RU2470304C2 - Method of determining homologous blood transfusion in doping control of athletes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to clinical chemistry and, in particular, to methods of determining blood composition and can be used in doping control of athletes. Essence of method: blood sample is divided into 10 aliquots, suspensions of erythrocytes of each aliquot are prepared. After that, erythrocytes are successively stained with primary and secondary antibodies with fluorescent labels with antigens C, c, E, Fy^a and Fy^b, Jk^a and Jk^b, S, P1 and N and fluorometric analysis by flow cytometry is carried out. Then, histograms of ratio of minor and major erythrocyte populations are built. If in all histograms of analysed sample single peak (population) is present, analysis results are interpreted as absence of signs of homologous blood transfusion. In case of presence of two and more histograms with two peaks, results of analysis are interpreted as positive. In case of presence of one histogram with two peaks (two populations), results of analysis are put into the category of suspicious, after which analysis are repeated after 30, 60 and 90 days, and indices of reduction or disappearance of minor population, final decision about homologous blood transfusion is made.

EFFECT: application of method makes it possible to increase accuracy and reliability of blood transfusion detection and provides possibility of carrying out additional control of athletes of risk group.

2 tbl, 4 dwg, 14 ex

Description

The invention relates to medicine, and more specifically to laboratory diagnostics and, in particular, to methods for determining the composition of blood, and can be used, for example, in doping control of athletes.

Known methods of blood analysis by analysis of infrared spectra of blood or luminescent analysis of blood components or chemoluminescent analysis to determine individual characteristics of the blood, such as the dynamics of the dispersion of physical parameters, platelet glow, biological compatibility of the red blood cell mass of the donor and recipient, respectively [1-3].

The disadvantage of these technical solutions is low information content and limited scope.

There are also known methods for analyzing cells and, in particular, blood by flow cytometry, in which radiation passing through a stream of single cells is recorded and the results of transmission are further interpreted [4-5].

The disadvantage of these technical solutions is also low information content and limited scope.

There is also known a method of conducting a blood test and a blood analyzer for its implementation, according to which a blood sample is mixed with a diluting and / or solvent reagent, a blood sample stream is formed, which is exposed to coherent polarized radiation with a wavelength of 330-680 nm, directing it along the flow axis , determine multi-angle scattering by blood cells in a stream, detect and count blood cells by multi-angle light scattering by them [6].

The disadvantage of this method is also the low information content of the results: obtaining generalized data mainly on the quantitative composition of blood without determining the differences between homogeneous cells and other blood components.

The closest to the claimed object in its technical essence and the technical result achieved is a method for determining transfusion of homologous blood by blood analysis, including the preparation of a suspension of red blood cells (EC), staining of minor EC antigens according to Duffy and Kidd primary and secondary antibodies with fluorescent labels and subsequent fluorometric analysis EC suspensions by flow cytometry with registration of light scattering and fluorescence intensity and analysis of histograms for the presence of a population EC [7].

The disadvantage of this method is the low reliability of detection of homologous blood transfusion, associated with a high probability of obtaining a false-positive result, since a very narrow group of coverage of the likely signs of transfusion and, accordingly, associated antigens is involved, which makes it difficult to correctly interpret the analysis results.

The technical result, the achievement of which the creation of this invention is aimed, is to increase the accuracy and reliability of determining blood transfusion by involving additional groups of coverage of signs of probable transfusion and determining indicators of decrease or disappearance of a minor population of EC in time, while reducing the duration of the analysis and its cheapening.

The technical result is achieved by the fact that in addition to eight antigens (C, c, E, Fy ^a , Fy ^b , Jk ^a and Jk ^b and S), ECs are additionally stained with primary and secondary antibodies with fluorescent labels in antigen groups “P” and “N "and then, based on the recorded intensities of scattered light and fluorescence, histograms of the ratio of minor and major populations of EC are plotted and, in the presence of a single peak (population) on all histograms of the analyzed sample, the results of the analysis are interpreted as the absence of signs of transfusion logical blood, in the presence of one histogram with two peaks (two populations), the analysis results are translated into the category of suspicious, and if there are two or more histograms with two peaks, the results of the analysis are interpreted as positive and then, in the category of suspicious, repeat analyzes are carried out after 30, 60 90 days, and in terms of a decrease or disappearance of the minor population, they make the final decision on transfusion of homologous blood.

