Method for detecting adhesive properties of blood leukocytes

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

 

The invention relates to medicine, in particular to studies of blood, and can be used in the study and treatment of inflammatory diseases, autoimmune and degenerative origin.

The ability of blood leukocytes to the adhesion (sticking) has been known for a long time. However, the significance of this property for a long time been underestimated.

Currently adhesive interactions of various types between cells and their interaction with the extracellular matrix play a leading role in many processes, developing both in physiological conditions and in pathology. These include such as the activation and differentiation of immune cells, overcoming the cells of the blood-tissue interfaces barriers, sending all sorts of signals and others. The adhesion (or raspletanie cells on the substrate) is one of the manifestations of their functional activation. In the whole organism adhesion of cells develops in relation to other cells, vascular endothelium, extracellular matrix, and in vitro it is played against nonspecific substrates, in particular, to glass, to plastic surface, it is assumed that this phenomenon reflects the ability of cells to adhesion to the endothelium.

As the material of the substrate that defines the adhesiveness the notches, now consider adhesion molecules (AM). Identified more than 5 groups AM, including integrins, selectins, the immunoglobulin superfamily, etc. Used to determine adhesion molecules methods involve differential detection of certain forms AM and is based on two principles.

One of them is associated with a detection membrane forms AM, i.e. the expression of a certain type AM on the cell surface in a dedicated suspension of leukocyte or lymphocyte. It is carried out using monoclonal antibodies (MAB). Another form of existence of adhesion molecules soluble (RAM).

Currently, the developed test systems for the detection frames (Firm R&D systems, USA). However, their extremely high cost limits their use in clinical and experimental conditions. The test system of domestic production intended for the determination of soluble forms AM, is still not produced.

The closest technical solution of the invention is a method for determining the adhesive properties of leukocytes, based on inhibition of adhesion of leukocytes after their contact with specific antigens reaction suppressing adhesion of leukocytes, which allows to determine the cellular reactivity (DAT, Soult. Micromodification test braking preliberalisation using plastic microplate. Oncology issues, t, No. 7, S. 17, 1985).

This method includes the following operations: drawing blood with heparin, obtaining the suspension of leukocytes by blood sedimentation, centrifugation for separation of leukocytes, laundering the last of the admixture of red blood cells and serum, preparation of suspensions of leukocytes, a fence from a particular scope, the incubation of a suspension of cells in the wells of plastic microplate, and counting the number of cells after incubation. In this way the adhesive properties of cells are determined only after their contact with specific antigens. It does not involve accounting and computing the ratio of the number of adhesive cells to the non-adhesive in the composition of the studied sample of cells. Also, do not take into account the influence of autoseparate adhesion of leukocytes in the form of a possible increase adhesion of blood leukocytes.

The technical result of the invention is to enhance the functionality of the method and increase the information content at the expense of additional indicators. Namely, indicators, allowing to take into account and calculate the ratio of the number of adhesive cells to the non-adhesive in the composition of the studied sample of cells, and to consider the impact of autoseparate adhesion of leukocytes.

It is achieved by the fact that in the known method lies in the fence of heparinisation the th blood, defending her to get plasma to the cells, and centrifuged for separation of the latter, which are washed from the admixture of red blood cells and serum, as well as in the counting of cells in a sample from the suspension of leukocytes after incubation (C) for 1.5 hours at 37°in the wells of the plastic microwell additionally prepare two samples, one of them is used for counting the total number of cells prior to incubation (A), the second is subjected to incubation in the same mode with the addition of autoseparate and count the number of cells after incubation (C), the adhesive properties of leukocytes in the blood are judged index spontaneous adhesion (D), whereand the effect of enhancing the adhesion of cells under the influence of autoseparate determined by the value ofwhen It≥30%, where In the number of cells subjected to further adhesion due to the addition of autoseparate.

THE METHOD IS AS FOLLOWS.

