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Temperature-dependent threshold shear stress of red blood cell aggregation
ARTICLE IN PRESS Journal of Biomechanics 43 (2010) 546–550
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Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Temperature-dependent threshold shear stress of red blood cell aggregation Hyun-jung Lim, Yong-Jin Lee, Jeong-Hun Nam, Seok Chung, Sehyun Shin School of Mechanical Engineering, Korea University, Anam-dong Seongbuk-gu, Seoul 136-713, South Korea
a r t i c l e in f o
a b s t r a c t
Article history: Accepted 6 September 2009
Red blood cell (RBC) aggregation is becoming an important hemorheological parameter, which exhibits a unique temperature dependence. However, further investigation is still required for understanding the temperature-dependent characteristics of hemorheology that includes RBC aggregation. In the present study, blood samples were examined at 3, 10, 20, 30, and 37 1C. When the temperature decreases, the whole-blood and plasma viscosities increase, whereas the aggregation indices (AI, M, and b) yield contrary results. Since these contradictory results are known to arise from an increase in the plasma viscosity as the temperature decreases, aggregation indices that were corrected for plasma viscosity were examined. The corrected indices showed mixed results with the variation of the temperature. However, the threshold shear rate and the threshold shear stress increased as the temperature decreased, which is a trend that agrees with that of the blood viscosity. As the temperature decreases, RBC aggregates become more resistant to hydrodynamic dispersion and the corresponding threshold shear stress increases as does the blood viscosity. Therefore, the threshold shear stress may help to better clarify the mechanics of RBC aggregation under both physiological and pathological conditions. Crown Copyright & 2009 Published by Elsevier Ltd. All rights reserved.
Keywords: RBC Aggregation Temperature Threshold Shear stress
1. Introduction At stasis or under low shear flows, red blood cells (RBCs) tend to form two-dimensional rouleaux. The rouleaux can grow end-toend or side-to-end; upon completion, they form 3D networks. RBC aggregation is influenced by intrinsic cellular properties (i.e., the deformability and surface charge of RBCs) (Rampling et al., 2004) and by extrinsic properties of the suspending medium (i.e., the pH, temperature, and protein concentration of plasma) (Weng et al., 1996). RBC aggregation plays an important role in the blood flow, particularly in the microvascular system. In fact, increased RBC aggregation has been observed in various pathological diseases, such as diabetes (Cloutier et al., 2008), sepsis (Baskurt et al., 1997), thrombosis (Baskurt and Meiselman, 2003), myocardial infarction (Ami et al., 2001), renal failure (Hein et al., 1987), and microcirculatory diseases (McHedlishvili et al., 1999). Thus, the degree of RBC aggregation is accepted widely as an important determinant of the hemorheological characteristics of blood. Thus, the study of RBC aggregation continues to be of basic scientific and clinical interest. Red-cell aggregation (RCA) is known to be the main determinant of the whole-blood viscosity (WBV) at low shear rates. RCA causes shear-thinning behavior of WBV. In addition, WBV and RCA are affected greatly by the temperature as are most hemorheological properties (Uyuklu et al., 2009). In general, the blood
Corresponding author.
E-mail address: [email protected] (S. Shin).