Currently, to achieve high results, athletes are again returning to blood doping. The end result of blood doping is an increase in hemoglobin in the blood and an increase in the supply of muscle tissue with oxygen and, as a result, an increase in stamina of an athlete.

Blood doping is diverse and involves the introduction of blood, red blood cells (EC), related blood products (including plasma), as a rule, shortly before the start. The use of blood doping, including autologous or homologous blood transfusion (blood transfusion), is prohibited in all sports in both competitive (In-competition) and non-competitive periods (Out-of-competition). Homologous blood transfusion means that an athlete receives blood transfusions with similar characteristics: blood type and Rh factor.

In practice, there are no two completely identical people in terms of the antigenic composition of EC, with the exception of identical twins. This is due to the large number of antigens of blood groups of EC and their variability between individuals. Currently, about 236 EC antigens are known, which form 29 blood group antigen systems. That is why the EC of homologous blood of a donor almost always differs from the recipient's EC in the expression of several minor antigens of blood groups due to differences in phenotypes. (The phenotype of EC is a collection of antigens present or absent on the erythrocytes of an individual). Thus, in practice, the definition of transfusion of homologous blood consists in identifying the EC phenotype of the studied blood sample of the athlete.

According to the authors, in order to obtain reliable results for determining the transfusion of homologous blood, it is necessary and sufficient to identify the EC phenotype for 10 antigens of moderate occurrence. The average incidence implies that the incidence of antigen in the population is more than 25%, but less than 85%. For phenotyping of EC, Rhesus antigens C, c, E, Fy ^a and Fy ^b antigens (Duffy system), Jk ^a and Jk ^b antigens (Kidd system) and S antigen (MNSs system), as well as "P" antigens can be selected and "N".

The authors note that the choice of antigens "P" and "N" is due to the fact that they are present in the blood of 50% of the human population, and therefore it is highly likely that the donor and recipient will also differ by 50% in these antigens (almost to the maximum).

When staining EC with antibodies, IgG and IgM class antibodies specific to the selected antigens can be used. As primary antibodies, -F (ab ') ² specific antibody fragments of murine antibodies conjugated with fluorescein isothiocyanate (FITC) can be used. First, ECs are stained with antibodies to the antigens studied, and then they are labeled with secondary antibodies with a fluorescent dye directed against the primary antibodies.

An ISOTON II Diluent flow cytometer buffer (Beckman Coulter, USA) can be used as a working buffer. ID-Cell Stab reagent (DiaMed, Switzerland) can be used as a stabilizing solution for preparing an EC suspension. As additional reagents - Phosphate-buffered saline PBS (Amresco, Canada), bovine serum albumin BSA (Sigma, USA), sodium azide NaN ₃ (Sigma, USA).

As equipment for conducting cytofluorimetric analysis, for example, a Beckman-Coulter FC500 series flow cytometer can be used, and CXP 2.0 program can be used as software. The systems described in [6] and [8] can also be used.

As equipment for counting the number of red blood cells, for example, a Sysmex XT-2000i automatic hematology analyzer (Sysmex Corporation, Japan) can be used.

As auxiliary equipment can be used, for example:

- a centrifuge for microtubes "Eppendorf-5417R" (Eppendorf, Germany);

- centrifuge with a bucket rotor "Rotina 46R" (Hettich, Germany);

- centrifuge for glue cards ID (DiaMed, Switzerland);

- analytical balance "Analytical Plus" (Ohaus, USA);

- Milli-Q water treatment system (Millipore, France);

- contact mixer "Vortex-Genie 1" (Scientific Industries Inc., USA);

- magnetic stirrer “RCT basic” (IKA, Germany);

- Roller-mixer “Stuart SRT9D” (Barloworld Scientific, Great Britain).

