Comparison of three commercially available ... - CiteSeerX

Biorheology 46 (2009) 251–264 DOI 10.3233/BIR-2009-0536 IOS Press

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Technical report

Comparison of three commercially available ektacytometers with different shearing geometries Oguz K. Baskurt a,∗ , M.R. Hardeman b , Mehmet Uyuklu a , Pinar Ulker a , Melike Cengiz a , Norbert Nemeth c , Sehyun Shin d , Tamas Alexy e and Herbert J. Meiselman e a

Department of Physiology, Akdeniz University, Faculty of Medicine, Antalya, Turkey Department of Physiology, Academic Medical Center, Amsterdam, The Netherlands c Institute of Surgery, Department of Operative Techniques and Surgical Research, Medical and Health Science Center, University of Debrecen, Hungary d Department of Mechanical Engineering, Korea University, Seoul, Korea e Department of Physiology and Biophysics, University of Southern California, Keck School of Medicine, Los Angeles, CA, USA b

Received 23 February 2009 Accepted in revised form 3 April 2009 Abstract. In December 2008, the International Society for Clinical Hemorheology organized a workshop to evaluate and compare three ektacytometer instruments for measuring deformability of red blood cells (RBC): LORCA (Laser-assisted Optical Rotational Cell Analyzer, RR Mechatronics, Hoorn, The Netherlands), Rheodyn SSD (Myrenne GmbH, Roetgen, Germany) and RheoScan-D (RheoMeditech, Seoul, Korea). Intra-assay reproducibility and biological variation were determined using normal RBC, and cells with reduced deformability (i.e., 0.001–0.02% glutaradehyde (GA), 48◦ C heat treatment) were employed as either the only RBC present or as a sub-population. Standardized difference values were used as measure of the power to detect differences between normal and treated cells. Salient results include: (1) All instruments had intra-assay variations below 5% for shear stress (SS) > 1 Pa but a sharp increase was found for Rheodyn SSD and RheoScan-D at lower SS; (2) Biological variation was similar and markedly increased for SS < 3–5 Pa; (3) All instruments detected GA-treated RBC with maximal power at 1–3 Pa, the presence of 10% or 40% GA-modified cells, and the effects of heat treatment. It is concluded that the LORCA, Rheodyn SSD and RheoScan-D all have acceptable precision and power for detecting reduced RBC deformability due to GA treatment or heat treatment, and that the SS range selected for the measurement of deformability is an important determinant of an instrument’s power. Keywords: Erythrocyte deformability, ektacytometry

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Address for correspondence: Dr. Oguz K. Baskurt, Department of Physiology, Akdeniz University, Faculty of Medicine, Antalya, Turkey. Tel.: +90 242 310 1560; Fax: +90 242 310 1561; E-mail: [email protected]. 0006-355X/09/$17.00 © 2009 – IOS Press and the authors. All rights reserved

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O.K. Baskurt et al. / Comparison of three commercially available ektacytometers