viscosity increases as the temperature decreases, whereas the aggregation index (AI) decreases as the temperature decreases (Dobbe, 2002). The temperature dependence of these two hemorheological parameters seems to be contradictory since an increase in the low-shear blood viscosity should accompany the increase in the AI as the temperature decreases. The paradoxical phenomenon can be clarified easily by considering the effect of the temperature-dependent plasma, which is an attenuating medium of RBC suspensions. As the plasma viscosity increases when the temperature decreases, the rate of formation of RBC aggregates is retarded. The effect of the temperature on the rate of formation of RBC aggregates can be eliminated by multiplying AI with the plasma viscosity that corresponds to the temperature. As reported in a previous study (Neumann et al., 1987), the rate of formation of RCA increased as the temperature increased but the corrected value of the rate decreased as the temperature increased. Thus, one should use the corrected rate of formation of RCA, while interpreting the temperature-dependent characteristics of whole-blood viscosity. However, the determination of the corrected indices would be difficult since it requires additional measurements of the plasma viscosity. It would be better to find an alternative aggregation index for interpreting directly the temperature-dependent characteristics of blood viscosity. Therefore, it is greatly necessary to develop a new aggregation index for representing RCA. The index should be informative and directly indicative of the temperature-dependent characteristics of blood viscosity without entailing the measurement of other hemorheological properties. Thus, one needs to examine the
0021-9290/$ - see front matter Crown Copyright & 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2009.09.031
ARTICLE IN PRESS Hyun-jung Lim et al. / Journal of Biomechanics 43 (2010) 546–550
temperature-dependent characteristics of various aggregation indices and to compare them with those of blood viscosity. Neumann et al. (1987) measured RBC aggregation using a coneplate aggregometer (Myrenne GmbH, Roetgen, Germany) and reported that the threshold shear rate (TSR) and the threshold shear stress (TSS) tended to increase as the temperature decreased. Thus, these two parameters are possible candidates for representing RCA and interpreting the temperature dependence of WBV without correction of the plasma viscosity. Recently, a critical shear stress (CSS) was proposed as a measure of RBC aggregation. In fact, the CSS was found to be another type of threshold shear stress whereby RBCs start to reaggregate in a decreasing-shear-flow environment. Therefore, the objectives of the present study are to investigate the temperature dependence of RCA with regard to various parameters and to correlate RCA in terms of the temperature-dependent characteristics of blood viscosity. The aggregation indices were measured with two aggregometers, LORCA (Hardeman et al., 2001) and a new microfluidic aggregometer (Shin et al., 2009).
2. Materials and methods Blood was obtained from healthy volunteers (five males, aged 267 2.7), who were not on any medication and who provided informed consent. The blood group of two of the participants was A and that of the remaining three was B. A venous blood sample was drawn from the antecubital vein and collected in vacutainers that contained (K2) EDTA (1.8 mg/ml) as the anticoagulant. The hematocrit was determined by centrifugation at 13,000g for 5 min. For the analysis of RCA and the apparent whole-blood viscosity, the hematocrit was adjusted to 4571%. This was achieved by either adding a calculated amount of autologous plasma to an aliquot of the sample or withdrawing plasma from the supernatant after centrifugation at 3000g for 5 min. Unless otherwise stated, the hematocrit values of the samples were adjusted to 0.45 L/L by either the addition or the removal of autologous plasma. For each experiment, five repeated measurements were conducted for each sample. All measurements complied with the new guideline provided by ISCH (Baskurt et al., 2009). Red-cell aggregation (RCA) was analyzed by two aggregometers (LORCA and a microfluidic aggregometer) at 4, 10, 20, 30, and 37 1C. Fig. 1 describes the schematics of two aggregometers (LORCA and the microfluidic aggregometer). The whole-blood sample was used directly for the measurement of RBC aggregation without the removal of WBCs. The details of LORCA (Laser-assisted Optical Rotational Cell Analyzer, RR Mechatronics, Hoorn, The Netherlands) have been described elsewhere (Hardeman et al., 2001). In brief, the system consists of a Couette geometry that is composed of a glass cup and a precisely fitting bob with a gap of 0.3 mm between the cylinders, as shown in Fig. 1; the RBC suspension is contained in the gap. The beam from a laser that is built into the bob is directed onto the sheared sample. The reflected light is recorded by two photodiodes that are located in the bob and is analyzed by a microcomputer. The sample is first sheared at a user-selectable shear rate for disaggregating the pre-existing RBC aggregates (600 s 1 in the present study), following which the shearing is stopped abruptly. The reflection of light from the suspension is recorded for a user-selectable period (120 s in the present
547
