J Biorheol (2015) 29:36–41DOI 10.17106/jbr.29.36

ORIGINAL ARTICLE

Feasibility study of a sinusoidal shear flow generator for using counter-oscillating flow fields in monitoring of individual red blood cells under shear flow conditions

Nobuo Watanabe*, Tatsuya Tsuzuki, Yusuke Suzuki

Dept. of Bio-Science and Engineering, College of Systems Engineering and Science, Shibaura Institute of Technology, Room-102, 6th Building, Ohmiya-Campus, 307 Fukasaku, Minuma-Ku, Saitama-City, Saitama 337-8570, Japan

Received: 21 February 2014 / Accepted: 18 December 2014 / Published online: 17 September 2015© Japanese Society of Biorheology 2015

Abstract Aim: To develop a prototype device that allows direct observation of the deformation of individual red blood cells (RBCs) in an oscillating shear flow field. Method: A counter-oscillation mechanism composed of two parallel glass plates was constructed to keep RBCs floating at the centerline of a 30 μm fluid gap. RBCs in the suspen-sion fluid were observed using a high-speed camera with 40-fold magnification. Results: RBCs remained within the camera’s field of view when exposed to a shear force field that oscillated at 2 Hz. Moreover, glutaraldehyde-treated, hardened RBCs always tumbled and low-density RBCs had a larger elongation than high-density RBCs when exposed to the same shear field. Conclusion: The feasibility of this counter-oscillating mechanism for evaluating RBC deformability has been demonstrated.

Keywords erythrocyte’s deformability, sinusoidal shear flow, the slider-crankshaft mechanism

1. Introduction

Red blood cells (RBCs) can exhibit various types of deformations depending on the physical conditions of the surrounding flow: a tank-treading motion [1–4], an umbrella-like shape [5], a greatly elongated shape [6], and an ellipsoidal shape [7–9]. The deformability of RBCs has been linked to lifestyle-related diseases such as diabetes [10–14]. However, no diagnostic method based on observing such RBC behavior has been established to date. Several methods, focusing on indirect evaluations such as filterabil-ity [15], relative viscosity of whole blood [16], and laser

diffractometry [17–21], have been proposed. However, direct observation would be the most reliable and precise method for clinical diagnosis. Using this approach, many studies have focused on RBCs passing through microchan-nels [22, 23] or measurement of direct tension using a micropipette [24, 25]. Additionally, several studies have investigated RBCs exposed to shear forces without wall contact by using an in vitro shear chamber [26–28]. From the results of those experiments, we speculated that improved observation of fully suspended RBCs would be the most promising approach to achieve the reproducibility required for deformability diagnostics.

To realize such an improvement, we adopted previous observation techniques utilized for cyclically reversing shear flow [28–30]. However, those methods lack the ability to monitor the shape changes of individual cells over time and the shear flow was generated by one-sided oscillation only. Any dynamic shape changes are analyzed by averaging the deformations of all observed RBCs. This leads to a large variation of test results, as shown previously [28].

We hypothesized that a test setup allowing continuous observation of an individual RBC would enable its deform-ability to be evaluated, leading to a more accurate and more reliable diagnostic method. To examine this hypothesis, we developed an oscillating shear flow generator, using the counter-oscillation of two parallel plates: the upper and bottom plates oscillate in opposite directions at equal dis-tances and speeds. RBCs located at the center of the gap should theoretically remain in this position regardless of any change in shear force magnitude.

The purpose of this study was to assess the feasibility of the proposed oscillating shear flow generator and to determine whether the counter-oscillation mechanism enabled obser-vation of the dynamic deformation of individual RBCs.

*E-mail: nobuo@sic.shibaura-it.ac.jp

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2. Materials and Methods

Design and prototyping of sinusoidal oscillating shear flow generator

On the basis of a previously published design of a slider-crankshaft mechanism [28–30], we developed a counter-oscillation mechanism composed of two plates. A schematic diagram of the mechanism is shown in Fig. 1a. A side view of the prototype setup is shown in Fig. 1b.

The plate traveling distance is designed to be 2.50 mm using a crankshaft with 1.25 mm eccentricity (Fig. 1a). The motion of the upper plate motion is always opposite to the motion of the bottom plate, generating a sinusoidal Couette shear flow in the narrow gap between the plates. Therefore, in theory, the velocity at the centerline between the two parallel plates is always zero, which may allow for optical tracking of an individual RBC.