1. Introduction Deformability of red blood cells (RBC) is an important parameter frequently used for hemorheological evaluations in experimental and clinical studies. A variety of methods have been used to quantitate RBC deformability, including filtration techniques, micropipette aspiration and ektacytometry [9]. Ektacytometry is a technique using diffraction patterns produced by laser light traversing a sheared low hematocrit RBC suspension; the geometry of the diffraction pattern reflects the deformed cell shape and is usually expressed as a dimensionless elongation index (EI). This technique has become the most utilized method in the field to test for RBC deformability, mainly due to its precision, sensitivity and convenience. Currently, there are several commercially available ektacytometers, all using the same laser-diffraction principle [10] but different shearing geometries (e.g., Couette, plate–plate, microchannel). All commercially-available instruments report EI measured over a range of shear stresses (SS), and in some devices this SS range is user selectable. Obviously, the value of an instrument used for research or clinical purposes is determined by its precision and sensitivity to alterations of RBC deformability. However, there are no generally-accepted standards for calibration or evaluation of hemorheological instruments, including devices to measure RBC deformability and aggregation. Therefore, a workshop was organized under the auspices of the International Society of Clinical Hemorheology to compare and evaluate three commercially-available ektacytometers. This report presents results from this workshop held in Antalya, Turkey, December 8– 13, 2008. 2. Materials and methods 2.1. Blood samples Venous blood samples were obtained from 10 healthy male volunteers, aged between 25 and 52 years. A tourniquet was applied to locate the antecubital vein prior to venipuncture and kept in place during the blood sampling. Thirty ml of blood was obtained from each donor using commercial vacuum tubes containing ethylenediamine-tetraacetic acid (EDTA, 1.5 mg/ml). All blood sampling was completed within two minutes after the application of the tourniquet. A five ml aliquot of each sample was saved for control measurements. The remaining 25 ml of each sample was centrifuged at 1400 × g for six minutes, the plasma aspirated and saved, and the buffy coat discarded. RBC were then washed twice with isotonic phosphate-buffered saline (0.01 M phosphate, PBS, pH = 7.4). 2.2. Glutaraldehyde treatment of RBC A portion of the washed RBC were re-suspended in PBS at 0.05 l/l hematocrit and divided into four aliquots. Glutaraldehyde (GA, Sigma Chemical Company, St. Louis, MO, USA) was added to achieve final concentrations of 0.001, 0.003, 0.005 and 0.02% and the suspensions incubated at room temperature (22◦ C) for 30 minutes. Following incubation with GA the RBC were washed three times with PBS. The washed RBC previously incubated with 0.001, 0.003 and 0.005% GA were re-suspended in autologous plasma at 0.4 l/l hematocrit. Additionally, mixtures of normal (untreated) and GA-treated RBC were prepared as follows: (1) 10% GA-treated (at 0.005%) RBC + 90% normal RBC; (2) 40% GAtreated (at 0.005%) RBC + 60% normal RBC; (3) 10% GA-treated (at 0.02%) RBC + 90% normal RBC; (4) 40% GA-treated (at 0.02%) RBC + 60% normal RBC. These RBC mixtures were also suspended in autologous plasma at 0.4 l/l hematocrit.


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2.3. Heat treatment of RBC Washed RBC were re-suspended in PBS at 0.1 l/l hematocrit and 20 ml of the suspension was transferred to a 50 ml Erlenmeyer flask. The flask was immersed in a water bath at 48◦ C for 9 min with gentle mixing, following which the flask was immediately cooled in a water bath at room temperature and the RBC used for deformability measurements. 2.4. RBC deformability measurements All measurements were carried out using RBC suspended at low hematocrit in an isotonic, viscous polymer solution: 40 µl of blood or RBC suspension added to 8 ml of a 6% polyvinylpyrrolidone solution (PVP, 360 kDa, Sigma, 30 mPa s at 37◦ C). Two types of RBC suspensions were used for deformability measurements: (1) to evaluate reproducibility a whole blood sample from one donor was used to determine intra-assay variation for each device; for this purpose, 10 separate aliquots of the dilute RBC suspension were measured; (2) to evaluate the ability of each device to detect altered RBC rigidity, normal samples, GA-treated and heat-treated RBC, and mixtures of normal and treated cells were employed using blood from each of the 10 individual donors. 2.5. Evaluated ektacytometers Three commercially-available ektacytometers having different shearing geometries were evaluated. 2.5.1. LORCA – Laser-assisted Optical Rotational Cell Analyzer The details of the LORCA (RR Mechatronics, Hoorn, The Netherlands) have been described elsewhere [7]. The dilute RBC suspension is sheared in a Couette system composed of a glass cup and a precisely fitting bob, with a gap of 0.3 mm between the cylinders. A laser beam is directed through the sheared sample and the diffraction pattern produced by the deformed cells is captured by a CCD camera and analyzed by a microcomputer. Based upon the geometry of the ellipsoidal diffraction pattern, an elongation index is calculated as: EI = (L − W )/(L + W ), where L and W are the length and width of the diffraction pattern. An increased EI at a given SS indicates greater cell deformation and hence greater RBC deformability. EI values were measured for nine SS at 0.3–75 Pa. All measurements were carried out at 37◦ C. 2.5.2. Rheodyn SSD The Rheodyn SSD (Myrenne GmbH, Roetgen, Germany) utilizes two parallel, transparent circular discs separated by a 0.5 mm gap as the shearing system [11]. The bottom disc is stationary and the upper one rotates at eight different pre-determined speeds to generate eight levels of shear rate. A polarized laser beam is directed through the sample perpendicular to the discs, and the geometry of the diffraction pattern on the opposite side sensed using four light-sensing diodes arranged in a square pattern. The mean length and width of the diffraction pattern is determined from the light intensities recorded by the diode array using a calibration factor, and the elongation index calculated as described above. The predetermined rotational speeds of the upper disc yielded a SS range of 0.3–75 Pa with the 30 mPa s PVP solution used in this study. The instrument is not temperature controlled, but was placed in a temperature controlled box throughout the measurements with the temperature maintained at 37◦ C.