study). The resulting syllectogram is analyzed for calculating several indices that reflect both the magnitude and the duration of aggregation. In the syllectogram that was obtained from LORCA measurements, following the sudden cessation of stirring, the conventional mathematical representation of the syllectogram could be adopted, as per the single-exponential representation below: IðtÞ ¼ I0 þ I1 ebt :
ð1Þ
In Eq. (1), b denotes the rate constant of the aggregation process. Since aggregation often can be considered a multi-step process (i.e., doublet-, rouleaux-, and 3D-aggregate formation), the above equation can include two exponential terms rather than just a single exponential term. However, in the present study, we intentionally adopt a single-exponential function in the equation for comparing the findings with those of prior research (Neumann et al., 1987). Through an analysis of the syllectogram, various indices were obtained as follows (Hardeman et al., 2001): (1) the amplitude (AMP), which pertains to the total extent of aggregation and the total change in the intensity of reflected light during a time period of 120 s; (2) the aggregation half time (T1/2), which is the time for AMP to decrease by 50%; (3) the surface area (SA), which is the area above the syllectogram over the first 10 s; (4) the aggregation index (AI), which is the ratio of the area above the syllectogram to the total area (i.e., the sum of the areas above and below the syllectogram curve) over the first 10 s; (5) b, which is the rate constant of RCA formation in stasis for fitting the syllectogram curve using Eq. (1); and (6) the threshold shear rate (gthr), which is the minimal shear rate that is needed for preventing RBC aggregation. The present microfluidic aggregometer (Shin et al., 2009) consists of a disposable element, a laser, two photodiodes, a pressure sensor, and a vacuumgenerating mechanism, as shown in Fig. 1. The disposable element, which is made of transparent plastic, consists of a microchannel and reservoirs at each end; the microchannel is 0.2 mm high, 4.0 mm wide, and 40 mm long. The whole-blood sample (of 0.5 ml) is sheared in the microchannel under continuously decreasing pressure differentials. As the pressure differential exponentially decreases over time, the RBC aggregates tend to break down and disperse at high shear flows and the corresponding backscattered light (BSL) increases. When the pressure differential further decreases, the dispersed RBCs tend to re-aggregate and the BSL starts to decrease. Thus, the maximum value of BSL over the running index for time, t, indicates the termination of RBC disaggregation and the commencement of RBC aggregation. The time and shear stress values that correspond to the maximum value of the BSL intensity are defined as the critical time (tc) and the critical shear stress (tc), respectively. Since both aggregometers cannot operate below room temperature, they were placed in a temperature-controlled chamber and therefore, all measurements were conducted in the chamber. The thermostatic control of the measurement chamber was achieved by a fan-mounted heat exchanger that was connected to a water bath; the chamber temperature was maintained by a thermostat. The temperature was also checked continuously by thermocouples in the instruments. After the specified temperature was reached, 30 min was allowed for full thermal equilibrium between the measurement chamber and the instruments. As stated earlier, in order to correct the effect of the temperature on RCA, the plasma viscosity is to be measured at each temperature. Furthermore, it is also necessary to measure the whole-blood viscosity in a range of shear rates for determining the threshold shear stress. Therefore, the plasma and whole-blood viscosities were measured at 4, 10, 20, 30, and 37 1C using a pressure-scanning capillary viscometer (Shin et al., 2004) and a rheometer (AR2000, TA Instruments, USA). At each temperature, the dimensions of the glass capillary were measured with a digital microscope (Sometech, Seoul, Korea) and the instrument was calibrated using standard oil (Brookfield Corporation) of known viscosity. The whole-blood viscosity was measured over a range of shear rates (1–1000 s 1). All
Fig. 1. Schematics of two aggregometers (LORCA and a microfluidic aggregometer).
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rheological measurements were performed within four hours after the withdrawal of blood. In addition, the threshold shear stress (TSS, tthr) was determined by the product of the threshold shear rate (TSR, gthr) and the corresponding whole-blood viscosity (Zthr). Thus, the TSR was measured with LORCA and the whole-blood viscosity was measured with a pressure-scanning capillary viscometer. Then, the TSS was obtained by multiplying the TSR with the corresponding blood viscosity. The TSS represents the minimal shear stress for keeping RCAs dispersed. This parameter yields a quantitative measure of the aggregative strength of RCAs in a dimensional unit (Pa). A similar parameter, the critical shear stress (CSS, tcr), was introduced recently (Shin et al., 2009). The CSS is defined as the threshold shear stress for keeping RCAs dispersed in a channel flow. The CSS can be determined through a simple microfluidic aggregometer with simultaneous measurements of the pressure and the optical intensity. The detailed principle of CSS measurement can be found elsewhere (Shin et al., 2009).
increased. Due to the strong temperature dependence of the SA index, the corrected SA (SA m) still showed the same trend as the SA index.
