Mehmet Uyuklu

Parameterization of red blood cell elongation index – shear stress curves obtained by ektacytometry

Abstract Measurement of red blood cell (RBC) deformability by ektacytometry yields a set of elongation indexes (EI) measured at various shear stresses (SS) presented as SS-EI curves, or tabulated data. These are useful for detailed analysis, but may not be appropriate when a simple comparison of a global parameter between groups is required. Based on the characteristic shape of SS-EI curves, two approaches have been proposed to calculate the maximal RBC elongation index (EImax) and the shear stress required for one-half of this maximal deformation (SS1/2): (i) linear Lineweaver-Burke (LB) model; (ii) Streekstra-Bronkhorst (SB) model. Both approaches have specific assumptions and thus may be subject to the measurement conditions. Using RBC treated with various concentrations of glutaraldehyde (GA) and data obtained by ektacytometry, the two approaches have been compared for nine different ranges of SS between 0.6–75 Pa. Our results indicate that: (i) the sensitivity of both models can be affected by the SS range and limits employed; (ii) over the entire range of SS-data, a non-linear curve fitting approach to the LB model gave more consistent results than a linear approach; (iii) the LB method is better for detecting SS1/2 differences between RBC treated with 0.001–0.005% glutaraldehyde (GA) and for a 40% mixture of rigid cells but is equally sensitive to SB for 10% rigid cells; and (iv) the LB and SB methods for EImax are equivalent for 0.001% and 0.003% GA and 40% rigid, with the SB better for 0.005% GA and the LB better for 10% rigid.

Effects of storage duration and temperature of human blood on red cell deformability and aggregation

Blood samples used in hemorheological studies may be stored for a period of time, the effects of storage have yet to be fully explored. This study evaluated the effects of storage temperature (i.e., 4 degrees C or 25 degrees C) and duration on RBC deformability and aggregation for blood from healthy controls and from septic patients. Our results indicate that for normal blood, RBC deformability over 0.3-50 Pa is stable up to six hours regardless of storage temperature; at eight hours there were no significant differences in EI but SS1/2 calculated via a Lineweaver-Burk method indicated impaired deformability. Storage temperature affected the stable period for RBC aggregation: the safe time was shorter at 25 degrees C whereas at 4 degrees C aggregation was stable up to 12 hours. Interestingly, blood samples from septic patients were less affected by storage. Blood can thus be stored at 25 degrees C for up to six hours for deformability studies, but should be limited to four hours for RBC aggregation; storage at 4 degrees C may prolong the storage period up to 12 hours for aggregation but not deformability measurements. Therefore, the time period between sampling and measurement should be as short as possible and reported together with results.

Time Course of Electrical Impedance During Red Blood Cell Aggregation in a Glass Tube: Comparison With Light Transmittance

Red blood cells (RBC) in normal human blood undergo reversible aggregation at low flow or stasis. The extent and kinetics of this phenomenon have been studied using various optical and electrical methods, yet results using such methods are not always in concordance. This study employed a horizontal glass tube in which blood flow could be established, then abruptly stopped. Normal blood and RBC suspensions with enhanced or decreased aggregation were studied. Light transmittance (LT) and electrical impedance at 100 kHz were recorded during high-shear flow and for 120 s after flow was abruptly stopped during which RBC aggregation occurs. Capacitance values were also obtained based on the imaginary part of impedance data and recorded. Various aggregation parameters were calculated, using the time course of LT, impedance, and capacitance, then compared with each other and with results from laboratory aggregometers. RBC aggregation parameters were calculated, using the time course of impedance data often failed to correlate with known changes of aggregation, even reporting aggregation for cells in nonaggregating media (i.e., RBC in buffered saline). Alternatively, RBC aggregation parameters based upon the time course of capacitance data are in general agreement with those derived from LT data and with RBC aggregation indexes, measured using commercial instruments.

