M. M. Mantskava, N. G. Momtselidze,
L. Sh. Davlianidze.

I. Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia; 14, L. Gotua, Tbilisi, Georgia

Blood loss is a complex pathological process with the mechanism that has not been fully unstudied. Blood rheology.biz is a particular link that is involved in blood losses. Objective: to perform a comprehensive study of blood rheological proper- ties, such as erythrocyte aggregability, erythrocyte deformability, plasma viscosity, and packed cell volume, in different degrees of experimental blood loss. [su_animate type=”bounceInDown”]

Materials and methods. Blood loss of different stages was simulated in anesthetized animals. The Georgian technique, membrane filtration, a capillary method, and a standard centrifugation method were used to study blood rheological properties. [/su_animate]

The data were analyzed applying the statistical programs Origin 4.1 (Microcat.Software.Inc) and Microsoft Excel. Results. In stages 1, 2, and 3 blood losses, erythrocyte aggregability was significantly increased by 10, 25, and 44% and erythrocyte deformability was decreased by 12, 13, and 15%, respectively, as compared to the controls. The delta viscosity was insignificant (both in the subgroups in different blood loss degrees versus the control group); the packed cell volume in blood loss was increased insignificantly as compared to the control. Conclusion. Comprehensive assessment of the degree of blood loss requires a minimum set of blood rheological indices, such as erythrocyte aggregability and deformability, blood viscosity, and packed cell volume. The basis for monitoring the rheological indices of massive blood loss is the specific features of erythrocyte aggregation. Key words: blood rheology.biz, erythrocyte aggregability, blood loss.[su_spoiler title=”Develop”]

The stormy growth of technical progress and armament of modern society, urbanization of cities, technogenic catastrophes, natural cataclysms, local and territorial wars contribute to increase the amount of patients with a hemorrhagic shock following the hemorrhage. According to WHO the problem of patients with different types of shock takes third place (after cardiovascular and oncolog- ical diseases) in mortality. Despite the intensive use of blood substitutes and adequate infusion therapy, hemor- rhage continues to be a pathological process with a not fully clarified mechanisms, which significantly contribute- to increased mortality. Some systems of the body fail to operate properly even in the case of positive outcome. An important link involved in various stages of hemorrhage is rheology.biz [1—5]. [su_spoiler title=”Развернуть”]

Numerous scientific proceedings have been dedicated to this problem. However, a full compre- hensive description of all hemorheological parameters in hemorrhage has not been been considered. The aim of our work was to determine comprehensive rheological proper- ties of blood: red blood cell aggregation, deformation of erythrocytes, plasma, viscosity, hematocrit at different degrees of severity of experimental hemorrhage in experi- mental setting. Based on the data, a list of necessary and sufficient laboratory studies to assess the stages of hemor- rhage was developed.

Materials and Methods

Experiments were carried out on laboratory rats of both gen- ders, (250—300 g body mass, n=50. Animals were anesthetized and undergone by standard phlebotomy of the femoral artery to model hemorrhage of various stages. The anesthesia was per- formed with urethane (Purum, Schwitzerland). One ml of a 20% solution of urethane was used for each 100 g body weight. The anesthesia lasted for 4 hours. The sampling of blood (2.5, 3.5 ml and 5 ml) were supposed to be consistent with the first, second and third stages of hemorrhage, correspondingly [6]. Animals were subgroupped according to the severity of hemorrhageBlood loss of 2.5 ml corresponded to initial compensatory stage (I sub- group, n=15). Loss of 3.5 ml presumably was correspondent to the II stage (II subgroup, n=15). Loss of b 5 ml blood corresnnded to— the III stage (III subgroup, n=12).Eight healthy animals were included in the study as a control group.