3. Results The plasma viscosity exhibited a strong temperature dependence, as seen in Fig. 2. The plasma viscosity decreased monotonically as the temperature increased. As the temperature increased from 4 to 37 1C, the plasma viscosity decreased from 3.2 to 1.7 mPa s (a reduction of 48%). In addition, the whole-blood viscosity decreased as the temperature increased. For instance, at a shear rate of 10 s 1, the whole-blood viscosity decreased from 21.4 to 13.1 mPa s (a reduction of 40%) when the temperature increased from 4 to 37 1C. The aggregation index (AI) exhibited a strong temperature dependence, as shown in Fig. 3. AI increased monotonically as the temperature increased. As the temperature decreased from 37 to 4 1C, AI decreased from 65 to 41 (a reduction of 32%). However, the corrected AI (AI m) was quite different from the original AI. There was no significant change in the value of AI m over the whole range of the temperature. This phenomenon was due primarily to the decrease in the plasma viscosity as the temperature increased. Depending on the temperature dependence of the plasma viscosity, the corrected AI could decrease as the temperature increased (Neumann et al., 1987). The surface area (SA) also exhibited a strong dependence on the temperature, as shown in Fig. 4. There was a doubling of the SA index as the temperature increased from 4 to 30 1C. However, above 30 1C, the SA index decreased slightly as the temperature
Fig. 3. Variation of AI and the corrected AI with the temperature using LORCA.
Fig. 4. Variation of SA and the corrected SA with the temperature using LORCA.
Fig. 2. Variation of the viscosity of plasma and whole blood with the temperature at three different shear rates (measured with a pressure-scanning capillary viscometer) (Shin et al., 2004).
ARTICLE IN PRESS Hyun-jung Lim et al. / Journal of Biomechanics 43 (2010) 546–550
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of the b index, the corrected b index (b m) still showed the same trend as the b index. In contrast to the above indices, the threshold shear rate (TSR, gthr) significantly decreased as the temperature increased from 4 to 20 1C. Beyond 20 1C, the TSR reached an asymptotic value and became temperature independent (Fig. 6). The results on the TSR are quite the opposite to those of either AI or b. In addition, the threshold shear stress (tthr), which is the product of the TSR and the corresponding blood viscosity (gthr Zthr), showed similar results to the TSR (Fig. 7). The threshold shear stress (TSS) decreased monotonically as the temperature increased from 4 to 30 1C. Above 30 1C, the TSS slightly increased as the temperature increased. 4. Discussion Fig. 5. Variation of b and the corrected b with the temperature using LORCA.
Fig. 6. Variation of the threshold shear rate with the temperature using LORCA.
Fig. 7. Comparison of the threshold shear stress (TSS) and the critical shear stress (CSS) at different temperatures. The TSS was determined through the LORCA aggregometer and a blood viscometer, whereas the CSS was measured directly through a microfluidic aggregometer. (The results on the TSS reported in Neumann et al. (1987), are also plotted for the sake of comparison.)
Similarly, the b index (the rate constant of RCA) increased monotonically as the temperature increased from 4 to 30 1C (Fig. 5). The b index approximately tripled when the temperature increased from 4 to 30 1C. In fact, the b index exhibited the strongest temperature dependence among the measured aggregation indices. Similar to the SA index, the corrected b index (b m) still showed the same trend as the b index. Above 30 1C, the slope of the b index curve significantly changed and there was a slight increase in the b index. In addition, the slope of the corrected b index (b m) became opposite to that of the b index above 30 1C. However, due to the strong temperature dependence
Conventional aggregation indices, such as the AI, SA, and b, increase as the temperature increases, which are opposite trends to that of the blood viscosity. As mentioned earlier, since RBC aggregation is the main determinant of low-shear blood viscosity, the temperature dependence of aggregation indices seem to be contradictory to that of the blood viscosity. These results were caused by the temperature-dependent viscosity of the suspending medium, viz., the plasma. Neumann et al. (1987) reported that the opposite trend of temperature-dependent aggregation indices could be corrected by multiplying the aggregation indices with the plasma viscosity. In the present study, however, the corrected indices could not yield straightforward interpretations for the temperature dependence of the blood