Effect of hemoglobin oxygenation level on red blood cell deformability and aggregation parameters

Measurements of red blood cell (RBC) deformability and aggregation can be subject to influence by pre-analytical handling procedures, with the degree of hemoglobin oxygenation having the potential to affect the results. To examine such effects, RBC deformability and aggregation were studied before and after oxygenation or deoxygenation of human blood samples. RBC deformability was assessed using a laser-diffraction ektacytometer having Couette geometry. RBC aggregation was assessed using the same system by monitoring light backscattering after a sudden cessation of high shear; aggregation was also measured by monitoring light transmittance through RBC suspensions. RBC deformability was found to be significantly increased after equilibrating RBC with ambient air (pO2: 142.0+/-3.1 mmHg) compared to the non-oxygenated sample (pO2: 42.4+/-1.8 mmHg). In contrast, equilibration with 100% nitrogen resulted in significant impairment in RBC deformability. RBC aggregation parameters were also affected by oxygenation if measured based on light backscattering, but not if measured using light transmittance. It is thus recommended that blood samples be oxygenated by repeated exposure to ambient air prior to the measurement of hemorheological parameters.

Role of hemoglobin oxygenation in the modulation of red blood cell mechanical properties by nitric oxide

It has been previously demonstrated that both externally generated and internally synthesized nitric oxide (NO) can affect red blood cell (RBC) deformability. Further studies have shown that the RBC has active NO synthesizing mechanisms and that these mechanisms may play role in maintaining normal RBC mechanical properties. However, hemoglobin within the RBC is known to be a potent scavenger of NO; oxy-hemoglobin scavenges NO faster than deoxy-hemoglobin via the dioxygenation reaction to nitrate. The present study aimed at investigating the role of hemoglobin oxygenation in the modulation of RBC rheologic behavior by NO. Human blood was obtained from healthy volunteers, anticoagulated with sodium heparin (15 IU/mL), and the hematocrit was adjusted to 0.4 L/L by adding or removing autologous plasma. Several two mL aliquots of blood were equilibrated at room temperature (22+/-2 degrees C) with moisturized air or 100% nitrogen by a membrane gas exchanger, The NO donor sodium nitroprusside (SNP), at a concentration range of 10(-7)-10(-4)M, was added to the equilibrated aliquots which were maintained under the same conditions for an additional 60 min. The effect of the non-specific NOS inhibitor l-NAME was also tested at a concentration of 10(-3)M. RBC deformability was measured using an ektacytometer with an environment corresponding to that used for the prior incubation (i.e., oxygenated or deoxygenated). Our results indicate an improvement of RBC deformability with the NO donor SNP that was much more pronounced in the deoxygenated aliquots. SNP also had a more pronounced effect on RBC aggregation for deoxygenated RBC. Conversely, l-NAME had no effect on deoxygenated blood but resulted in impaired deformability, with no change in aggregation for oxygenated blood. These findings can be explained by a differential behavior of hemoglobin under oxygenated and deoxygenated conditions; the influence of oxygen partial pressure on NOS activity may also play a role. It is therefore critical to consider the oxygenation state of intracellular hemoglobin while studying the role of NO as a regulator of RBC mechanical properties.

Photometric measurements of red blood cell aggregation: light transmission versus light reflectance

Red blood cell (RBC) aggregation is the reversible and regular clumping in the presence of certain macromolecules. This is a clinically important phenomenon, being significantly enhanced in the presence of acute phase reactants (e.g., fibrinogen). Both light reflection (LR) and light transmission (LT) from or through thin layers of RBC suspensions during the process of aggregation are accepted to reflect the time course of aggregation. It has been recognized that the time courses of LR and LT might be different from each other. We aim to compare the RBC aggregation measurements based on simultaneous recordings of LR and LT. The results indicate that LR during RBC aggregation is characterized by a faster time course compared to simultaneously recorded LT. This difference in time course of LR and LT is reflected in the calculated parameters reflecting the overall extent and kinetics of RBC aggregation. Additionally, the power of parameters calculated using LR and LT time courses in detecting a given difference in aggregation are significantly different from each other. These differences should be taken into account in selecting the appropriate calculated parameters for analyzing LR or LT time courses for the assessment of RBC aggregation.

Measurement of red blood cell aggregation in disposable capillary tubes

A new method is described in this paper that allows measurement of red blood cell (RBC) aggregation indexes in disposable glass tubes within minutes. Light transmission through the RBC suspension filled into a microhematocrit capillary at stasis is recorded during RBC aggregation; a novel method assures an initial dispersion of aggregates in the capillary. The resulting light transmittance-time data are analyzed to calculate various parameters. Measurement of erythrocyte sedimentation rate (ESR) and RBC aggregation using well established methods and the newly developed capillary tube aggregometer in blood samples with a wide range of RBC aggregation indicated significant correlations between these parameters. Additionally, light transmittance during complete disaggregation allows estimating hematocrit, thereby enabling hematocrit correction of the measured and calculated parameters. The newly developed capillary tube RBC aggregometer is suitable for use as a method to rapidly monitor disease activity and the acute phase response, especially at the point-of-care (e.g., health care facilities, physicians office) and for field studies.

