Method for activation of white-cell-rich suspension growth and complex correction of blood composition in acoustic field in vitro

FIELD: medicine.

SUBSTANCE: blood or its fractions are exposed to broadband oscillating acoustic signals at a temperature of 4°C to 38°C within the frequency range of 16-20000 Hz with a dominating level of noise pressure more than 45 dB, and less than 60 dB at 60-80 beats per minute, or to sequentially alternating acoustic signals with the dominating level of noise pressure ≤45 dB at <60 beats per minute and acoustic signals with the dominating level of noise pressure more than 65 dB, and less than 90 dB at >80 beats per minute.

EFFECT: method enables activating the white-cell-rich suspension growth and providing the complex correction of the blood composition.

5 cl, 4 tbl, 2 dwg

The invention relates to medicine and biology, relates to a method of enhancing growth leukocyte mass and comprehensive correction of the blood in the acoustic field of in vitro and can be used in research centers, laboratories, blood transfusion stations, biotechnological plants and in various medical institutions.

The closest is a way of correcting the coagulation system of the blood (EN 2336098, IPC A61M 21/00, published on 20.10.2008).

There is a method of musical acoustic correction of the blood coagulation system is directional change in a patient condition of the blood coagulation system by means of musical and acoustic influence on him as a musical influence use specially prepared musical-acoustic program, and before the session musical and acoustical diagnose the performance of the blood coagulation system and psycho-physiological condition of the patient, and the results form the program of musical influences, encoded by different combinations of the elements of musical language and acoustic signals in the frequency range of 20 Hz - 20000 Hz, while in the implementation of music-acoustic correction of the blood coagulation system use two methodological under the ode direct impact of acoustic signals in the patient's blood in vitro with the aim to improve the performance of the blood coagulation system in individuals with low or normal levels to reduce the intensity or stop bleeding in the case of traumatic or post-surgical wounds and immediate impact on patient receptive method for the treatment and prevention of decrease of the blood coagulation system in individuals who are prone to blood clots, or to enhance the performance of the blood coagulation system in individuals with reduced performance.

The disadvantage of this method is that when the direct influence of acoustic signals in the patient's blood in vitro aim is to improve the performance of the blood coagulation system in individuals with low or normal levels to reduce the intensity or stop bleeding in case of traumatic or post-surgical wounds, while intended clinical purpose is narrowly limited, are not taken into account quantitative and qualitative changes in the cellular composition of blood in vitro, produced in response to musical-acoustic effects, and consequently, biomedical potential changes that may occur underestimated and unjustifiably narrows the possibilities for therapeutic and prophylactic application of the method.

Achievable technical result is the activation of growth leukocyte mass and comprehensive correction of the blood in the acoustic field in vitro.

The method is as follows.

In the past previously the examination of patients taking venous blood, by venepuncture from the cubital vein in special jars, vials or plastic bags filled with a stabilizing solution, with a capacity of 5 ml and above, but not more than 500 ml, If necessary, from whole blood, using existing hardware methods, receive, separate fractions of blood cells, such as cells or hematopoietic stem cells, which before the procedure also placed in sterile containers with a stabilizing solution. Acoustic impact on the capacity (capacity) with blood or blood fractions is carried out at a temperature of 4^aboutC38^aboutC for a time from 10 to 300 minutes through 2 or more translator (headphones or speakers)that may be located closely (figure 1) to the tanks or at a distance of 1 m, but not less than two parties, whereby the effect can be: a) open (1) or (b) by closed method, under the solid sphere (figure 2) or within a vessel such as a thermostat (for greater concentration of acoustic waves). To activate the life and growth of biomass blood cells in Vitro are used broadband oscillating acoustic signals in the frequency range 16-20000 Hz, which is characterized by the dominant sound pressure level of more than 45 dB, but less than 60 dB, with a rate of 60-80 beats/min, or sequential rotations AK the statistical signals with dominant sound pressure level ≤45 dB, rate <60 BPM with acoustic signals, characterized by the dominant sound pressure level of 65 dB, but less than 90 dB and the rate of >80 beats/min

For activation of growth leukocyte mass and comprehensive correction of the blood in the acoustic field of in vitro can be used the blood of healthy individuals with the subsequent application of biomaterials in clinical practice and experiments. The readings can also be used in the blood of patients for subsequent reinfused, autohemotherapy or for experimental research.

