Diagnostic technique for erythrocyte stability in pregnant women suffering aggravated cytomegaloviral infection

IPC classes for russian patent Diagnostic technique for erythrocyte stability in pregnant women suffering aggravated cytomegaloviral infection (RU 2513456):

G01N33/68 - involving proteins, peptides or amino acids

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FIELD: medicine.

SUBSTANCE: blood serum is examined for TNFα; erythrocyte membrane lipids are analysed for ω-6 and ω-3 polyunsaturated fatty acids; that is followed by calculating the relation K=Σω-6/Σω-3, wherein Σω-6 is an amount sum of linoleic and arachidonic acid, %, Σω-3 is an amount sum of α-linolenoic, eicosapentanoic and docosahexaenoic acid, %. If blood serum TNFα is 110±1.97 pg/ml, while K is more than 0.67, developing hemic hypoxia is predicted.

EFFECT: method has enabled the high-accuracy prediction of developing unstable erythrocytes in the pregnant women suffering aggravated cytomegaloviral infection at the early stages of the clinical manifestations of hemic hypoxia.

1 ex

The invention relates to medicine, namely to methods of medical diagnostics, and can be used to diagnose the stability of erythrocytes in pregnant women with viral diseases such as cytomegalovirus infection.

It is known that phospholipids and included in their composition of saturated and polyunsaturated fatty acids are the main component of the membrane of erythrocytes (2, 6). Destruction of the phospholipid layer and the fatty acid residue, due to the high phospholipase activity, increases microviscosity membranes, transformation of red blood cells, which in turn causes premature hemolysis of the cells, the development of stagnation in the microvasculature and, as a result, hypoxia(1, 3, 8).

It is especially important to highlight the unstable pretheoretically state of erythrocytes in pregnant women with viral infections herpes group (HSV-1, 2, CMV) to prevent the development of intrauterine fetal hypoxia, when you want to preserve the health of the unborn child.

Identification of unstable pretheoretical status of red blood cells is crucial for the prevention himicheskoi hypoxia. However, the real evaluation of this condition is limited by the lack of adequate diagnostic methods. Known methods of evaluating the stability of erythrocyte membrane core is designed on the analysis of peripheral blood, for example, osmotic, acid and(or) mechanical resistance of erythrocytes. However, all known methods can only detect already formed morphofunctional changes of erythrocytes, which are the criteria for pathological conditions, including hypoxia. This leads to hypoxia in pregnant enters clinical form, accompanied by the risk of intrauterine fetal hypoxia, can cause pregnancy complications and requires adequate therapy.

There is a method of evaluation of osmotic resistance of erythrocytes (4)to evaluate the morphological and functional state of erythrocytes, based on the determination of the kinetics hypoosmotic (0,55% NaCl) hemolysis of erythrocytes. This assessment of the functional state of erythrocytes in the known method is the basis for the diagnosis of some forms of hemolytic anemia.

The disadvantage of this method is that it does not allow for early diagnosis of unstable pretheoretical status of erythrocytes and thus to diagnose the disorder of the structure of erythrocyte membranes and to predict the development himicheskoi hypoxia.

We offer a way to diagnose the stability of erythrocyte membranes, including the detection in serum of pregnant TNFα and calculation of the ratio ω-6/ω-3 fatty acid is t, what has value to determine the hidden damage of cell membranes and the nature of the metabolic processes of the organism as a whole.

ω-3 and ω-6 fatty acids belong to the class of polyunsaturated fatty acids with opposite effect on effect on metabolic processes. ω-3 fatty acids (α-linolenic, eicosapentaenoic, docosahexaenoic) prevent the development of thromboembolic complications, the threat of termination of pregnancy, improve uteroplacental circulation, ω-6 fatty acids (linoleic, arachidonic), as precursors of Pro-inflammatory eicosanoids (leukotrienes, thromboxanes), increased microcirculatory disorders in the tissues, causing hypoxia and metabolic disorders (7, 10).

Our studies have shown that the herpes virus infection in peripheral blood erythrocytes of pregnant women increases the number of ω-6 polyunsaturated fatty acids while reducing the content of ω-3 acids, indicating a conformational rearrangement of cell membranes (5). An important consequence of changes in the content in peripheral blood erythrocytes of pregnant women with herpes viral infection of ω-3 and ω-6 fatty acids is a modification of viscous properties of membranes, increased cell transformation and reinforced their destruction, but for diagnostic purposes stability er is trozitos in cytomegalovirus infection and the effect on membrane TNFα revealed regularities in the form of calculation of the ratio ω-6 (linoleic, arachidonic acid) and ω-3 (α-linolenic, eicosapentaenoic, docosahexaenoic) polyunsaturated fatty acids has not yet been used.

The objective of the invention was to develop a way to diagnose the stability of erythrocytes in pregnant women with acute cytomegalovirus infection.

The basis of the proposed method is more accurate evaluation of reduction of the stability of red blood cells to the toxic effects of CMV on the level of the membrane complex, which is achieved by determining the serum of pregnant TNFα and calculation of the ratio ω-6/ω-3. The sensitivity of the method to detect the unstable state of erythrocyte membranes is increased and allows the early clinical signs to predict violations of the structure of cells and the development of himicheskoi hypoxia in pregnant women. The practical significance of the method is determined by the capabilities of the formation of risk groups and determine the indications for early prevention himicheskoi hypoxia in pregnant women with acute cytomegalovirus infection.

The method is as follows.