viscosity, since the temperature dependence of aggregation indices showed mixed patterns of variation even after the plasma viscosity was corrected for. These different results between the two studies were due to the degree of temperature dependence of the plasma viscosity. In other words, the plasma viscosity in the present study was slightly less temperature-dependent than that in the previous study (Neumann et al., 1987). In the present study, the threshold shear rate and shear stress of RBC aggregates decreased as the temperature increased, which showed good agreement with the results on the blood viscosity. Thus, the temperature dependence of whole-blood viscosity may be interpreted through the threshold shear rate and shear stress. However, since the threshold shear rate (TSR) is a strong function of the hematocrit (Shin et al., 2009), it is necessary to adjust the hematocrit for comparing TSR with other results. Furthermore, the determination of the threshold shear stress requires measurements of both the threshold shear rate (gthr) and the threshold blood viscosity (Zthr), even though the threshold shear stress is hematocrit independent (Eldwood et al., 1993; Shin et al., 2009). However, the critical shear stress (CSS), which has the same physical definition as the TSS, can be measured directly through a transient microfluidic aggregometer (Shin et al., 2009). In Fig. 7, the CSS (tcr) is compared with the TSS (tthr). Additionally, the TSS obtained in a prior study (Neumann et al., 1987) is plotted in Fig. 7 for the purpose of comparison. Both threshold shear stresses were at least twice the CSS. This difference is attributed to the lower effectiveness of the shear stress, the direction of which is parallel to the rouleaux interface if there is no alignment with the rouleaux interface (Chien et al., 1977). The difference between the two threshold shear stresses may be caused by the measurements of the whole-blood viscosity. However, the CSS showed the same trend as the TSS when the temperature varied between 4 and 30 1C. In addition, CSS and TSS were found to be hematocrit independent; hence, there is no need to adjust the hematocrit value for comparing CSS with TSS (Razavian et al., 1992; Shin et al., 2009). Thus, the CSS may provide a direct and intuitive understanding of the hemorheological characteristics of whole blood.
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The threshold (or critical) shear stress significantly increased as the temperature decreased. This fact indicates that increasingly high shear stresses are required for dispersing RCAs at low temperatures. Hence, under hypothermic conditions, the mechanical stability of RCAs appears to be increased, which may cause a significant increase in the blood-flow resistance, especially in microcirculation. The significant increase of the threshold shear stress may be correlated with the seasonal rise of circulatory diseases in winter. In general, the seasonal variation of the rate of occurrence of ischemic circulatory diseases is strongly correlated with elevated blood pressure, lipids, and other factors related to thrombosis and hemostasis (Eldwood et al., 1993). Since red-cell aggregation is also closely related to thrombosis and hemostasis, it is worth investigating the possible correlation between seasonal ischemic heart disease and red-cell aggregation.
5. Conclusion In summary, various aggregation indices were examined for their temperature dependencies on the basis of the interpretation of the variation of the blood viscosity with the temperature. Even though the aggregation indices were corrected with reference to the temperature-dependent plasma viscosity, they did not show the same trend with regard to the temperature dependence of the blood viscosity. The threshold shear rate and the threshold shear stress yielded the same trends as that of the whole-blood viscosity with regard to temperature variations. The threshold shear rate requires the adjustment of the hematocrit, whereas the threshold shear stress that is measured in a Couette-flow-type aggregometer requires the additional measurement of the viscosity. However, the critical shear stress, which can be directly measured in a microchannel flow, yielded the same trend as for the wholeblood viscosity with temperature variations. As the temperature decreases, RBC aggregates become more resistant to hydrodynamic dispersion and the corresponding threshold (or critical) shear stress becomes large and the blood becomes viscous. Thus, the threshold (or critical) shear stress may help to better clarify the mechanics of RBC aggregation under both physiological and pathological conditions.
Conflict of interest None.