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2.5.3. RheoScan-D slit-flow ektacytometer The RheoScan-D (RheoMeditech, Seoul, Korea) consists of a disposable element, a laser, a CCDcamera, a screen, a pressure sensor and a vacuum-generating mechanism [12]. The disposable element, which is made of transparent plastic, consists of a microchannel and reservoirs at each end; the dimensions of the microchannel are 0.2 mm high × 4.0 mm wide × 40 mm long. The dilute RBC suspension is sheared in the microchannel under continuously decreasing pressure-differentials. A laser beam is directed through the sheared sample and the diffraction pattern produced by the deformed cells is captured by a CCD camera and analyzed by a computer. Based upon the captured elliptical diffraction patterns, an elongation index is determined as described above. EI values are given for sixteen SS levels at 0.5– 20 Pa, with SS calculated using an average shear rate (i.e., one-half wall shear rate, assuming a parabolic velocity profile); SS values utilized herein were selected to be comparable to those from the other two instruments. The RheoScan-D instrument is not temperature controlled, but in the present study the plastic disposable elements were placed in a 37◦ C temperature controlled box prior to use. Due to the short test time (i.e., ∼30 s) and the use of pre-warmed suspensions, the sample temperature was assumed to be maintained at 37◦ C during the measurement period. 2.6. Other methods Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values were determined using a hematology analyzer (Cell-Dyn 3500R, Abbott Diagnostic Division, IL, USA). RBC shape was examined in dilute wet-mount, unstained preparations under light microscopy. The viscosity of the PVP solution was measured at 37◦ C using a Wells–Brookfield cone–plate viscometer (DV II + Pro, Brookfield Engineering Labs, Middleboro, MA, USA; shear rate = 750 s−1 ). 2.7. Calculations and statistics Intra-assay variation was calculated as the coefficient of variation (CV) of 10 repeated measurements on the same sample. Biological variation was expressed as the CV of the data obtained using the blood samples from 10 donors. The results for RBC suspensions with reduced deformability (i.e., GA or heat-treated) are expressed as mean ± standard error (SE); statistical comparisons were done using a two-way ANOVA for EI data measured at various SS followed by appropriate statistical post-tests. Additionally, standardized differences from control values were calculated by dividing the mean difference of EI between normal and the treated RBC at a given SS by the “pooled standard deviation” of the normal plus treated data. That is [mean normal (10 donors) − mean treated (10 donors)]/pooled standard deviation. The pooled standard deviation is calculated as the square root of the mean of squared standard deviations of the two groups being compared [5]. The standardized difference has been accepted as a measure of the power of each measurement condition to detect a difference between two groups [14].