To obtain diagnostic criteria was investigated in peripheral blood of 25 pregnant women with acute exacerbation of cytomegalovirus infection and 25 pregnant, not ill throughout gestation. The examined pregnant made the fence of a blood from a vein in a test tube with heparin (1:25), centrifuged, the selected plasma, in which enzyme-linked immunosorbent assay determined the titer of IgM and IgG antibodies to cytomegalovirus, the content of TNFα. Sediment RBC mass used for identification by gas chromatography of ω-6 and ω-3 polyunsaturated fatty acids. Methylation of fatty acids was carried out according to the method of Carren J.P. and Dubacy J. (9). Counting and identification of the obtained peaks was performed using a hardware-software complex "Hromatek Analyst 2,5" as holding using the standards of the company "Supelco" (USA). Quantitative calculation of the chromatograms was performed by the method of internal standardization by determining the peak areas of the analyzed components and their share in % to the total peak areas methylated products of higher fatty acids. The results of the content of ω-6 and ω-3 were used to calculate their correlation coefficient (K) by the formula: K=Σω-6/Σω-3, where Σω-6 - the sum of linoleic and arachidonic acids, %; 2 Σω-3 - the sum of α-linolenic, eicosapentaenoic and docosahexaenoic acids, %. When values of TNFα 110±1,97 PG/ml and the ratio Σω-6/Σω-3 (K) is greater than 0.67 and diagnose unstable state of erythrocyte membranes and with probability 95% forecast development himicheskoi hypoxia.

The proposed method allowed us to predict the development nestabilnoj the state of erythrocytes in pregnant women with acute exacerbation of cytomegalovirus infection in the early stages of clinical manifestations himicheskoi hypoxia.

The correctness of the conclusions and the ability to diagnose unstable state of erythrocyte development assessment himicheskoi hypoxia was confirmed by the results of a comprehensive survey of 25 pregnant women with CMV and 25 pregnant women without symptoms of the disease during pregnancy. In pregnant women with acute exacerbation of cytomegalovirus infection, which was confirmed by the presence of IgM and dynamics within 10 days high 1:1600 titer of IgG antibodies, showed a higher content in serum TNFα to 110±1,97 PG/ml and in erythrocyte membranes ω-6 fatty acids: linoleic to 5.43±0.22% and arachidonic to 3.26±0.12%, reduction of ω-3 fatty acid: α-linolenic acid to 0.13±0,04%, eicosapentaenoic 1,10±0,05% and docosahexaenoic 6,10±0.18%, the ratio Σω-6/Σω-3 (K) was 1.19±0,07 that helped with 95% probability to diagnose unstable condition of erythrocytes. In pregnant women without symptoms of disease during the period of gestation content in serum TNFα was 20±0,86 PG/ml and in erythrocyte membranes ω-6 fatty acids linoleic did not exceed values to 4.81±0,16% and arachidonic to 2.94±0,10%. Indicators of ω-3 fatty acids α-linolenic acid was determined at the level 0,22±0,02%, eicosapentaenoic 1,19±0.08% and docosahexaenoic 8,54±0,27%. The ratio Σω-6/Σω-3 (K) was 0.67±0,04.

Thus, the presented results show that when containing the AI in serum TNFα 110±1,97 PG/ml and the values of the ratio K=Σω-6/Σω-3 more 0,67 determine the unstable condition of red blood cells, which confirms the diagnostic value of determination of this ratio to predict himicheskoi hypoxia.

The presented method improves the sensitivity of the estimation methods to the unstable state of red blood cells and allows early, before clinical manifestations, to predict with a probability of 95% the development himicheskoi hypoxia in pregnant women with acute exacerbation of cytomegalovirus infection.

Example

Pregnant B., 25 years old, was admitted in the obstetric Department of pregnancy pathology at the fgbi "center FPD" SB RAMS on the gestation of 12 weeks with exacerbation of cytomegalovirus infection. According to the results of an initial survey in the peripheral blood revealed IgM and dynamics within 10 days defined fourfold increase in titer of IgG antibodies to cytomegalovirus to 1:1600. The content of TNFα in the serum at the time of admission was 111 PG/ml In the study of fatty acid composition of erythrocyte membrane lipids showed that the content of representatives of ω-3 fatty acids α-linolenic, eicosapentaenoic and docosahexaenoic amounted to 0.14%, of 1.05% 6,07%, respectively. Indicators ω-6 fatty acids linoleic and arachidonic was determined at the level of 5.4% and 3.2%, respectively. In accordance with the proposed formula for calculation of the ratio Σω-6/Σω-3, which amounted to K=5,4+3,2/0,14+1,05+6,07=1,18. D. anastasovna unstable condition of erythrocytes and forecasted development himicheskoi hypoxia, which was confirmed when conducting additional clinical studies and was determined to reduce the total number of red blood cells to 3.4·10¹²number of discocytes to 77% and increase the share of echinocytes and degenerative forms, respectively, to 14% and 6%, decrease in pO₂arterial blood to 74 mm Hg, which confirmed the accuracy of the applied method.

Literature

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A method for predicting the development himicheskoi hypoxia in pregnant women with acute cytomegalovirus infection, including blood sampling, determination of serum TNFα, identification of the erythrocyte membrane lipids ω-6 and ω-3 polyunsaturated fatty acids, and then calculate the ratio K=Σω-6/Σω-3, where Σω-6 - the sum of linoleic and arachidonic acids, %, Σω-3 - the sum of α-linolenic, eicosapentaenoic and docosahexaenoic acids, %, and when the content in serum TNFα 110±1,97 PG/ml and the values of K more 0,67 predict the development himicheskoi hypoxia.