The most frequent indications for the use of the proposed methodology are: 1) clinical or experimental the need to increase leukocyte mass of blood; 2) clinical or experimental necessity of increasing the mass of individual populations of leukocytes: granulocytes, neutrophils, lymphocytes, etc. 3) clinical or experimental the need to increase the number of hematopoietic stem cells.

For objective evaluation of the efficiency of the method activation of growth leukocyte mass and comprehensive correction of the blood in the acoustic field of in vitro was carried out experimental research program.

Materials and methods research.

For research were selected 30 practically healthy persons p who lichnogo age and gender.

All examined individuals on an empty stomach from the cubital vein was performed blood sampling in 20 ml 3-5 ml of blood was used for morphological and biochemical analysis of the initial samples. The remaining blood was divided into 3 parts, 5 ml, each of which was placed in the appropriate sterile containers with °°stabilizing solution.

In the experimental series blood (30 tubes) have been voiced over 90 min at a temperature of 23°C in an open way broadband oscillating acoustic signals in the frequency range 16-20000 Hz, which is characterized by the dominant sound pressure level of more than 45 dB, but less than 60 dB, with a rate of 60-80 beats/min, which are sequentially alternated with acoustic signals with dominant sound pressure level less than 45 dB, a rate of less than 60 BPM, as well as acoustic signals, characterized by the dominant sound pressure level of 65 dB, but less than 90 dB and rate of more than 80 beats/min Music acoustic treatment programme was recorded on CD and broadcast using a hardware-software complex through Studio headphones, which are adjoined to the vessels with blood.

In the control series No. 1 the blood of those same respondents (30 tubes) have been voiced for 90 min at 23°C in an open way, used rostie ascending modulated tones to 22 kHz.

In the control series No. 2 the blood of individuals (30 tubes) were in the same conditions for 90 min but without sound.

Upon completion of the experiment, all samples were subjected to repeated morphological and biochemical study.

Methods.

For acoustic measurements experimental musical-acoustic software was used generic instrument Akathisia-110A (Russia).

For studies of the morphological composition of the blood used Hematology analyzer Sysmex XE-2100, Sysmex Corporation (Japan).

For studies of biochemical parameters of blood used biochemical analyzer Stat Fax 3300 (USA).

The research results were processed by methods of variation statistics. When comparing the distributions of the various parameters used U-test Mann-Whitney.

The results of the study

The effects of musical and acoustic effects on the elements of leukocyte blood system are presented in table 1.

In accordance with the data of table No. 2, after the MAV in the experimental series of morphological blood tests revealed significant changes in the following indicators of white blood:

WBC - total number of leukocytes increased 4.7 times, amounting to 20.6±of 10.73 10⁹/L (the empirically obtained value U_EMF(9) is the area of significance of p≤0,01).

NE is the number of neutrophils increased from 3.0±1,17 10⁹/L 4.7 times (the empirically obtained value U_EMF(10.5) is in the area of significance of p≤0,01).

SEG is the number of segmented leukocytes with level 2,94±1,16 10⁹/L increased 4.5 times (the empirically obtained value U_EMF(11.5) is in the area of significance of p≤0,01).

BA - the percentage of basophils in relation to the original level of 0.4±0,29% increased 18.5 time (the empirically obtained value U EMF(1) is in the area of significance of p≤0,01).

LY is the number of lymphocytes increased 3.9 times, reaching 2,84 10⁹/L (the empirically obtained value U_EMF(12.5) is in the area of significance of p≤0,01).

Significantly increased performance the percentage of immature cells:

ImGr - 18, 3 times with the initial level of 0.42±0.31 in (the empirically obtained value U_EMF(1) is in the area of significance of p≤0,01);

BAND - in Android 4.04 times with the initial level of 0.92±0,30 (the empirically obtained value U_EMF(7) is in the area of significance of p≤0,01).

Changes in monocytes and eosinophils were false.

