Olga M. Ryaboshapka

Aggregation and Disaggregation of Erythrocytes in Whole Blood: Study by Backscattering Technique

The aggregation phenomenon is of great importance for the evaluation of performance of the microcirculation system because of its influence on the blood viscosity at low shear stresses. Some important features and consequences of this phenomenon in vivo can be predicted in the in vitro experiments using optical methods. These methods are considered to be the most informative and applicable not only for the basic study of the aggregation phenomenon, but also for the diagnosis of a number of diseases and for the monitoring of therapeutic treatment in clinics. Results presented in this paper prove that the backscattering technique allows one to detect different changes of aggregational ability and deformability of erythrocytes and to get reliable and reproducible results distinguishing normal blood and blood with different pathologies.

Heat-induced changes in optical properties of human whole blood in vitro

The effect of anomalous optical behavior of biological tissue at high-intensity laser irradiation can be caused by heat- induced changes in optical properties of consisting components, mainly muscle tissue and blood. We registered the spectral transmission of fresh human whole blood and serum samples in the wavelength range of 300 - 700 nm at the heating of samples in the temperature range of 35 - 65 degrees Celsius. The results showed an increase of 10 - 15% in the transmission of blood serum at the temperature rising up to 50 - 60 degrees Celsius. In the case of diluted whole blood a sharply enhanced transmission was observed at the temperature of 56 - 60 degrees Celsius, while further heating resulted in a decreased transmission down to the initial level. The significant changes (of a three orders of magnitude) in the transmission of whole blood at the wavelength of Nd:YAG laser (1064 nm) were observed. The obtained results can be considered as one of the possible explanations of the anomalous light distribution in certain tissues.

Aggregation kinetics of erythrocytes in whole blood: comparison of data processing algorithms

Samples of erythrocyte suspensions of normal and pathological blood were studied by light back-scattering technique. This method allows to register the kinetics of the formation of erythrocyte aggregates in stasis and the disruption of large and small aggregates in shear flow of aggregated blood in a Couette chamber. The output signal proportional to backscattered light intensity was processed by different algorithms to quantitatively determine the aggregational properties of erythrocytes. We suggest that the registered time dependence of the signal representing the aggregation kinetics can be approximated as a sum of exponentials with parameters T1 and T2 that reflect the characteristic times of linear and clump or network aggregates formation respectively. On the other hand the hyperbolic approximation of the aggregation kinetics enables to determine the characteristic constant of the whole aggregation process. We show that the parameters obtained with both exponential and hyperbolic data processing algorithms are sensitive to the concentration of erythrocytes in blood sample, mode of scattered light detection and other factors. The advantages of each algorithm and latest results of their comparative analysis will be discussed in this paper.

Problems of the optimum design of erythronephelometer

The photometric studies of blood aggregation characteristics were started by the works of Dognon et.al. [1,2] who registered the deviation of the intensity of light scattered from a layer of blood under mixing and after its halt. They have shown the dependence ofthe signal (scattering intensity) on orientational aggregation ofthe erythrocytes. The detailed study of the intensity variation oflight passing through a blood layer in a rheoscope coneplate viscometer was carried out by H.SchmidSchonbein et.al. [3,4,5]. A simplified modification of the registration technique in case of light passing through a special couvette under lOOjn thick after the halt of stochastic mixing of blood, has been elaborated by R.Tukhvatulin (1986) [6]. Backscattering from a shear Couette flow was registered by S.Usami and S.Chien (1973) [7]. This registration mode allowed to considerably raise the probed volume of blood. Detailed analysis has shown that there exists a strong connection between the intensity of the transmitted or backscattered light with the aggregation characteristics ofthe erythrocytes suspension. This has made the technique a major one in clinical and experimental hemorheology (J.F.Stoltz, M.Donner, 1987) [8]. In accord with the primary experiments two approaches have been developed in the aggregometry. The first is based on the registration of light transmission through the StUdied thin (25pi) blood layer in a coneplate type viscometer [3,4,5]. Thesecond is based on the registration ofthe backscattered light from a blood layer in a Couette type viscometer [9, 1 0]. The corresponding commercial apparatuses most commonly used are: the Myrenne aggregometer and the Sefam erythroaggregometer. Anyhow the differences in geometry of the flow and in the detection modes of the outcome light yield different results in the study of the hyperaggregational syndrome even in the in vitro experiments [11]. Attempts of theoretical analysis of multiply scattered light from blood [12,13,14] have not yieldod any important results in the estimation of aggregates dimensions and morphology, and thus could not form the basis to substantiate the optimum construction of the aggregometer. Such optimization includes the optimal choice: of the shear blood layer thickness, ofthe registration mode of scattered light (transmitted or backscattered), of the scattering angle and detection aperture. The ideal design of aggregometer must give reliable information about the sizes of aggregates in each moment of the spontaneous aggregation of erythrocytes and enable to estimate the velocity of aggregation process and durability of aggregates. In this article we shall characterize each of these problems in more detail.