First taking heparinised blood, then defend it to obtain plasma with leukocytes. Last centrifuged for separation of leukocytes, which are cleaned from impurities of erythrocytes and serum. Next, prepare a suspension of cells from which take 3 samples in the form of a certain volume. In the first sample to produce the miscalculation of the total number of cells prior to incubation (A). The second sample is incubated for 1.5 hours at t=37° With the well plastic microplate and count the number of non-adhesive cells (In) - the number of cells remaining after incubation. A third sample of leukocytes put with the addition of autoseparate, incubated under the same conditions, and then count the number of non-adhesive cells (C), which allows to determine the number of cells subjected to additional adhesion under the influence of autoseparate (b-C).

Index spontaneous adhesion D reflects the expression of the membrane form AM on leukocytes, and it is calculated by the formula.

Another parameter is associated with the possibility of revealing the effect of enhancing the adhesion of blood cells during incubation in conditions add autoseparate. In case of detection of a specified effect in the given reaction will receive a reduction in the number of unattached cells after incubation. The difference between the number of unattached cells in the sample without autoseparate and their number in the sample with the addition of autoseparate indicates the number of cells subjected to additional adhesion due to the addition of autoseparate (b-C), and their ratio to the number of unattached cells in the third sample (C), expressed as a percentage, indicates the degree of amplification of cell adhesion under autoseparate (K).

The reaction in which the value of K is 30% or more, is selected as the criterion for the amplification of ADH is Ziya cells. This result reflects the higher level frames in the serum.

Studies carried out using the proposed method allowed us to obtain new information, reflecting the significant differences of the values D and the amplification of adhesion in patients with multiple sclerosis (PC) compared with healthy controls, and to determine the relationship of these parameters with the characteristics of the disease.

Were surveyed 81 sick PC and a control group of 23 healthy persons.

The study was set to the range of fluctuations of values of D, in the control group values ranged from 9 to 38%, in the group of patients PC - all values were higher - up to 63%, i.e. the difference was dominated by high values of D in the group of patients with PC. In 14.8% of patients with PC from among persons with high levels of adhesion found very high values of D (over 41%). In the control group such indicators of adhesiveness were not registered in any observation.

Thus, performance On more than 38% should be regarded as pathological.

Were also found significant differences in the rate of adhesion of leukocytes when comparing subgroups of patients with different nature of the current PC, and subgroups with different duration and severity of disease.

The effect of amplification of cell adhesion under dei is the journey of autoseparate several were seen more frequently in the group of PC patients compared with the control group, however, the reliability of the higher frequency of detection of this effect are established only in subgroups of patients with primary progredient course over PC, and in patients with the severity of neurological deficit at 3.5-4.5 points.

The findings suggest that increased level of membrane and soluble forms of adhesion molecules in PC. These results contribute to a better understanding of the pathogenesis of the disease, consistent with the published data of a number of foreign authors about the increased levels of adhesion molecules in the blood of patients with PC, obtained by other methods, and should be considered when the search and development of new and appropriate therapeutic methods.

In the pathogenesis of PC immune mechanisms and adhesion molecules play a very important role. However, the study of the adhesive properties of leukocytes and blood serum, including, and with the help of the proposed method can be very useful and give new information and the study of inflammatory diseases, degenerative and other causes.

The advantage of the proposed method for determining the adhesive properties of blood leukocytes, in comparison with the existing other ways of determining moms and frames, is its simplicity, availability and extremely low cost. In addition, the proposed method gives information reflecting summary the level of adhesion molecules, unlike other methods, suggesting differential identification of individual representatives of different classes AM, what more is necessary and justified for research purposes.

The method for determining the adhesive properties of blood leukocytes, namely, that taking heparinised blood, defend it to obtain blood plasma with leukocytes, which then centrifuged for separation of cells, the latter washed from the admixture of red blood cells and serum, prepare a suspension of cells from which to take samples, incubated for 1.5 h at 37C in the wells of the plastic microwell plate and count the number of cells after incubation (B), characterized in that the obtained suspension of leukocytes additionally prepare two samples, one of them is used for counting the total number of cells prior to incubation (A), the second sample is subjected to incubation in the same mode with the addition of autoseparate and then count the number of cells remaining after incubation (C), the content of adhesion molecules on leukocytes of the blood is measured by an index of spontaneous adhesion (D), where

and the effect of enhancing the adhesion of cells under the influence of autoseparate determine the value To≥30%, where

where-S - the number of the notches, subjected to further adhesion due to the addition of autoseparate.



 

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