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3. Results 3.1. Hematological parameters and red blood cell shape MCV, MCH and MCHC values were 84.7 ± 1.2 fl, 31.8 ± 0.5 pg and 37.3 ± 0.1 g/dl respectively. RBC shape was observed to be normal in normal and treated samples, except for heat-treated RBC where some stomatocyte formation and fragmentation were noted. 3.2. Intra-assay variation The CV values of the 10 repeated EI measurements on the same sample were below 5% at all SS for the LORCA (Fig. 1). CV results exceeded 5% only at lowest SS for the Rheodyn SSD and RheoScan-D, being markedly increased for the Rheodyn SSD at 0.38 Pa. 3.3. Biological variability Biological variability for normal samples from the 10 donors were similar and SS dependent, being less than 10% for all three instruments if the SS was above 0.5 Pa (Fig. 2). 3.4. Deformability measurements for glutaraldehyde-treated RBC Figure 3 presents EI versus shear stress data for RBC treated with GA at 0.001%, 0.003% and 0.005% concentrations. Inspection of these results indicates: (1) Data obtained for the three instruments have essentially similar shapes over comparable SS ranges; (2) The concentration-dependent effect of GA was observed with all three instruments, with EI progressively decreased with increased GA concentrations at most SS. Treatment of RBC with 0.001% GA resulted in a very slight decrease of deformability, with the differences statistically significant over SS ranges of 1.2–4.8 Pa for the LORCA, 1.5–3.8 Pa for the Rheodyn SSD and 0.5–15 Pa for the RheoScan-D. Differences between normal and GA-treated RBC were significant (p < 0.001) over a wider range of SS for RBC treated with 0.003% and 0.005% GA: (1) LORCA at all SS except the lowest (0.3 Pa) for both concentrations; (2) Rheodyn SSD at SS between 1.5 and 15 Pa for 0.003% and at all SS except the lowest (0.4 Pa) for 0.005% GA; (3) RheoScan-D at all SS over its range of 0.5–20 for both 0.003 and 0.005% GA.

Fig. 1. Coefficients of variation (CV) of 10 repeated measurements using the same blood sample.

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Fig. 2. Coefficients of variation (CV) of the measurements on normal samples from the 10 donors (i.e., biological variability). Note the similar dependence on shear stress with the three instruments.

Fig. 3. Elongation indexes of RBC treated with 0.001, 0.003 and 0.005% glutaradehyde. Data are presented as mean ± standard error, n = 10. Standard error lines remain inside the symbol for most data points. See Section 3.4 for statistical comparisons.


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Fig. 4. Standardized differences calculated using the data presented in Fig. 3. Each graph corresponds to RBC treated with GA concentrations of 0.001% (A), 0.003% (B) and 0.005% (C).

Figure 4 presents standardized differences for GA-treated RBC, where it is obvious that they increase with GA concentration and hence with increasing cell rigidity (Fig. 3). It is also obvious from Fig. 4 that the level of SS is also a determinant of standardized difference, with peak values at about 0.5–1.5 Pa and significantly lower levels at higher SS. Standardized differences for all three instruments in their lower SS range were similar for RBC treated with GA at the two lower concentrations (i.e., 0.001 and 0.003%). At 0.005% GA, the RheoScan-D had the highest standardized differences over the SS range for this device (0.5–20 Pa). Standardized differences for all GA concentrations were lowest for the Rheodyn SSD at SS > 10 Pa. 3.5. Mixtures of GA-treated and normal RBC EI–SS data for mixtures of normal RBC and RBC treated with 0.005% GA are presented in Fig. 5. Inclusion of 10% GA-treated RBC resulted in a very slight decrease of EI, with the LORCA and Rheodyn SSD not detecting significant differences and the RheoScan-D yielding significant alterations (p < 0.05 only at 15 and 17 Pa). All three devices were more sensitive to the presence of 40% of GA-treated cells (Fig. 5): LORCA EI values were significantly decreased at SS > 1.2 Pa; Rheodyn SSD results were significantly decreased between 1.5 and 75 Pa (p < 0.001 between 3.8 and 15 Pa), and RheoScan-D indicated significant EI decreases over its entire SS range (p < 0.001).