Aggregation and hemolysis of human erythrocytes at photodynamic therapy

The goal of this work is to comparatively study the side effect PDT on blood with the use of two Russian photosensitizers -- photohem and photosense. These effects were studied at in vitro and in vivo conditions. The technique of backscattering nephelometry was used to measure the parameters of aggregation kinetics and the strength of aggregates of whole blood erythrocytes. The conventional method of absorption spectrophotometry was used to monitor the level of erythrocytes hemolysis. It was shown that sensitization of blood leads to the rise of the characteristic time of Rouleax formation, of the strength of aggregates, and of the level of hemolysis. The revealed tendency to increased strength of aggregates and level of hemolysis as a result of photodynamic action can be considered as a side effect of PDT.

Dependences of erythrocytes aggregation and disaggregation parameters on suspension hematocrit: study by backscattering nephelometry

The backscattering nephelometry technique is used to simultaneously obtain the aggregation and disaggregation parameters of erythrocytes in whole blood: characteristic times T1 of linear aggregates formation and T2 of network formation; hydrodynamic durability of aggregates (beta) , and the coefficient of deformability of erythrocytes D. We have previously shown that these parameters are sensitive to different diseases, external conditions and stimuli. Samples of blood have different values of hematocrit H. So we have studied dependences of parameters T1, T2, and (beta) on suspension hematocrit. The obtained results show that the values of T1 are inversely proportional to H2 for both blood of healthy donors and blood of patients suffering hereditary glomerulonephritis. With hematocrit growing towards 100% (H equals 1) these aggregation parameters asymptotically aspire to certain values which determine the natural limit of the aggregation rate. The decrease of hematocrit causes the intensification of shear-induced aggregation at shear rates up to 150 s-1.

Aggregation and disaggregation kinetics of erythrocytes in whole blood under low-energy laser irradiation

Therapeutic applications of low—energy laser irradiation (LELI) of human patients suffering different diseases is currently experiencing fast progress in a number of countries including Russia (1] . This can hardly be clearly justified without efficient diagnostic technique which can provide objective and quantitative registration and monitoring of patients individual responses to the phototherapeutic treatment. This is particularly true in relation to the LELI of blood which is considered a very efficient way of treatment of a number of pathologies, including pulmanory, cardio-vascular, ischaemic, etc. A number of diagnostic techniques currently in clinical practice include, in addition to the conventional clinical blood tests, the measurements of refraction indeces and absorbance of whole blood and plasma which have been shown sensitive to LELI in vitro and in vivo at different wavelengths including 632.8 and 1234 nm [2,33, alternations in leycocyte metabolism under He-S-Ne 1 aser irradiation [4] , index of erythrocytes deformabi 1 ity (5], etc. We have tested the technique of laser backscattering nepheloznetry (LBN) which we have been developing for the diagnosis of blood pathologies [6,7] in respect to its sensitivity to laser irradiation of blood at in vitro and in vivo experimental conditions. The advantages of the LBN technique is that we deal with the whole blood but not with delute solutions or single blood cells and, also, this technique yeilds two characteristics of blood: deformability of erythrocyte which was previously shown sensitive to laser irradiation [3] and aggregation and disaggregation kinetics. In this paper we present preliminary results of our experiments showing the sensitivity of aggregation— disaggregation kinetics to LELI at certain experimental conditions and pathologies.

Study of erythrocyte-aggregation kinetics in shear flow in vitro by light-scattering technique

The kinetics of aggregation and disaggregation of red blood cells in whole blood subjected to shear stress is different with normal and pathological blood. To measure this kinetics, we have applied a backscattering nephelometric technique, and quantitatively registered the alteration of the scattered intensity due to appearance or disappearance of different types of cell aggregates under controlled shear stresses. Fresh samples of normal and pathological blood from 25 patients suffering psoriatic arthritis were tested and showed differences in the RBCs (dis)aggregation kinetics. Relaxation times, index of hydrodynamic durability, and anisotropy of light scattering along and across to flow were discussed.

Aggregation properties of erythrocytes of whole blood under shear stress by backscattering nephelometry

The kinetics of aggregation and disaggregation of red blood cells in whole blood subjected to shear stress is different with normal and pathological blood. To measure the kinetics we have applied a backscattering nephelometric technique, and quantitatively registered the alternation of the scattered intensity due to appearance or disappearance of different types of cell aggregates under controlled shear stresses. The latter were obtained in a Couette flow in a thin gap between two concentrical cylinders, the inner one rotating at controlled velocities. The peculiarities of backscattered light signal formation and obtained results were discussed.

Spectrophotometry of biological fluids under visible and near-infrared irradiation

Transmittance spectra and spectra of optical loses due to absorption and scattering of different biofluids were registered by spectrophotometer DU-64 Beckman in the range of wavelength from 200 to 900 nm. The alteration of spectra of blood serum and whole blood were observed after irradiation of the samples by laser light (633 or 850 nm). The preliminary results indicating that the laser effects depend on the initial biophysical and biochemical state of blood are discussed.