Acknowledgement This research was supported by the Seoul R&BD Program Grant (NT080573). References Ami, R.B., Barshtein, G., Zeltser, D., Goldberg, Y., Shapira, I., Roth, A., Keren, G., Miller, H., Prochorov, V., Eldor, A., Berliner, S., Yedgar, S., 2001. Parameters of red blood cell aggregation as correlates of the inflammatory state. Am. J. Physiol. Heart Circ. Physiol. 280, H1982–H1988. Baskurt, O.K., Meiselman, H.J., 2003. The hyperviscosity syndromes. Semin. Thromb. Hemostasis 29, 435–450. Baskurt, O.K., Temiz, A., Meiselman, H.J., 1997. Red blood cell aggregation in experimental sepsis. J. Lab. Clin. Med. 130, 183–190. Baskurt,, O.K., Boynard, M., Cokelet, G.C., Connes, P., Cooke, B.M., Forconi, S., Liao, F., Hardemanh, M.R., Jung, F., Meiselman, H.J., Nash, G., Nemeth, N., Neu, B., Sandhagen, B., Shin, S., Thurston, G., Wautier, J.L., 2009. New guidelines for hemorheological laboratory techniques. Clin. Hemorheol. Microcirc. 42, 75–97. Cloutier, G., Zimmer, A., Yu, F.T.H., Chiasson, J.L., 2008. Increased shear rate resistance and fastest kinetics of erythrocyte aggregation in diabetes measured with ultrasound. Diabetes Care 31, 1400–1402. Chien, S., Sung, L.A., Kim, S., Burke, A.M., 1977. Determination of aggregation force in rouleaux by fluid mechanical technique. Microvasc. Res. 13, 327–333. Dobbe, I., 2002. Engineering developments in hemorheology, Ph.D. Dissertation, University Amsterdam, pp. 39–41. Eldwood, P.C., Beswick, A., O’Brien, J.R., Renaud, S., Fifield, R., Limb, E.S., Bainton, D., 1993. Temperature and risk factors for ischaemic heart disease in the Caerphilly prospective study. Heart 70, 520–523. Hardeman, M.R., Dobbe, J.G.G., Ince, C., 2001. The laser-assisted optical rotational cell analyser (LORCA) as red blood cell aggregometer. Clin. Hemorheol. Microcirc. 25, 1–11. Hein, H.J., Bauersachs, R.M., Feinstein, E.I., Meiselman, H.J., 1987. Hemorheological abnormalities in chronic renal failure patients. Clin. Hemorheol. 7, 430–436. McHedlishvili, G., Gobejishvili, L., Mamaladze, A., Momtselidze, N., Varazashvili, M., 1999. Microcirculatory stasis induced by hemorheological disorders: further evidence. Microcirculation 6, 97–106. Neumann, F.J., Schmid-Schonbein, H., Ohlenbusch, H., 1987. Temperature dependence of red cell aggregation. Pflugers Arch. 408, 524–530. Rampling, M.W., Meiselman, H.J., Neu, B., Baskurt, O.K., 2004. Influence of cellspecific factors on red blood cell aggregation. Biorheology 41, 91–112. Razavian, S.M., Del Pino, M., Simon, A., Levenson, J., 1992. Increase in erythrocyte disaggregation shear stress in hypertension. Hypertension 20, 247–252. Shin, S., Hou, J.X., Suh, J.S., 2009. Transient, a transient, microfluidic approach to the investigation of erythrocyte aggregation: the threshold shear-stress for erythrocyte disaggregation. Clin. Hemorheol. Microcirc. 42, 117–125. Shin, S., Ku, Y.H., Park, M.S., Suh, J.S., 2004. Measurement of blood viscosity using a pressure-scanning capillary viscometer. Clin. Hemorheol. Microcirc. 30, 467–470. Uyuklu, M., Cengiz, M., Ulker, P., Hever, T., Tripette, J., Connes, P., Nemeth, N., Meiselman, H.J., Baskurt, O.K., 2009. Effects of storage duration and temperature of human blood on red cell deformability and aggregation. Clin. Hemorheol. Microcirc. 41, 269–278. Weng, X., Cloutier, G., Beaulieu, R., Roederer, G.O., 1996. Influence of acute-phase proteins on erythrocyte aggregation. Am. J. Physiol. Heart Circ. Physiol. 271, H2346–H2352.
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Temperature-dependent threshold shear stress of red blood cell aggregation Hyun-jung Lim, Yong-Jin Lee, Jeong-Hun Nam, Seok Chung, Sehyun Shin School of Mechanical Engineering, Korea University, Anam-dong Seongbuk-gu, Seoul 136-713, South Korea
a r t i c l e in f o
a b s t r a c t
Article history: Accepted 6 September 2009
Red blood cell (RBC) aggregation is becoming an important hemorheological parameter, which exhibits a unique temperature dependence. However, further investigation is still required for understanding the temperature-dependent characteristics of hemorheology that includes RBC aggregation. In the present study, blood samples were examined at 3, 10, 20, 30, and 37 1C. When the temperature decreases, the whole-blood and plasma viscosities increase, whereas the aggregation indices (AI, M, and b) yield contrary results. Since these contradictory results are known to arise from an increase in the plasma viscosity as the temperature decreases, aggregation indices that were corrected for plasma viscosity were examined. The corrected indices showed mixed results with the variation of the temperature. However, the threshold shear rate and the threshold shear stress increased as the temperature decreased, which is a trend that agrees with that of the blood viscosity. As the temperature decreases, RBC aggregates become more resistant to hydrodynamic dispersion and the corresponding threshold shear stress increases as does the blood viscosity. Therefore, the threshold shear stress may help to better clarify the mechanics of RBC aggregation under both physiological and pathological conditions. Crown Copyright & 2009 Published by Elsevier Ltd. All rights reserved.