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Fig. 5. Elongation indexes of mixtures of normal RBC and 10% or 40% RBC treated with 0.005% glutaradehyde. Data are presented as mean±standard error, n = 10. Standard error lines remain inside the symbol for most data points. See Section 3.5 for statistical comparisons.

An increase of GA concentration to 0.02% resulted in almost total loss of cellular deformability as evidenced by EI values very close to zero regardless of the magnitude of the applied SS (data not shown). Mixtures of these rigid RBC with normal RBC at 10% and 40% were characterized by more pronounced decreases of EI at all SS (Fig. 6). EI results for mixtures of these rigid cells exhibited similar patterns for the three instruments, and thus were consistent with data for only GA-treated RBC (Fig. 3) and for mixtures of normal RBC with cells treated with 0.005% GA (Fig. 5). For mixtures containing 10% of these very rigid cells, EI values were significantly decreased (p < 0.01 or p < 0.001) between 2.4 and 75 Pa for LORCA, 7.5 and 75 Pa for the Rheodyn SSD and for SS > 2 Pa for RheoScan-D. As expected, 40% of rigid RBC in the suspensions resulted in more pronounced changes in EI–SS curves, with decreases of EI were statistically significant for all instruments at all SS > 0.8. Figure 7 presents standardized differences for mixtures of normal cells and RBC treated with 0.005% or 0.02% GA. These differences were larger for mixtures containing a greater percent of GA-treated RBC (i.e., 40% vs. 10%), and more pronounced if the modified RBC were made very rigid by treatment with 0.02% GA. For mixtures of RBC with modestly reduced deformability caused by 0.005% GA, all instruments had similar low standardized differences, especially for the suspensions containing 10%


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Fig. 6. Elongation indexes of mixtures containing 10 or 40% RBC treated with 0.02% glutaraldehyde. Data are presented as mean±standard error, n = 10. Standard error lines remain inside the symbol for most data points. See Section 3.5 for statistical comparisons.

GA-treated RBC (Fig. 7A). Standardized differences for suspensions containing 40% of these cells were slightly increased with a rank order of RheoScan-D > LORCA > Rheodyn SSD (Fig. 7B). The pattern for standardized difference versus SS for mixtures containing 10% of RBC treated with 0.02% GA continued to increase with SS for each instrument (Fig. 7C). However, for suspensions containing 40% of RBC treated with 0.02% GA, maximum values were reached at 1–2 Pa followed by decreases at higher SS (Fig. 7D). 3.6. Heat-treated RBC Heating RBC at 48◦ C for 9 minutes resulted in a marked decrease of deformability, with this change detected similarly by all three instruments (Fig. 8). Differences between heat-treated and normal RBC were statistically significant at all SS (p < 0.001), with the exception of the lowest levels (0.3–0.8 Pa) for the LORCA and Rheodyn SSD. Standardized differences for heat-treated RBC (Fig. 9) demonstrated a pattern similar to that for RBC treated with 0.005% GA (Fig. 7B): The LORCA and Rheodyn SSD had a maximum at around 1 Pa that gradually declined at higher SS, with the RheoScan-D exhibiting a lower degree of dependence on SS.

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Fig. 7. Standardized differences calculated using the data presented in Figs 5 and 6 for mixtures of normal and GA-treated cells. Panels A and B are for 10 or 40% RBC treated with 0.005% GA, and panels C and D are for 10 or 40% RBC treated with 0.02% GA.