In the control series No. 1 and No. 2, identified minor variation of the white blood cells (p≥0,05).

Response indicators of erythrocyte blood system after the mAh presented in table 2.

As follows from the data table is s 2, erythrocyte system turned out to be quite inert to acoustic impacts, because of the 7 studied parameters, significant changes were detected only in the experimental series on two indicators:

MCHC is the average concentration of hemoglobin in erythrocytes decreased by 2% 336.6±4.6 to 328.8±5.1 g/L (the empirically obtained value U_EMF(14) is in the area of significance of p≤0,05);

RDW is the coefficient of variation of the average volume of erythrocytes, in contrast, grew by 15% to 12.73±0.91 to 14.69±1.28% (the empirically obtained value U_EMF(10,5) is in the area of significance of p≤0,01).

In the control series No. 1 and No. 2, identified minor variation of erythrocyte blood (p≥0,05). The dynamics of individual biochemical indices of blood after the mAh presented in table No. 3.

In accordance with the data of table 3, the experimental is Stalnoy series of tests failed with the following significant changes of biochemical parameters of blood:

Total Protein - total protein from the original value 68.52±1.16 gl increased by 5.5% (the empirically obtained value U_EMF(0) is in the area of significance of p≤0,01);

HDL - HDL-cholesterol (high density lipoprotein) from the original value of 1.41±0,03 umoll increased by 5% (the empirically obtained value U_EMF(1,5) is in the area of significance of p≤0,01);

Total Bilirubin - total bilirubin decreased from 4.13±0.68 umoll 1.9 times (the empirically obtained value U_EMF(5,5) is in the area of significance of p≤0,01);

ALT - aminotransferase level increased by 12.6% from the original value 13.26±0.52 Ul (the empirically obtained value U_EMF(7) is in the area of significance of p≤0,01);

AST - is aminotransferase increased from 21.37±3.22 Ul 29.9% (the empirically obtained value U_EMF(7) is in the area of significance of p≤0,01);

LDH is an indicator of lactate dehydrogenase from the value 188.02±16.06 Ul increased by 83% (the empirically obtained value U_EMF(0) is in the area of significance of p≤0,01);

Glucose - glucose level decreased from 4.09±0.11-2.35±0.23 mmoll, which accounted for 43% (the empirically obtained value U_EMF(0) is in the area of significance of p≤0,01).

In the control series No. 1 and No. 2 revealed minor variations of the above biochemical parameters of blood (p≥0,05).

Dynamics of the level of trace elements in blood plasma in the Le mAh presented in table No. 4.

As follows from the data presented in table 4, the level of trace elements in blood plasma in experimental series of studies has undergone significant changes.

Iron is an indicator of iron content increased by 13% to 8.81±0.17 to level 9.97±0.35 mmoll (the empirically obtained value U_EMF(0) is in the area of significance of p≤0,01).

Phosphorus - phosphorus level increased by 8% from 1.34±0.06 to 1.45±0.09 mmoll (the empirically obtained value U_EMF(7,5) is in the area of significance of p≤0,01).

Magnium - magnesium levels increased by 12% from 0.93±0.04 to 1.04±0.05 mmoll (the empirically obtained value U_EMF(3) is in the area of significance of p≤0,01).

Na is the decrease in sodium 146.58±0.47 to 133.68±0.55 mmoll amounted to 9% of the original level (the empirically obtained value U_EMF(0) is in the area of significance of p≤0,05).

K - potassium is increased from 5.19±0.12 to 8.77±0.21 mmoll, which accounted for 69% of the original level (the empirically obtained value U_EMF(0) is in the area of significance of p≤0,01).

In the control series of studies No. 1 significant changes occurred only in the level of iron content by 6.3% from baseline with 8.23±0.13 to 8.79±0.19 mmoll (the empirically obtained value U_EMF(16,5) is in the area of significance of p≤0,05).

In to trolley series of studies No. 2, significant changes are not installed.

The discussion of the results

As a result of researches it is established that the musical-acoustic effects on blood in vitro, used in the experimental series of experiments, have a significant impact on the cellular composition and blood biochemical parameters. So the total number of leukocytes increased 4.7 times the number of neutrophils is 4.7 times the number of segmented leukocytes 4.5 times, the percentage of basophils in relation to the original level increased 18.5 times the number of lymphocytes - 3.9 times.