Keywords: RBC Aggregation Temperature Threshold Shear stress
1. Introduction At stasis or under low shear flows, red blood cells (RBCs) tend to form two-dimensional rouleaux. The rouleaux can grow end-toend or side-to-end; upon completion, they form 3D networks. RBC aggregation is influenced by intrinsic cellular properties (i.e., the deformability and surface charge of RBCs) (Rampling et al., 2004) and by extrinsic properties of the suspending medium (i.e., the pH, temperature, and protein concentration of plasma) (Weng et al., 1996). RBC aggregation plays an important role in the blood flow, particularly in the microvascular system. In fact, increased RBC aggregation has been observed in various pathological diseases, such as diabetes (Cloutier et al., 2008), sepsis (Baskurt et al., 1997), thrombosis (Baskurt and Meiselman, 2003), myocardial infarction (Ami et al., 2001), renal failure (Hein et al., 1987), and microcirculatory diseases (McHedlishvili et al., 1999). Thus, the degree of RBC aggregation is accepted widely as an important determinant of the hemorheological characteristics of blood. Thus, the study of RBC aggregation continues to be of basic scientific and clinical interest. Red-cell aggregation (RCA) is known to be the main determinant of the whole-blood viscosity (WBV) at low shear rates. RCA causes shear-thinning behavior of WBV. In addition, WBV and RCA are affected greatly by the temperature as are most hemorheological properties (Uyuklu et al., 2009). In general, the blood
Corresponding author.
E-mail address: [email protected] (S. Shin).
viscosity increases as the temperature decreases, whereas the aggregation index (AI) decreases as the temperature decreases (Dobbe, 2002). The temperature dependence of these two hemorheological parameters seems to be contradictory since an increase in the low-shear blood viscosity should accompany the increase in the AI as the temperature decreases. The paradoxical phenomenon can be clarified easily by considering the effect of the temperature-dependent plasma, which is an attenuating medium of RBC suspensions. As the plasma viscosity increases when the temperature decreases, the rate of formation of RBC aggregates is retarded. The effect of the temperature on the rate of formation of RBC aggregates can be eliminated by multiplying AI with the plasma viscosity that corresponds to the temperature. As reported in a previous study (Neumann et al., 1987), the rate of formation of RCA increased as the temperature increased but the corrected value of the rate decreased as the temperature increased. Thus, one should use the corrected rate of formation of RCA, while interpreting the temperature-dependent characteristics of whole-blood viscosity. However, the determination of the corrected indices would be difficult since it requires additional measurements of the plasma viscosity. It would be better to find an alternative aggregation index for interpreting directly the temperature-dependent characteristics of blood viscosity. Therefore, it is greatly necessary to develop a new aggregation index for representing RCA. The index should be informative and directly indicative of the temperature-dependent characteristics of blood viscosity without entailing the measurement of other hemorheological properties. Thus, one needs to examine the
0021-9290/$ - see front matter Crown Copyright & 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2009.09.031
ARTICLE IN PRESS Hyun-jung Lim et al. / Journal of Biomechanics 43 (2010) 546–550
temperature-dependent characteristics of various aggregation indices and to compare them with those of blood viscosity. Neumann et al. (1987) measured RBC aggregation using a coneplate aggregometer (Myrenne GmbH, Roetgen, Germany) and reported that the threshold shear rate (TSR) and the threshold shear stress (TSS) tended to increase as the temperature decreased. Thus, these two parameters are possible candidates for representing RCA and interpreting the temperature dependence of WBV without correction of the plasma viscosity. Recently, a critical shear stress (CSS) was proposed as a measure of RBC aggregation. In fact, the CSS was found to be another type of threshold shear stress whereby RBCs start to reaggregate in a decreasing-shear-flow environment. Therefore, the objectives of the present study are to investigate the temperature dependence of RCA with regard to various parameters and to correlate RCA in terms of the temperature-dependent characteristics of blood viscosity. The aggregation indices were measured with two aggregometers, LORCA (Hardeman et al., 2001) and a new microfluidic aggregometer (Shin et al., 2009).