4. Discussion The three ektacytometers evaluated in this study were characterized by a very low intra-assay variation (i.e., CV for repeated measurements of the same sample), with intra-assay variations below 5% for SS > 1 Pa. However, for the RheoScan-D and Rheodyn SSD instruments, there were sharp increases of intra-assay variation at SS < 1 Pa; CV values were approximately 10% at 0.5 Pa and reached ∼40% at 0.38 Pa (Fig. 1). This low reproducibility may thus limit the value of these two devices for measurements at less than 1 Pa. The dependence of intra-assay variation on SS observed herein for the LORCA is consistent with previous reports indicating a CV of about 4% at 0.5 Pa and 0.4% at 50 Pa [7]. Wang et al. report that for 6 repeated measures on the same normal blood sample, the reproducibility for both the LORCA and Rheodyn SSD was less than 5% but unfortunately do not indicate the shear stress range for these results [15]. The biological variation of EI for 10 normal donors (Fig. 2) also reflects a dependence on SS, in that all instruments indicated a markedly increased variation at SS less than about 3–5 Pa. Wang et al. also indicate a SS-dependence of biological variability for the LORCA and Rheodyn SSD: 28–33% CV at 0.5 Pa and 3–10% at 30 Pa. Note, however, that their studies used a 30 mPa s PVP solution for the LORCA and a 25%, 24 mPa s solution of 40 kDa dextran for the Rheodyn SSD. In addition, unequal


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Fig. 8. Elongation indexes of RBC heated at 48◦ C for 9 minutes. Data are presented as mean± standard error, n = 10. Standard error lines remain inside the symbol for most data points. See Section 3.6 for statistical comparisons.

Fig. 9. Standardized differences calculated using the data presented in Fig. 8.

temperatures were employed (22◦ C for Rheodyn SSD, 37◦ C for LORCA) and thus direct comparisons are inappropriate. In addition to determining reproducibility as an index to the merit of a hemorheological device, the use of standardized differences provides a parameter that allows comparison of instruments. Note that this parameter is determined by both the sensitivity of the instrument to a given change in deformability

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from normal and the variation within the two groups being compared [14] and is thus similar to a power analysis of a method [5]. Two separate approaches were utilized to compare the power (i.e., standardized difference) of each instrument for detecting differences between two RBC populations: (1) Comparisons between untreated RBC and RBC treated with 0.001, 0.003 or 0.005% GA in which all cells being tested had been exposed to this agent. GA at these low concentrations results in very slight and gradual alterations of deformability [1,14]; (2) Comparisons between untreated RBC and mixtures of untreated and GA-treated RBC using mixtures containing either 10% or 40% cells with slightly reduced deformability (0.005% GA) or with very rigid RBC (0.02% GA). The second approach was designed to model samples that may be obtained from patients where only a sub-population of RBC have impaired deformability [13,14]. Both approaches have been previously used to evaluate ektacytometers and alterations of EI, with concentrations of GA and percentages of rigid cells similar to those utilized herein [1,8,15]. GA treatment rigidifies RBC by cross-linking membrane proteins. It should be noted that this approach may not be representative for all clinically relevant mechanisms of deformability alterations (e.g., change in surface area/volume ration, change in cytoplasmic viscosity). In samples where all cells were treated with GA, the three instruments detected alterations induced by GA even at the lowest concentration (0.001%) and showed dose-dependent depression of SS–EI curves (Fig. 3). These measurements were all characterized by low coefficients of variation, with standard error bars being within the symbols used in Fig. 3. The CV values for these suspensions varied with SS (e.g., for the LORCA, 1.3–57% for the highest and lowest SS levels) and were similar to biological variability results for untreated, normal cells (Fig. 2); they also tended to increase with GA concentration and hence with increased differences between untreated and normal samples. Although the three instruments demonstrated very similar standardized differences between the untreated and GA-treated RBC at 0.001 and 0.003% concentrations, the RheoScan-D had a higher power (i.e., higher standardized difference) for 0.005% GA-treated RBC (Fig. 4C). It is also clear from Fig. 4 that standardized differences reached a maximum at about 1–2 Pa SS then gradually decreased with increased SS. This decreasing standardized difference with increasing SS is due to smaller differences between treated and untreated RBC at high stress levels, and not to poorer performance; the CV levels for these modified cell suspensions were smaller at higher SS than at lower SS. The instruments evaluated in this study also yielded similar results for samples with GA-treated RBC sub-populations. However, the changes caused by the presence of 10% of 0.005% GA-treated RBC could only be detected by the RheoScan-D at high SS (15–17 Pa, Fig. 7A); this instrument exhibited somewhat greater power for detecting the presence of 40% of 0.005% GA-treated RBC (Fig. 7B). All three instruments had similar power in detecting the presence of 10% or 40% RBC treated with 0.02% GA. EI–SS relations were remarkably similar for heat-treated RBC (Fig. 8) and are consistent with the known increased rigidity of such cells [3,8,15]. Standardized differences were also very similar, although the LORCA values were slightly higher (Fig. 9). It follows from the above discussion that the LORCA, Rheodyn SSD and RheoScan-D all had acceptable precision and power for detecting the various degrees of alterations induced by GA treatment and heat treatment. It is also obvious that the SS range selected for the measurement of EI is an important determinant of the power. Measurements at low SS are characterized by higher variation which may significantly decrease the power of the measurement. Further, differences between normal and altered RBC become smaller at higher SS, especially if the alteration of deformability is relatively small (Fig. 4). However, this is not true for RBC suspensions containing non-deformable RBC. Rather, in the presence