The gap between the two glass plates was set at 30 μm using shim tapes (FGSM0.03, Misumi Corporation, Tokyo, Japan). An inverted microscope (IX71, Olympus Corporation, Tokyo, Japan) was installed above the device, as shown in Fig. 1c. For testing, a dilute suspension of RBCs was placed between the two glass plates. RBCs suspended at the center of the narrow gap were selected for observation using the microscope.

Preparation of RBC suspension

Porcine arterial blood taken from the carotid artery was acquired at a slaughterhouse (Tokyo Shibaura Zouki Co., Ltd., Tokyo, Japan). An anticoagulant (3.24% sodium acid citrate solution) was mixed with the whole blood at a volume ratio of 10%. The RBC suspension for the experiment was prepared by mixing 10 μL of blood sample with 1990 μL of 31 wt% dextran phosphate-buffered saline (PBS) solution. Dextran powder with a molecular weight of 60,000–90,000 (dextran, low fraction, Acros Organics, Fairlawn, NJ) was used for making this fluid. The PBS solution was a mixture of PBS powder (#168-19323, Wako Pure Chemical Indus-tries, Ltd, Osaka) and 1 L of ion-exchanged water from a pure water production system (Pure Port PP-101, Sibata Scientific Technology Ltd., Soka, Japan).

The viscosity of the 31 wt% dextran PBS solution, meas-ured with a rotational viscometer (Brookfield DV-II+Pro, Brookfield Engineering Laboratories, Inc., Middleborough, MA), was 192 mPa·s at 23.5°C, so an approximate hemato-crit value of 0.4% was assumed. All experiments were con-ducted within 2 days of blood collection.

Feasibility study by microscopic flow visualization

We conducted a feasibility study of the prototyped oscil-lating flow generator by microscopic flow visualization. The test fluid was porcine blood mixed with a high-viscosity

Fig. 1 (a) Schematic drawing of the mechanism for generating counter-oscillating motions of upper and lower slider blocks. Two glass plates are attached to each slider block. Between those plates, a flow field is sandwiched in a 30 μm gap. (b) The sinusoidally oscillating shear flow generator prototype, incorporating the counter-oscillation mechanism. The cam’s eccentric structure (top and bottom) makes it possible to generate opposing slider-crankshaft motions. (c) View of the experi-mental setup. An inverted microscope is mounted below the oscillating shear flow generator.

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31 wt% dextran PBS solution [28]. The motor speed of the flow generator was set at 120 rpm (2 Hz). The flow field at the center of the 30 μm gap was photographed with a high-speed camera (Memrecam GX-1, Nac, Tokyo, Japan), using 40-fold magnification and a frame rate of 500 fps with an exposure time of 100 μs per frame. The observed flow area of 139.2 × 302.4 μm was recorded with a resolution of 464 × 1008 pixels.

By this method, the deformability of three types of RBCs was examined: RBCs hardened by glutaraldehyde treatment provided negative control data, and density-derived young and old RBCs, collected through a centrifugation process, were studied as highly deformable and less deformable cells, respectively. The relationship between cell density and cell age has been a subject of debate but, in a review paper, Piomelli and Seaman [32] concluded that the density separation would be a most useful technique to separate cells by age. The method for preparing RBCs with each deformability level is described below.

Preparation of glutaraldehyde-treated hardened RBCs

Porcine blood was centrifuged and the plasma removed. To remove all plasma proteins, which glutaraldehyde would bind to, the remaining RBCs were washed with PBS solution

by the following procedure. The RBCs were suspended in PBS with a volume equal to that of the removed plasma and then centrifuged. The supernatant was removed and replaced with an equal volume of PBS. Washing was performed three times.

Glutaraldehyde was gradually added to the RBC suspen-sion over 15 min via a syringe pump (SPE-1, AS ONE Cor-poration, Osaka, Japan). The final concentration of glutar-aldehyde was 1% by weight in the RBC/PBS suspension. Fixation took about 2 h by a previously published protocol [31].

Preparation of density-fractionated intact cells

Intact cells were fractionated by density-gradient centrif-ugation. The low-density RBCs, representing young cells, were taken from the top 10% layer and the high-density RBCs, representing old cells, were taken from the bottom 10% layer. Each layer was pipetted into 31 wt% dextran PBS solution to give a 200-fold dilution.