Wavelength selection in measuring red blood cell aggregation based on light transmittance.

The reversible aggregation of red blood cells (RBC) is of current basic science and clinical interest. Using a flow channel and light transmittance (LT) through RBC suspensions, we have examined the effects of wavelength (500 to 900 nm) on the static and dynamic aspects of RBC aggregation for normal blood and suspensions with reduced or enhanced aggregation; the effects of oxygenation were also explored. Salient observations include: 1. significant effects of wavelength on aggregation parameters reflecting the extent of aggregation (i.e., number of RBC per aggregate); 2. no significant effects of wavelength on parameters reflecting the time course of RBC aggregation; 3. a prominent influence of hemoglobin oxygen saturation on both extent and time-course related aggregation parameters measured at wavelengths less than 700 nm, but only on the time-course at 800 nm; and 4. the power of parameters in detecting a given alteration of RBC aggregation is affected by wavelength, in general being greater at higher wavelengths. It is recommended that light sources with wavelengths around 800 nm be used in instruments for measuring RBC aggregation via LT.

Effect of lanthanides on red blood cell deformability and response to mechanical stress: Role of lanthanide ionic radius

Prior studies exploring the effects of lanthanides (Ln) on red blood cells (RBC) have primarily focused on ion transport, cell fusion, and membrane protein structure. Our previous report [Biorheology 44 (2007), 361-373] dealt only with lanthanum (La) and cell rigidity; the present study extends these observations to other lanthanides (Nd, Sm, Eu, Dy, Er) and to RBC response to mechanical shear. Deformation-shear stress behavior of normal human RBC was measured at Ln concentrations up to 200 μM. In another series of experiments, RBC were exposed to mechanical stress (190 Pa, 300 s) at 50 μM Ln and deformation-stress data obtained prior to and after this stress. Data were fitted to a Lineweaver-Burke model to obtain the shear stress at one-half maximum deformation (SS1/2). Our results include: (1) lanthanides cause decreased cell deformability with the magnitude of the decrease dependent on concentration and shear stress; (2) this decrease of deformability is affected by Ln ionic radius such that La>Nd>Sm>Eu>Dy>Er and is reversible for cells in Ln-free media; (3) mechanical stress decreases deformability (i.e., increases SS1/2) such that compared to control, La and Sm reduce and Dy and Er enhance the mechanical stress effect; (4) the decrease of deformability consequent to mechanical stress scales inversely with Ln ionic radius. These results indicate a reciprocal relation between cell rigidity and sensitivity to mechanical stress that is mediated by Ln ionic radius. Additional studies are clearly warranted, particularly those that explore membrane-glycocalyx and intracellular mechanisms.

Diffuse reflectance spectroscopy for the measurement of tissue oxygen saturation

Tissue oxygen saturation (StO2) is a useful parameter for medical applications. A spectroscopic method has been developed to detect pathologic tissues, due to a lack of normal blood circulation, by measuring StO2. In this study, human blood samples with different levels of oxygen saturation have been prepared and spectra were acquired using an optical fiber probe to investigate the correlation between the oxygen saturation levels and the spectra. A linear correlation between the oxygen saturation and ratio of the intensities (760 nm to 790 nm) of the spectra acquired from blood samples has been found. In a validation study, oxygen saturations of the blood samples were estimated from the spectroscopic measurements with an error of 2.9%. It has also been shown that the linear dependence between the ratio and the oxygen saturation of the blood samples was valid for the blood samples with different hematocrits. Spectra were acquired from the forearms of 30 healthy volunteers to estimate StO2 prior to, at the beginning of, after 2 min, and at the release of total vascular occlusion. The average StO2 of a forearm before and after the two minutes occlusion was significantly different. The results suggested that optical reflectance spectroscopy is a sensitive method to estimate the StO2 levels of human tissue. The technique developed to measure StO2 has potential to detect ischemia in real time.