Rainer Bayer

The laser diffractoscope – a new and fast system to analyse red blood cell flexibility with high accuracy

Abstract Red blood cell (RBC) flexibility is one of the determining parameters of the microcirculation. As capillaries are smaller in diameter than RBC, RBC can only pass through by deformation. Reduced flexibility is associated with a reduced microcirculation. In obstetrics, pre-eclampsia and intrauterine growth retardation are regarded as diseases exhibiting a reduced microcirculation. With this new system it might be possible to achieve new data concerning the microcirculation in these diseases. The laser diffractoscope is based on laser light diffraction and computer-assisted image analysis. It offers the ability to determine the flexibility of RBC in whole blood with high accuracy between 0 and 30 Pa. Two hundred microlitres of whole blood are required, and the analysis is carried out in less than 5 min, with a standard deviation ≤1%. The laser diffractoscope is a new system which offers the ability to measure RBC deformability with high accuracy in a short time. Evaluation of reproducibility and validity, as well as comparison with data from other measuring systems, proves the excellence of the laser diffractoscope. An example of its application is shown by analysing the deformability of RBC in 10 uncomplicated pregnancies at term.

Stress-induced rigidification of erythrocytes as determined by laser diffraction and image analysis

An apparatus to measure red cell deformability, the laser diffractoscope, is presented. As in ektacytometry, the apparatus generates diffraction patterns using a laser beam that passes through a red blood cell (RBC) suspension in a viscosimeter. Introducing a CCD camera to record the diffraction pattern and computer-aided image analysis has reduced the measurement error to below 1% (variation coefficient). Employing this apparatus we have studied the effects of mechanical stress on RBC deformability in vitro. We submitted the erythrocytes to different shear stresses of various magnitudes (260 to 2620/s, viscosity of suspending medium 24 cP) and variable duration 1 to 16 mm). We demonstrate that a high mechanical stress reduces deformability at low shear rates but does not influence elongation at high shear rates and that the rigidification is related to mechanical stress in a dose-dependent manner. The period of exposure as well as the degree of mechanical stress influences the extent of deformability loss. We also show that RBC rigidification accumulates if mechanical stress is applied repetitively, the cells could not recover from this stress, below a certain threshold (1100/s, 24 cP) shearing does not produce any loss of flexibility, and the decrease of deformability is not accompanied with detectable hemolysis. It is suggested that the shear-induced rigidification is due to rearrangements in the cytoskeleton of the erythrocyte.

Discrimination between orientation and elongation of RBC in laminar flow by means of laser diffraction

The elongation curve of RBC as determined by rheoscopy or ektacytometry (laser diffraction) resembles a rectangular hyperbola. The experimental data obtained so far included too large errors of measurement to allow precise mathematical description. The combination of laser diffraction with image analysis has improved ektacytometry considerably, such that the error of measurement is reduced to less than 0.5%. In laminar flow RBC of healthy donors are elongated elliptically (p <EQ 0.001). Using the precise data of elliptical deformation, the elongation curve can be described to be hyperbolic. Hence, the double reciprocal plot gives a linear curve which -- over a wide range of shear stress (15 to 500 dyn/cm2) -- fits well the experimental data (r >= 0.99: p <EQ 0.001). The stress strain characteristics (i.e. elongation curve) can be described by two parameters: maximum elongation (Emax) and the shear stress needed for half-maximum elongation (KE).

Carrier systems in PDT II: accumulation strategies of biotin-avidin coupled photosensitizers developed on cultured tumor cells

Two possibilities to accumulate photosensitizing drugs at the surface of cultured tumor cells were exploited: biotinylation of the cell surface followed by avidin and dibiotin, and reaction with a biotinylated antibody followed by avidin and biotinylated sulfophthalocyanin. Both reactions were performed successfully. The latter leads to enhanced phototoxicity of the accumulated drug.

Erythrocyte shape analysis by means of laser diffraction

In quite a large number of disorders, reduced flexibility of red blood cells (RBC) can be detected. In cardiovascular diseases it is supposed that rigidification of RBC may be regarded as a pathogenetic factor aggravating ischemia by disturbing capillary perfusion. Most methods established so far to estimate RBC deformability are hard to standardize and include large measurement errors. We present a low-cost system to determine RBC shape and flexibility. It combines laser diffraction of RBC in Couette flow with automated computer assisted image analysis. Effortless handling allows the system to be used for RBC elongation measurements even in routine diagnostics. Analysis of the whole information content of diffraction patterns reduces errors due to noisy diffraction patterns of working a little off axis. The system allows detection of very small changes in flexibility (less than 5%). The accuracy of measurement is not affected by variation of hematocrit or the intensity of transmitted light. Using the newly developed system it is demonstrated (1) that mechanically induced RBC rigidification may occur without hemolysis; (2) that in photodynamic therapy (e.g., pheophorbide A) RBC rigidification occurs during irridation; and (3) that in-vitro aging of conserved blood may partly be inhibited by calmodulin antagonists (e.g., fendiline).