The invention can be implemented as follows.

An EC suspension is prepared by diluting a blood sample with a working buffer to a selected final concentration (N × 10 ⁶ EC in 100 μl), 100 μl of the prepared EC suspension are taken, washed with a working buffer from the plasma, and during washing, the EC is precipitated by centrifugation and resuspended in the working buffer after washing. . Primary antibodies diluted in a working buffer to a pre-selected working concentration are added to the reconstituted suspension, intensively mixed and incubated. After incubation with primary antibodies, erythrocytes are washed three times in the working buffer, and then secondary antibodies are added to the EC suspension at the working concentration, mixed and incubated. Next, the EC is washed three times with a working buffer, resuspended in a certain volume of the working buffer and mixed. The resulting suspension of EC is subjected to cytofluorimetric analysis. The results of the analysis are displayed in the form of a series of histograms (in accordance with the number of antigens of the coverage group for signs of homologous blood transfusion) and based on the histogram readings, it is concluded that there are signs of homologous blood transfusion.

It should be noted that the inventive method for determining transfusion of homologous blood can significantly reduce the duration of doping control and significantly reduce the cost of its implementation.

For a better understanding, the invention can be illustrated, but not exhausted by the following examples of its specific implementation.

Example 1

10 antigens were selected for staining during the analysis: Rhesus antigens C, c, E, Fy ^a and Fy ^b antigens (Duffy system), Jk ^a and Jk ^b antigens (Kidd system), S antigen (MNSs system), and antigens "P" and "M".

A blood sample is taken from an athlete (biathlon) of obviously not transfused homologous blood in compliance with the rules for such an operation prescribed by WADA. An EC suspension is obtained by diluting a blood sample with a working buffer (PBS with 0.1% BSA and 0.02% sodium azide, pH 7.4) to a final concentration of 5 × 10 ⁶ EC in 100 μl. The amount of EC in the test sample was previously measured on a Sysmex XT2000i automated hematology analyzer. Then, the resulting suspension is divided into 10 aliquots. From the first aliquot, 100 μl of the prepared suspension of EC are taken and washed twice with the working buffer to remove the plasma. In the process of washing, ECs are precipitated by centrifugation for 2 minutes at 450 g. Next, EC are resuspended in 100 μl of working buffer to prevent agglutination. To the resulting suspension, add 50 μl of primary antibodies (Anti-C monoclonal, human IgM, clone MS-24), specific for antigen C, diluted in working buffer to a working concentration of 1:10, intensively mixed and incubated in the dark for 30 minutes at room temperature . After incubation with primary antibodies, the EC is washed three times with 500 μl of the working buffer, after which 50 μl of fluorescently labeled secondary antibodies (Goat F (ab ') ² Anti-Human IgM-, FITC) specific for the primary antibody used above at the working concentration are added to them 1:20. Stirred on a Vortex contact mixer and incubated for 30 minutes in the dark at room temperature. Next, the EC is washed three times with 500 μl of the working buffer. Then, the EC is resuspended in 300 μl of the working buffer with stirring. The resulting suspension of EC is subjected to cytofluorimetric analysis, the results are recorded in the form of a histogram, and the histogram is analyzed for signs of transfusion of homologous blood. Exactly the same as described above, the remaining nine aliquots of the sample are manipulated, except that each time the next antigen in the selected row is stained. Accordingly, each time the results are recorded in the form of a histogram and analyze it.

Figure 1 shows histograms of the analyzed EC samples without blood transfusion, labeled with: Rhesus C antigens, Rhesus C antigens, Rhesus antigens E, Fy ^a antigens, Fy ^b antigens, Jk ^a antigens, Jk ^b antigens, S antigen, antigens "P" and antigens "N".

As can be seen from the presented histograms, only one peak appeared on all histograms (major population of EC is 100%).

Examples 2-9

Analysis of the studied blood samples is carried out as in Example 1, except that the blood is examined after transfusion with a known content of major and minor populations of EC and the staining of antigens is varied (see the column "Antigens" of Table 1).