3. Results

Fig. 2 shows the shear flow field observed under the

Fig. 2 (a) Tracking a single RBC during one cycle of flow oscillation (0.5 s). Scale bar: 10 μm. (b) Example of raw image data and the scaled-up view of an extracted, single RBC. The major and minor axis lengths L and W, respectively, are quantified through image analysis after testing.

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microscope during the visualization tests. The time step between successive images (Δt) is 0.056 s. The images clearly show RBCs remaining within the high-speed camera’s field of view throughout the observation period, even though the flow was continuously oscillating. In response to the oscillating shear flow, RBCs change their shape cyclically into an ellipsoidal shape, aligning their major axis parallel to the flow direction, as shown in Fig. 2b. This phenomenon has been observed previously [28].

Some RBCs moved periodically back and forth within the viewer: the arrows in Fig. 2a point to an individual RBC and indicate its micro-movements during the observation period. Fig. 3 shows the tracks of several RBCs, which remained within the field of view throughout one oscillation cycle, with respect to both the parallel direction (Y-axis) and the perpendicular direction (X-axis) of the flow field. The coordinate origin is the position of the RBC at the start of tracking. The time step between each data point is 0.023 s. The figure shows a movement of about 70 μm along the flow direction and about 20 μm perpendicular to the flow.

We analyzed the dynamics of the shape changes of an individual RBC during one cycle period. The changes in major axis length (L), minor axis length (W), and major-to-minor axis ratio (L/W), indicating the degree of deforma-tion, are shown in Fig. 4a, b, and c, respectively. In the fig-ure, three time series corresponding to the deformations of five individual RBCs are shown.

Fig. 5a shows the dynamic deformation of low- and high-density RBCs during one cycle of flow oscillation (0.5 s). Here, each plot is made by taking the average measurements of three individual RBCs. The mean maximal values of L/W for low- and high-density RBCs were 2.66 and 2.44, respectively.

Glutaraldehyde-treated RBCs always exhibited tumbling, as shown in Fig. 5b. The time steps between frames are 0.02 s. The image of the individual RBC was manually extracted from the raw image and scaled to 41 × 41 pixels.

The RBC’s edge is blurred because of its rapid tumbling.

4. Discussion

Microscopic observation confirms that individual RBCs can be monitored in an alternating shear field. RBCs sus-pended at the center of the 30 μm gap between the plates of the shear flow generator remain within the field of view. However, a small amount of movement has been recorded. The movement along the flow direction is in line with our expectations based on the focus depth characteristics of the camera lens. The measured oscillating motion of 70 μm parallel to the flow direction suggests that the observed

Fig. 3 Analyzed displacement of an individual RBC during one cycle of flow oscillation (0.5 s).

Fig. 4 Dynamic change of a single RBC’s (a) major axis length L, (b) minor axis length W, and (c) major-to-minor axis ratio L/W during one cycle flow oscillation at 2 Hz (0.5 s). Image analysis was performed for five individual RBCs; cell numbers are the same in each figure.

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RBCs float within ±0.42 μm of the true centerline of the gap.

However, there was also unexpected movement perpen-dicular to the flow direction. Possible causes for this are the surface roughness of the plates and positional tolerance issues such as the parallelism and distance between the plates and mechanical joints. Further optimization of the bench setup is necessary.

Within the viewing area of 302.4 × 139.2 μm, an RBC’s motion parallel to the flow is 70.3 μm, which is 2.83% of the glass plate oscillation (2.5 mm), and the lateral movement perpendicular to the flow is 17.8 μm (0.71% of the plate oscillation). The lateral motion quantified through image analysis is negligible. Thus, the flow field was well con-trolled.

The periodic shape changes of RBCs in response to sinu-soidal shear stress could be observed. The major axis of all RBCs remained parallel to the flow throughout the cycle (except for membrane hardened tumbling RBCs). The length of the major and minor axes as well as the major-to-minor axis ratio (L/W ratio) of individual RBCs was suc-cessfully quantified through a single flow oscillation cycle as shown in Fig. 4a–c. In this study, the estimated maximal shear rate and shear stress were 1047 s–1 and 201 Pa, respectively. There is a clear difference between the levels of deformation of low-density and high-density RBCs, as shown in Fig. 5a: Compared with the high-density RBCs (old), the low-density RBCs (young) exhibited more elon-gation. The larger variation in deformation observed in the old RBCs may be attributed to larger variation in their

membrane flexibility. The glutaraldehyde-treated hardened RBCs that served as the negative control (no deformation) for the deformability evaluation always showed tumbling. In the current setup it is difficult to focus on the cell’s edge with an exposure time of 0.02 s, so their shape variations could not be analyzed. Their tumbling frequency was too high to be captured with the maximum exposure time achievable at 500 fps.