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of rigid RBC subpopulations, differences at higher SS became larger, thereby increasing the standardized differences (Fig. 7C and D). It is thus essential that each of these ektacytometers be operated in a shear stress range that optimizes their performance and ability to detect differences; it is especially important to avoid very low stress levels where instrument reproducibility is low (Fig. 1). While similar in performance, there are important differences between the three instruments: 1. The RheoScan-D operates over a smaller SS range (0.5–20 Pa) than the LORCA or Rheodyn SSD (0.35–75 Pa). However, this difference in measurement range does not impair the value of RheoScan-D since the highest sensitivity, and hence the most useful SS region for detecting differences, is available with all three instruments; 2. While all three devices were operated at 37◦ C, both the RheoScan-D and the Rheodyn SSD required the use of a temperature controlled box since neither presently have built-in temperature regulation. Since measurement temperature does affect sensitivity to changes of deformability [2] as well as the viscosity of the suspending medium, temperature control for all devices is warranted; 3. In the disc–disc Rheodyn SSD system, shear rate is uniform across the gap and is determined by the rotational speed of one disc and by the radial position of the laser beam. The LORCA uses a bob-in-cup Couette geometry in which variation of shear rate across the gap is proportional to the ratio of each radii squared. The nominal variation is approximately 5% and an average value is used. The RheoScan-D uses a parallel plate flow system having a parabolic flow velocity across the gap, and hence the shear is maximum at the walls and zero in the center. An average value of shear rate is also used in this instrument; 4. Both the LORCA and RheoScan-D use a computer-controlled video system to capture and analyze the diffraction image, with the LORCA obtaining repeated images at each shear rate and the RheoScan-D continuously recording images as the shear rate decreases. The Rheodyn SSD uses a set of diodes arranged parallel and perpendicular to the long axis of the diffraction pattern and obtains light intensity data at each of several shear rates. Given the above differences it is remarkable that each instrument provides very similar results for normal and modified RBC. In particular, it is curious that systems with small variations of shear rate throughout the sample (i.e., LORCA, Rheodyn-D) are in agreement with a device in which shear rate varies from maximum to zero across the suspension being measured. The resolution of this apparent dilemma must lie in an optical averaging process in which all cells in the path of the laser beam contribute to the resulting diffraction pattern, and an average shear rate is appropriate for computing shear stress [4]. It would thus be of potential value to incorporate an optical and image system in which individual RBC deformation is measured and a population distribution of RBC deformability determined. Such an approach has been utilized for counter-rotating rheoscopes in which the cells are deformed but remain stationery in the flow field [6]. If such an approach was automated and applied to the shear fields used in these ektacytometers, it would be possible to detect sub-populations of rigid cells and thus should supply additional information useful for research studies and hopefully improved patient care. In conclusion, the results of the current study indicates that all three evaluated instruments have acceptable sensitivity to alterations in deformability induced by GA treatment. Although GA treatment of RBC is a widely used model of deformability alterations, it should be noted that this approach may not be representative for all clinically relevant mechanisms of deformability alterations.