What resources and mechanisms so significantly increased the level of almost all types of Mature cells in vitro experimental series, not capable of normal conditions for cell division? Isn't that a contradiction?

Found that of all types of Mature blood cells only the white cells can acquire the ability to divide, which appear exclusively in cultures in vitro and again disappears when ingested into the bloodstream.

Moreover, under the conditions of our experiments themselves leukocytes could get both physical and chemical activating stimuli. Physical factors we consider ourselves broadband acoustic signals, accompanied by inputvalue, different frequencies which can directly affect the cells and intracellular article shall ucture, causing the activation of the activity, which is enhanced by the simultaneous arising of mechanical vibrations of the blood, which, as is known, liquid. These complex variations can lead to lysis of some cells of the blood, which promotes the release into the plasma internal chemical content of the cells, including the so-called growth factors contained in platelets. In our earlier studies it was shown that the acoustic signals affect blood clotting in vitro (EN 2336098, IPC A61M 21/00, published on 20.10.2008) and electrochemical performance of water, which is measured by changes in the concentration of superoxide and peroxide ion-radicals (EN 2195655, G01N 33/48 published 27.12.2002), component 90-92% of blood plasma. The whole complex of physical and chemical processes, obviously, enhances the permeability of cell membranes, which together with the lysis of cell parts, eventually leads to the increase in the content of total protein, minerals (iron, phosphorus, manganese, potassium, and others) and enzymes (ALT, AST, and others) in the blood, which we found in our experiments.

Such chemically active environment, located in the acoustic field, is a comprehensive incentive for increased division of Mature leukocytes of all forms.

However, in our opinion this is not the only source of increasing tile is offered by the mass. As you know, in the peripheral blood is constantly circulating a number of hematopoietic stem cells. We believe that stem cells present in the blood in vitro, has also contributed to this significant increase in the number of leukocytes. A direct proof of this hypothesis is to increase the percentage of immature cells: BAND - in Android 4.04 times, and immature granulocytes (ImGr) - 18, 3 times the initial level.

Indirect confirmation of a decrease in the average concentration of hemoglobin in erythrocytes (ICSU) by 2%, which can be explained by an increase in cell volume. In turn, the growth of the coefficient of variation of the average volume of erythrocytes (RDW) 15% can be explained by the presence of several populations of red cells, which is hardly possible in our experimental conditions without the involvement of stem cells.

And finally, lowering the level of glucose in the blood by 43% suggests that the increase in cell mass of cells required a significant energy resource.

In the control series No. 1, where the blood was affected by a simple upward modulated tones to 22 kHz, of the studied parameters was significantly changed only one, the increased level of iron content by 6.3% from baseline, the remaining variation were on the significance level p≥0,05.

In the control series No. 2, where the blood is not exposed acoustic effects, particularly the dynamics of blood to the initial state is not set.

The conclusions. The proposed method exposure in the acoustic field of in vitro causes the activation of leukocyte growth and mass °comprehensive correction of the blood.

1. A way to enhance growth leukocyte mass and comprehensive correction of the blood in the acoustic field in vitro, including acoustic effects on the blood or blood fraction is carried out at a temperature of 4^aboutC to 38^aboutC, broadband oscillating acoustic signals in the frequency range 16-20000 Hz with a dominant sound pressure level of more than 45 dB, but less than 60 dB and rate of 60-80 beats/min, or sequential alternation of acoustic signals with dominant sound pressure level ≤45 dB, rate <60 BPM with acoustic signals, characterized by the dominant sound pressure level of 65 dB, but less than 90 dB and the rate of >80 beats/min

2. The method according to claim 1, in which the acoustic effect is carried out for from 10 to 300 minutes.

3. The method according to claim 1, in which the acoustic effects carry through 2 or more translator.

4. The method according to claim 3, in which the translators use headphones or speakers.

5. The method according to claim 3, in which translatory are close to the tanks with irradiated blood or at a distance of 1 m, but not less than two sides.