Taken together, the findings of this work indicate that this method of evaluating the deformability of individual RBCs is feasible. Further research is needed to show the variation of deformability within a representative sample of different batches of normal whole blood. Furthermore, the correla-tion between applied shear force and deformation response needs to be qualitatively assessed next.

5. Conclusion

We have developed a novel oscillating flow generator that incorporates a counter-oscillation mechanism. Micro-scopic observation demonstrates that this apparatus allows visual monitoring of individual RBCs during one oscillation cycle. The proposed method can therefore be applied as a new technique for evaluating the deformability of individ-ual RBCs.

Acknowledgements The authors thank Mr. Hiroshi Miyagi and Mr. Kazuo Narushima for their assistance with mechanical design and manufacturing processes at the workshop of the Omiya Campus,

Fig. 5 (a) Change of mean L/W values for the low- and high-density RBCs acquired from the centrifuged top 10% and bottom 10% layers. The image analysis was performed through one oscillation cycle; each plot shows the averaged data obtained from three RBCs. (b) Dynamic view of a glutaraldehyde-treated hardened RBC floating in 31 wt% dextran PBS solution under a 2 Hz oscillating shear flow. The motion of one RBC is shown at 0.02 s intervals (time increasing from left to right). Each picture is cropped and magnified to 41 × 41 pixels. A length of 33.3 pixels corresponds to 10 μm in both height and width directions. Because of the continuous tumbling, which was observed for all hardened RBCs, the edge of the shape is blurred.

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Shibaura Institute of Technology. We are grateful to the Shibaura Institute of Technology for financial support through research funds awarded in 2011 and 2013.

References

1. Fischer T, Schmid-Schönbein H. Tank tread motion of red cell membranes in viscometric flow: behavior of intracellular and extracellular markers (with film). Blood Cells. 1977; 3: 351–65.

2. Fischer TM, Schmid-Schönbein H, Stöhr-Liesen M. The red cell as a fluid droplet: tank tread-like motion of the human erythrocyte membrane in shear flow. Science. 1978; 202: 894–6.

3. Sutera, SP, Pierre PR, Zahalak GI. Deduction of intrinsic mechan-ical properties of the erythrocyte membrane from observations of tank-treading in the rheoscope. Biorheology. 1989; 26: 177–97.

4. Sutera SP, Tran-Son-Tay R, Boylan CW, Williamson JR, Gardner RA. Study of variance in measurements of tank-treading frequency in populations of normal human red cells. Blood Cells. 1983; 9(3): 485–95.

5. Skalak R, Brånemark PI. Deformation of red blood cells in capil-laries. Science. 1969; 164: 717–9.

6. Schmid-Schönbein H, Wells R. Fluid drop-like transition of eryth-rocytes under shear. Science. 1969; 165: 288–91.

7. Tran-Son-Tay R, Sutera SP, Zahalak GI, Rao PR. Membrane stress and internal pressure in a red blood cell freely suspended in a shear flow. Biophys J. 1987; 51(6): 915–24.

8. Kon K, Maeda N, Shiga T. Erythrocyte deformation in shear flow: influences of internal viscosity, membrane stiffness, and hemato-crit. Blood. 1987; 69: 727–34.

9. Pfafferott C, Nash GB, Meiselman HJ. Red blood cell deforma-tion in shear flow. Effects of internal and external phase viscosity and of in vivo aging. Biophys J. 1985; 47: 695–704.

10. McMillan DE, Utterback NG, La Puma J, Barbara S. Reduced erythrocyte deformability in Diabetes. Diabetes. 1978; 27(9): 895-901.

11. Williamson JR, Gardner RA, Boylan CW, Carroll GL, Chang K, Marvel JS, Gonen B, Kilo C, Tran-Son-Tay R, Sutera SP. Micro-rheologic investigation of erythrocyte deformability in diabetes mellitus. Blood. 1985; 65(2): 283–8.

12. Brown CD, Ghali HS, Zhao Z, Thomas LL, Friedman EA. Asso-ciation of reduced red blood cell deformability and diabetic nephropathy. Kidney International. 2005; 67(1): 295–300.