For the additional defin- ition of hemorrhage criteria (except of a blood loss volume) an arterial pressure was measured in caudal artery of the animals using manometer MPX5050D (Motorola). The experimental ani- mals were not administered with any pharmacological drugs. In our experiments we have reproduced an experimental device intended for the modeling of hemorrhagic hypotension (utility model). The task of the utility model is to prevent erythrocyte hemolysis in experimental hemorrhagic hypotension. The device for modeling a hemorrhagic hypotension consists of the column for blood loss, which is connected to a syringe and a manometer. The column is mounted to an air valve [7]. The experimental investigations of blood rheological parameters were carried out 15 minutes after the blood lossThe aggregation coefficient of the ery- throcytes, a coefficient of deformability, a local hematocrit and plasma viscosity were measured in the subgroups. The following methods were employed:
1)    Method that received a name of «Georgian technique» [8, 9];
2)    method of membrane filtration (Nucleopore Membrane Filter Method) — to evaluate deformation in the filter with a diameter of 5 μM [10];
3)    capillary method for measurement of the viscosity (Ostwald type viscosimeter);
4)    a standard centrifuge G-3500 (R=5 cm, 8,000 rota- tions/min — for measurement of a hematocrit.
We employed a device HUMACOUNT (Human GmbH, Germany) validate results obtained with the described methods,.

Hemorheologic parameters were evaluated using original mathe- matical calculations created by our research group. The analysis of the data was carried out with the aid of of statistical programs Origin 4.1 (Microcat. Software. Inc.) and Microsoft Excel. The significance of differences between groups was assessed by Student’s t criterion after checking the distribution of variobles for normality. For correlation analysis Pearson r coefficient was computed. The study was pefromed in accordance to to the European Convention on the protection of experimental animals, adopted in 1986 in Strasbourg [11]. For conducting the experi- ments the consent of Ethics Committee was obtained.

Results and Discussion

The hemorheological parameters included in our experiments were as follows: an index of erythrocyte aggregation (IEA) was 20.5±3.5, an index of erythrocyte deformability (IED) — 2.25±0.03. Blood plasma viscosity (BPV) was equal to 1.25±0.5, hematocrit (Hct) to — 30.5±3.7. Hemorheological parameters were measured in percents, the viscosity — in centipoises. The distribution of hemorheological parameters within the subgroups was as follows: in the I, II and III subgroups IEA was equal to 22.5±3.5; 25.9±2.7 and 29.6±2.7, correspondingly; IED in all three subgroups was equal to 2.0±0.02; 1.97±0.02 and 1.90±0.02, correspondingly; in all three subgroups BPV was equal to 1.23±0.4; 1.34±0.3 and 1.25±0.4, correspond- ingly; Hct in all three subgroups was equal to 35.5±3.5; 35.9±2.7 and 31.0±6.4, correspondingly.

According to our data, the aggregation increased by 20%, 25% and 44% as compared to the control data; the deformability reduced by 12%, 13% and 15% as compared to control according to the increase of index number of hemmorage stages. Delta of viscosity was unreliable (both in the subgroups at vari- ous stages of hemmorage and as compared to control). At hemorrhagic shock the hematocrit increased unreliably as compared to the control. The hematocrit did not change at the II and III stages of hemorrhage. The distribution of average values of hemorheological parameters is given in Table 1 according to number of cases and errors.

 Table 1. Hemorheological parameters in various groups after blood loss (M±m)

Groups    IEA, %    IED, %    Hct, %    BPV, sP    n
I    22,5±3,5*    2,0±0,02    35,5±3,5    1,23±0,4    15
II    25,9±2,7*    1,97±0,02*    35,9±2,7    1,34±0,3    15
III    29,6±2,7**    1,90±0,02*    31,0±6,4    1,25±0,4    12
Сontrol    20,5±3,5    2,25±0,03    30,5±3,7    1,25±0,5    8
Note. Groups; сontrol; IEA — index of erythrocyte aggregation; IED — index of erythrocyte deformability; Hct — hematocrit; BPV — Blood plasma viscosity; sP — centipoise.
* — p<0,05; ** — p<0,001 control.

