying and surviving subgroups (two dashed lines with opposite dynamics in behavior in FIG. 2). In the surviving subgroup, there is an increase in the BOS of the SREPC during the whole RPP and after 24 hours the value is more than 7%. After 48 hours, more than 8%, and during the third day reaches 12%. For the dying subgroup, there was a significant decrease in the VOS of the SREPK 72 hours after radiation injury, more than 10% compared with the norm. It is important to note that, at a dose of 600P, the behavior pattern of the BOS SREPK in the RPP exhibits a dynamics of change specific for the sublethal (300 P) and absolutely fatal (1000 P) doses.

Thus, it can be argued that at an irradiation dose of LD50 / 30, dying animals behave similarly to an absolutely lethal dose, and surviving animals are similar to a sublethal radiation dose (Fig. 2). This fact is quite clearly demonstrated in figure 2 and it can be called the "splitting effect". Figure 3 shows that the "splitting effect" occurs when irradiated with a dose of 600 R. Thus, based on the constructed graph (figure 3), a forecast of the outcome of acute radiation syndrome is made, both for sublethal and absolutely lethal doses, and for half-fatal radiation doses.

The proposed method for early diagnosis of the severity and prediction of the outcome of acute radiation injury allows a quick and reliable separation of the irradiated organisms into subgroups according to the severity of the damage with the same dose of environmental radiation, especially when the radiation level is close to a half-fatal dose for this type of animal. Also, the inventive method improves the accuracy of early diagnosis of severity and predicting the outcome of acute radiation injury in the early post-radiation period, leads to a significant simplification of the implementation of the method, increased expressivity, labor productivity, reliability of the analysis results, as well as its unification and expansion of the scope.

Claims (1)

A method for early diagnosis of severity and prediction of the outcome of acute radiation injury by examining peripheral blood erythrocytes, characterized in that an integral indicator is introduced for the assessment — the relative shift of the average radius of peripheral blood erythrocytes (BOS SREPK) compared to the norm and an automated assessment of this indicator by computer processing digital information of a red blood cell microimage in a thin layer of peripheral blood using an optical microscope and CCD-k measures at different stages of the early post-radiation period, and if 72 hours after the total exposure, the BER of the SREDC increases compared to the norm by more than 10%, then they diagnose acute radiation injury of moderate severity and predict that the animal will survive if 72 hours after the total exposure of the BOC of the SREDC decreases compared with the norm by 10% or more, a lethal dose is diagnosed and the animal is predicted to die within 30 days.

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