13. Saito K, Kogawa Y, Fukata M, Odashiro K, Maruyama T, Akashi K, Fujino T. Impaired deformability of erythrocytes in diabetic rat and human: investigation by the nickel-mesh-filtration technique. Journal of Biorheology. 2011; 25(1-2): 18–26.

14. Sabo A, Jakovljević V, Stanulović M, Lepsanović L, Pejin D. Red blood cell deformability in diabetes mellitus: effect of phytomena-dione. Int J Clin Pharmacol Ther Toxicol. 1993; 31(1): 1–5.

15. Reinhart WH, Usami S, Schmalzer EA, Lee MM, Chien S. Evalu-ation of red blood cell filterability test: influences of pore size, hematocrit level, and flow rate. J Lab Clin Med. 1984; 104(4): 501–16.

16. Rogausch H. Modifications of the erythrocyte deformability alter the effect of temperature on the relative viscosity of human blood.

Biorheology. 1982; 19(1/2): 237–44.17. Presti RL, Caracciolo C, Montana M, Barone R, Catania A, Caimi

G. Erythrocyte deformability evaluated by laser diffractometry in polycythemia vera. Clin Hemorheol Microcirc. 2012; 50(3): 189–92.

18. Bessis M, Mohandas N. A diffractometric method for the meas-urement of cellular deformability. Blood Cells. 1975; 1: 307–13.

19. Bessis M, Mohandas N, Feo C. Automated ektacytometry: a new method of measuring red cell deformability and cell indices. Blood cells.1980; 6(3): 315–27.

20. Shin S, Ku Y, Park MS, Jang JH, Suh JS. Rapid cell-deformability sensing system based on slit-flow laser diffractometry with decreasing pressure differential. Biosens Bioelectron. 2005; 20(7): 1291–7.

21. Clark MR, Mohandas N, Shohet SB. Osmotic gradient ektacy-tometry: comprehensive characterization of red cell volume and surface maintenance. Blood. 1983; 61: 899–910.

22. Tajikawa T, Imamura Y, Ohno T, Muranishi F, Kubota M, Ohba K. Measurement and analysis of the shape recovery process of each erythrocyte for estimation of its deformability using the microchannel technique: the influence of the softness of the cell membrane and viscosity of the hemoglobin solution inside the cell. J Biorheol. 2013; 27: 1–8.

23. Kikuchi Y, Sato K, Ohki H, Kaneko T. Optically accessible microchannels formed in a single silicon substrate of blood rheol-ogy. Microvasc Res. 1992; 44: 226–40.

24. Markle DR, Evans EA, Hochmuth RM. Force relaxation and permanent deformation of erythrocyte membrane. Biophys J. 1983 April; 42(1): 91–8.

25. Hochmuth, RM, Worthy PR, Evans EA. Red cell extensional recovery and the determination of membrane viscosity. Biophys J. 1979; 26: 101–14.

26. Sutera SP, Gardner RA, Boylan CW, Carroll GL, Chang KC, Marvel JS, Kilo C, Gonen B, Williamson JR. 1985. Age-related changes in deformability of human erythrocytes. Blood. 65: 275–82.

27. Fischer TM. Shape memory of human red blood cells. Biophys J. 2004; 86: 3304–13.

28. Watanabe N, Kataoka H, Yasuda T, Takatani S. Dynamic defor-mation and recovery response of a red blood cell to cyclically reversing shear flow: Effects of frequency of cyclically reversing shear flow and shear stress level. Biophys J. 2006; 91(5): 1984–98.

29. Watanabe N, Arakawa Y, Sou A, Kataoka H, Ohuchi K, Fujimoto T, Takatani S. Alteration in human red blood cells’ deformability exposed to uniform shear stress: Evaluation using a cyclically reversing shear flow. Physiol Meas. 2007; 28: 531–45.

30. Watanabe N, Sakota D, Ohuchi K, Takatani S. Deformability of red blood cells and its relation to blood trauma in rotary blood pumps. Artif Organs. 2007; 31(5): 352–8.

31. Morel FM, Baker RF, Wayland H. Quantitation of human red blood cell fixation by glutaraldehyde. J Cell Biol. 1971; 48(1): 91–100.

32. Piomelli S, Seaman C. Mechanism of red blood cell aging: Rela-tionship of cell density and cell age. Am J Hematol. 1993; 42(1): 46–52.