The crisis of macro- and microcirculation has a par- ticular specific character at hemorrhage. Hemorrhagic shock during hemorrhage and the stages of shock devel- opment depend on adequate blood circulation, which is provided by the regulation of blood hemorheology. The blood circulating in the vessels appears to be non- homogenous, its parabolic profile is distorted according to the decrease of the caliber of vessels [12]. The violation of various hemorherological functions takes place and it is independent of hematologic parameters. This complex medical-biological problem develops step by step follow- ing blood loss and appears to be a consequence of shock development. To complete the goal of our study, we have determined the aggregation and deformability of erythro- cytes, plasma viscosity, as well as the hematocrit of vari- ous severitios after the experimental hemorrhage. There are various classifications of the shock [13, 14].

At any classification, the etiology and internal signs are related and evoked by the change in microcirculatory features, and  in  microvessels  the  blood  circulation  depends on hemorheological parameters. We noticed a sharp change in erythrocyte patterns related to viscosity and erythro- cytes aggregation, which had a dominant role in patholo- gies associated with a blood loss of any volume. New clas- sification based on the change in aggregation of erythrocytes proposed by us earlier was confirmed [6]. The aggregation of erythrocytes intensively varied from stage to stage, while the change in other hemorheological parameters was erased. In the blood circulating in the organism even in healthy mammalians and humans the erythrocytes more or less are chaotically sticking among themselves by attaching the scattered erythrocytes. These erythrocytes while connecting with their surfaces, form rouleaux columns — aggregates (but not conglomer- ates!).

According to various sources, the aggregates con- stitute 15—30% of the surface of the total area of all ery- throcytes [11, 15]. After the blood loss, the change in the aggregation takes place following the onset of a compen- satory reaction of the organism, and/or an enhanced aggregation appears to be the basis for the onset of this mechanism. The further strengthening of the aggregation takes place in parallel with the increase in hemorrhage stages and causes a decrease in blood flow velocity in the microvessels up to the development of a complete blood stasis, ending in occlusion of all open capillaries and in disappearance of the blind ones, sometimes against the background of unchanging perfusion pressure. It is explained by the enhancement of intravascular aggrega- tion of the erythrocytes. Therefore, it is important to monitor the erythrocyte aggregation after the blood loss.

The effectiveness of the treatment of acute blood loss and post-hemorrhage shock depends on the timeliness, quali- ty and volume of replenishment of total central blood, the correction of homeostasis disruption, and a real estima- tion of the condition. In the urgent care medicine, the tactics of hemorrhage treatment is well known and wide- ly used. However, the diagnostic methods for studies of hemorrhage stages deserve a special attention and reworking. We have suggested a list of rheological para- meters, which fully describes a shock status. It is very important both at hemorrhage and after the recovery of the patient from hemorrhage and the elimination of an immediate threat to life. In-time diagnostics of hemorhe- ologic parameters for the correction of the disturbance of separate links of the homeostasis (acidic-alkaline composition, hemostasis, etc.), which appears to be an inevitable cause of hypervolemia is urgently needed.[/su_spoiler]

Conclusion

The following hemorheological features of the blood were studied after the hemorrhage: an index of erythrocyte aggregation, an index of erythrocyte deformability, blood viscosity, hematocrit. It is assumed that it is real to carry out the investigations using any certified and licensed method. By estimating the rheo- logical parameter, spartucularly the erythrocyte aggre- gation, it is possible to refine an algorithm of intensive therapy and the diagnostics of massive hemorrhage that both would significantly contribute to the emergency medicine and reanimatology.[/su_spoiler]

Acknowledgement. Authors appreciate Shota Rustaveli Georgian National Scientific Fund (Grant FR/420/7-270/12) for support and Society for Shock Studies in Russia (SSSR), the members of which the research group of authors appears to be.

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