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Acknowledgements The workshop that provided data for this report was sponsored and financially supported by the International Society for Clinical Hemorheology. This study was also supported by NIH Research Grants HL15722, HL 70595 and HL 090511, by the Akdeniz University Research Projects Unit, and by a joint project supported by the Turkish Scientific and Technical Research Council (SBAG-MACAR-6; 106S251) and the National Office for Research and Technology of Hungary (BILAT TR-03/2006). References [1] O.K. Baskurt, T.C. Fisher and H.J. Meiselman, Sensitivity of the cell transit analyzer (CTA) to alterations of red blood cell deformability: role of cell size-pore size ratio and sample preparation, Clin. Hemorheol. 16 (1996), 753–765. [2] O.K. Baskurt and F. Mat, Importance of measurement temperature in detecting the alterations of red blood cell aggregation and deformability studied by ektacytometry: a study on experimental sepsis in rats, Clin. Hemorheol. Microcirc. 23 (2000), 43–49. [3] O.K. Baskurt and H.J. Meiselman, Cellular determinants of low-shear blood viscosity, Biorheology 34 (1997), 235–247. [4] C.F. Bohren and D.R. Huffman, Absorption and Scattering of Light by Small Particles, Wiley, New York, 1983. [5] J. Cohen, Statistical Power Analysis for the Behavioral Sciences, Lawrance Earlbaum Associates, Hillsdale, NJ, 1988. [6] J.G.G. Dobbe, M.R. Hardeman, G.J. Steekstra and C.A. Grimbergen, Validation and application of an automated rheoscope for measuring red blood cell deformability distributions in different species, Biorheology 41 (2004), 65–77. [7] M.R. Hardeman, P.T. Goedhart, J.G.G. Dobbe and K.P. Lettinga, Laser-assisted optical rotational cell analyzer (LORCA). I. A new instrument for measurement of various structural hemorheological parameters, Clin. Hemorheol. 14 (1994), 605–618. [8] M.R. Hardeman, P.T. Goedhart and N.H. Schut, Laser-assisted optical rotational cell analyzer (LORCA). II. Red blood cell deformability: elongation index versus cell transit time, Clin. Hemorheol. 14 (1994), 619–630. [9] M.R. Hardeman, P.T. Goedhart and S. Shin, Methods in hemorheology, in: Handbook Hemorheology and Hemodynamics, O.K. Baskurt, M.R. Hardeman, M.W. Rampling and H.J. Meiselman, eds, IOS Press, Amsterdam, 2007, pp. 242–266. [10] R.M. Johnson, Ektacytometry of red blood cells, Methods Enzymol. 173 (1989), 35–54. [11] H. Schmid-Schönbein, P. Ruef and O. Linderkamp, The shear stress diffractometer Rheodyne SSD for determination of erythrocyte deformability. I. Principles of operation and reproducibility, Clin. Hemorheol. 16 (1996), 745–748. [12] S. Shin, Y.H. Ku, M.S. Park and J.S. Suh, Laser diffraction in a slit rheometer, Cytometry 65B (2005), 6–13. [13] J. Stuart, A.J. Keidan, P.C.W. Stone and M.C. Sowter, Rheological study of erythrocyte sub-populations prepared by density-gradient fractionation, Clin. Hemorheol. 8 (1988), 467–475. [14] J. Stuart, P.C.W. Stone, G. Freyburger, M.R. Boisseau and D.G. Altman, Instrument precision and biological variability determine the number of patients required for rheological studies, Clin. Hemorheol. 9 (1989), 181–197. [15] X. Wang, H. Zhao, F.Y. Zhuang and J.F. Stoltz, Measurement of erythrocyte deformability by two laser diffraction methods, Clin. Hemorheol. Microcirc. 21 (1999), 291–295.