The objective of this work is to evaluate plasma membrane blebbing of peripheral blood lymphocytes for patients with local cold trauma at the background of using standard conservative therapy and systematic ozone therapy.

Methods and materials. The research included 60 patients with limbs congelation of degrees III-IV (early reactive period) who have been treated stationary in surgical division №2 of Municipal budgetary healthcare institution "City clinical hospital №7" of Krasnoyarsk during the period from 2011 to 2013. All of the studied have been split into 3 groups of 20 patients. The first group has been formed of patients who received traditional conservative therapy. Ozone therapy has been introduced to the treatment programme of the second group of patients along with the traditional therapy (infusions of ozonized solution in concentration of 2-4 mg/l in total volume of 200 ml, 4-5 sessions every other day). Physiological solution has been ozonized with ozone generator "YOTA 60-01" (Medozone, Moscow). Patients of the third group have been treated with a large autohemotherapy with ozone along with the traditional therapy (4-5 sessions every other day). The control group has been formed of healthy people of the same gender and age".

All works of registering plasmatic membrane blebbing of lymphocytes have been carried out at the base of the Center of Collective Usage of "Scientific-research institute of molecular medicine and pathobiochemistry" of Krasnoyarsk state medical university.

In order to discharge lymphocytic fraction of peripheral blood, we have used Lympholyte^® (CEDERLANE^®, Canada). The discharge method has been carried out according to the standard protocol of the producing company. Microscoping has been carried out on luminescent microscope OLYMPUS CX 41, equipped with video recorder DP 71, using phase-contrast attachment (x1000), calculates for 100 cells. 

The following types of lymphocytes have been differentiated in the microscope sight: 1) intact cells (with a visually-unaltered plasmatic membrane, round, smooth surface); 2) cells in condition of initial blebbing (small vesicles on membrane - up to 1/3 of a cell radius); 3) cells in condition of terminal blebbing (large vesicles on membrane - over 1/3 of a cell radius).

Calculation of lymphocyte blebbing index values has been carried out after microscoping. Lymphocyte blebbing index is received via multiplying lymphocytes in condition of initial blebbing by 100, divided by total lymphocyte blebbing.  Total lymphocyte blebbing has been found by adding lymphocytes in condition of initial and terminal blebbing.    

Statistic analysis of the results has been carried out according to the methods of descriptive and variational statistic via statistical pack of Microsoft Excel, operational system Windows XP, and Statistica 6.0.

The received results and conclusions. Persons of both genders have been among the studied, their age varied from 30 to 60 years. The main group of patients with local cold trauma has been formed of lowly-protected stratums. The share of working and employed persons has formed only 37,5%. 53,3% of cases consisted of feet damage, 33,3% of cases - hand damage, and 13,3% of patients have had combined injuries.   

The analysis of lymphocyte blebbing expression has shown that blebbing index (BI) indicators of the studied groups have differed reliably from those of the control group. The number of lymphocytes in terminal blebbing has not exceeded 5 among donors of the control group, while it could achieve 29 per 100 cells among the patients with local cold trauma.

The greatest expression degree of lymphocytic BI has been observed among patients of groups 2 and 3 at the moment of their admission. BI level of plasmatic membrane lymphocytes has had an unreliable trend to decrease by day-10 of hospitalization among the patients of group 2 at the background of the delivered treatment. BI indicators have been lower among patient group 3 by days 5 and 10 of hospitalization than those of amongst the 2nd group, reliability level p<0,05. BI indicator levels of the 1^st group have been similar to those of the 2^nd and 3^rd group at the moment of admission, but no decreases in level of the studied index have been registered at the background of standard conservative therapy. On the contrary, lymphocytic BI level has had a slight tendency to increase by day 10 of hospitalization. However, statistical analysis has shown that the received differences are unreliable.   

Thus, intensification of peripheral blood lymphocytes blebbing in case of local cold trauma characterizes an amplification of immune response. Evaluation of cytoplasmic membrane blebbing of peripheral blood lymphocytes among patients with frostbitten limbs allows one to monitor an efficiency of an implemented therapy.