The conditions for the analyzes of Examples 2-7 are shown in Table 1

As follows from Table 1, a blood test with a known minor population of red blood cells for all ten antigens (Examples 2, 3 and 4) shows the presence of blood transfusion when the content of minor populations of red blood cells is 5.0-0.5%. At the same time, under the same conditions, a blood test involving nine antigens (Comparative Examples 5 and 6) does not confirm the presence of blood transfusion with a content of minor red blood cell populations of 1.5-0.5%. Examples 7, 8 and 9 comparative on the prototype (analysis involving only the first eight antigens) show the presence of blood transfusion with a content of minor erythrocyte populations of 5.0%. The low (1.5-0.5%) content of minor populations in this case is not determined.

Examples 10-14

The analysis of the studied blood samples is carried out as in Example 1, while the blood is examined according to Examples 2, 3 and 4 30, 60 and 90 days after the initial analysis. The results are shown in Table 2.

From Table 2 it follows (see Examples 13 and 14 comparative in prototype) that, with a high content of minor erythrocyte populations (5.0%), an analysis with eight antigens allows us to determine the presence of these populations in the suspicious category after 30 days, but not Further. At the same time, analysis by the proposed method (with ten antigens) allows to determine the presence of minor populations at an initial concentration of 5.0% after 90 days, and at 0.5% after 60 days.

Figure 2 shows histograms of a blood test containing 5% of minor ECs after 30 days (see Table 2, Example 10),

Figure 3 shows histograms of a blood test containing 5% of minor ECs after 60 days (see Table 2, Example 10),

Figure 4 shows histograms of a blood test containing 5% of minor ECs after 90 days (see Table 2, Example 10),

As can be seen from the description, examples of a specific implementation of the method and comparative examples, the inventive method can significantly improve the accuracy and reliability of determining blood transfusion by attracting antigens with a high degree of occurrence in a population of people, and additional monitoring of athletes at risk, to reduce the duration of doping control and reduce the cost of its implementation.

Information sources

1. RU 02148267 C1, IPC G01N 33/49, 1999.06.01.

2. RU 02230322 C2, IPC G01N 33/49, 2004.06.10.

3. RU 0219 3197 C1, IPC G01N 33/49, 2002.11.20.

4. EP 1399728 A2, G01N 33/49, 03.24.2004.

5. WO 2004/019047, G01N 33/49, 03/04/2004.

6. RU 02347224 C2, IPC G01N 33/49, 2004.06.10.

7. Proof of homologous blood transfusion through quantification of blood group antigens, Haematologica, 2003; 88: 1284-1295, (http://www.haematologica.org/2003_11/1284.htm) - prototype.

8. EP 1297334 A1, G01N 33/49, 04/02/2003.

Claims (1)

A method for determining homologous blood transfusion during doping control of athletes by blood analysis, which includes taking a blood sample, dividing the sample into aliquots, preparing a suspension of red blood cells of each aliquot, staining the minor erythrocyte antigens with primary and secondary antibodies with fluorescent labels, and subsequent fluorometric analysis of the erythrocyte suspension with erythrocyte with registration of light scattering and fluorescence intensity, characterized in that the blood sample is shared and ten aliquots, and the precipitated red blood cells were stained with antibodies against one of the antigens: C, c, ^{E, Fy a, Fy b, Jk} a, Jk b, S, «P» and «N», performed flow cytometric analysis, record the results as histograms, build histograms of the ratio of minor and major erythrocyte populations and, if there is one peak (population) on all histograms of the analyzed sample, the analysis results are interpreted as the absence of signs of homologous blood transfusion, if there are two or more histograms with two peaks, the analysis results are interpreted they are positive, if there is one histogram with two peaks (populations), the analysis results are transferred to the category of suspicious ones and in this case, repeated analyzes are performed after 30, 60 and 90 days and, according to the indicators of the decrease or disappearance of the minor population, they make the final decision on the transfusion of homologous blood.

Images