Effects of oxidative stress on erythrocyte deformability

Hemolysis as a consequence of open heart surgery is well investigated and explained by the oxidative and/or mechanical stress produced, e.g. by the heart lung machine. In Europe O3 is widely used by physicians, dedicated to alternative medicine. They apply O3 mostly by means of the Major Autohematotherapy (MAH, a process of removing 50-100 ml of blood, adding O3 gas to it and returning it to the patients body). No controlled studies on the efficacy of O3 are available so far, but several anecdotal cases appear to confirm that MAH improves microcirculation, possibly due to increased RBC flexibility. Most methods established to estimate RBC deformability are hard to standardize and include high error of measurement. For our present investigation we used the method of laser diffraction in combination with image analysis. The variation coefficient of the measurement is less than 1%. Previous investigations of our group have shown, that mechanical stress decreases deformability, already at rather low levels of mechanical stress which do not include hemolysis. On the other hand exposure to O2, H2O2 or O3 does not alter the deformability of RBC and--except O3--does not induce considerably hemolysis. However this only holds true if deformability (shear rates 36/s - 2620/s) is determined in isotonic solutions. In hypertonic solutions O3 decreases RBC deformability, but improves it in hypotonic solutions. The results indicate that peroxidative stress dehydrates RBC and reduces their size. To explain the positive effect of O3 on the mechanical fragility of RBC we tentatively assume, that the reduction of RBC size facilitates the feed through small pore filters. In consequence, the size reduction in combination with undisturbed deformability at iso-osmolarity may have a beneficial effect on microcirculation.

Analysis of erythrocyte flexibility by means of laser diffraction: rigidification due to defined shearing

A newly developed system to determine red blood cell flexibility is presented. It combines ektacytometry (laser diffraction) with image analysis. Laser light is passed through erythrocyte suspensions which are sheared in a Cuette like viscosimeter. The laser diffraction patterns are photographed by a CCD camera, and further analyzed by a computer. Flexibility is characterized by the quotient of minor and major axes of isointensity lines of the elliptically transformed diffraction patterns. The variation coefficient of the measurement is less than 1%.

Subcellular storage compartments of bacteriopheophorbide sensitizers

Fluorescence colocalization with the Golgi specific stain, NBD-ceramide, and the mitochondrial localizing stain, Rhodamine 123, confirmed the earlier assumption that the Golgi apparatus is one of the prominent storage compartments for bacteriopheophorbide esters in OAT 75 SCLC cells and several amelanotic melanoma cell lines (A375, Melur SP18, SkAMel 25). Furthermore, a diffuse staining of mitochondria, of non-structured cytoplasm, and an additional storage in melanine vesicles of the amelanotic melanoma cells suggests further storage compartments with quantitatively different contributions to the phototoxicity of bacteriochlorophyll-derived photosensitizers. Independent observations of early phototoxic effects on microfilamentous networks, enzymatic activities (succinate dehydrogenase, lactate dehydrogenase), and redistribution phenomena following primary uptake of the sensitizers let us assume that only a part of the 108 molecules taken up by a cell contribute directly to phototoxicity. Thus it may be asked if a proper subcellular positioning of only a few sensitizer molecules may have similar phototoxic effects as the huge amounts stored at apparently ineffective sites.

Laser diffraction of RBC: the method and its pitfalls

The method to determine red blood cell deformability by laser diffraction is presented. It combines ektacytometry (laser diffraction) with image analysis. Laser light is passed through erythrocyte suspensions, which are sheared in a Searle like viscometer. The laser diffraction patterns are photographed by a CCD camera and further analyzed by a computer. Flexibility is characterized by the quotient of minor and major axes of isointensity lines of the elliptically transformed diffraction patterns. The variation coefficient of the measurement is less than 0.5%.

Analysis of erythrocyte flexibility by means of laser diffraction: effects of mechanical stress, photosensitization, and ozone

A newly developed system to determine red blood cell (RBC) flexibility is presented. It combines ektacytometry (laser diffraction) with image analysis. Laser light is passed through erythrocyte (RBC) suspensions which are sheared in a Searle like viscosimeter. The laser diffraction patterns are photographed by a linear CCD camera, and further analyzed by a computer. Flexibility is characterized by the quotient of minor and major axes of isointensity lines of the elliptically transformed diffraction patterns. The variation coefficient of the measurement is less than 1%. Exposing RBC to mechanical stress within the viscosimeter (shear rates from 130 to 2620/s, viscosity of the suspending medium 22 - 24 cpoise) is followed by a decrease of RBC flexibility. This effect is only detectable if elongation is measured at low shear rates (e.g., 260/s) and a threshold of stress (1100/s) is exceeded. It is not reversible and not accompanied with detectable hemolysis. Exposing whole blood to ozone (2 - 77 (mu) g/ml) does not alter RBC flexibility, but induces significant hemolysis at ozone > 7 (mu) g/ml. Thus the action of ozone can be regarded as an all or nothing effect either leaving the cell unaffected (flexibility) or destroying it (hemolysis).