2. Materials and methods Blood was obtained from healthy volunteers (five males, aged 267 2.7), who were not on any medication and who provided informed consent. The blood group of two of the participants was A and that of the remaining three was B. A venous blood sample was drawn from the antecubital vein and collected in vacutainers that contained (K2) EDTA (1.8 mg/ml) as the anticoagulant. The hematocrit was determined by centrifugation at 13,000g for 5 min. For the analysis of RCA and the apparent whole-blood viscosity, the hematocrit was adjusted to 4571%. This was achieved by either adding a calculated amount of autologous plasma to an aliquot of the sample or withdrawing plasma from the supernatant after centrifugation at 3000g for 5 min. Unless otherwise stated, the hematocrit values of the samples were adjusted to 0.45 L/L by either the addition or the removal of autologous plasma. For each experiment, five repeated measurements were conducted for each sample. All measurements complied with the new guideline provided by ISCH (Baskurt et al., 2009). Red-cell aggregation (RCA) was analyzed by two aggregometers (LORCA and a microfluidic aggregometer) at 4, 10, 20, 30, and 37 1C. Fig. 1 describes the schematics of two aggregometers (LORCA and the microfluidic aggregometer). The whole-blood sample was used directly for the measurement of RBC aggregation without the removal of WBCs. The details of LORCA (Laser-assisted Optical Rotational Cell Analyzer, RR Mechatronics, Hoorn, The Netherlands) have been described elsewhere (Hardeman et al., 2001). In brief, the system consists of a Couette geometry that is composed of a glass cup and a precisely fitting bob with a gap of 0.3 mm between the cylinders, as shown in Fig. 1; the RBC suspension is contained in the gap. The beam from a laser that is built into the bob is directed onto the sheared sample. The reflected light is recorded by two photodiodes that are located in the bob and is analyzed by a microcomputer. The sample is first sheared at a user-selectable shear rate for disaggregating the pre-existing RBC aggregates (600 s 1 in the present study), following which the shearing is stopped abruptly. The reflection of light from the suspension is recorded for a user-selectable period (120 s in the present
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study). The resulting syllectogram is analyzed for calculating several indices that reflect both the magnitude and the duration of aggregation. In the syllectogram that was obtained from LORCA measurements, following the sudden cessation of stirring, the conventional mathematical representation of the syllectogram could be adopted, as per the single-exponential representation below: IðtÞ ¼ I0 þ I1 ebt :
ð1Þ
In Eq. (1), b denotes the rate constant of the aggregation process. Since aggregation often can be considered a multi-step process (i.e., doublet-, rouleaux-, and 3D-aggregate formation), the above equation can include two exponential terms rather than just a single exponential term. However, in the present study, we intentionally adopt a single-exponential function in the equation for comparing the findings with those of prior research (Neumann et al., 1987). Through an analysis of the syllectogram, various indices were obtained as follows (Hardeman et al., 2001): (1) the amplitude (AMP), which pertains to the total extent of aggregation and the total change in the intensity of reflected light during a time period of 120 s; (2) the aggregation half time (T1/2), which is the time for AMP to decrease by 50%; (3) the surface area (SA), which is the area above the syllectogram over the first 10 s; (4) the aggregation index (AI), which is the ratio of the area above the syllectogram to the total area (i.e., the sum of the areas above and below the syllectogram curve) over the first 10 s; (5) b, which is the rate constant of RCA formation in stasis for fitting the syllectogram curve using Eq. (1); and (6) the threshold shear rate (gthr), which is the minimal shear rate that is needed for preventing RBC aggregation. The present microfluidic aggregometer (Shin et al., 2009) consists of a disposable element, a laser, two photodiodes, a pressure sensor, and a vacuumgenerating mechanism, as shown in Fig. 1. The disposable element, which is made of transparent plastic, consists of a microchannel and reservoirs at each end; the microchannel is 0.2 mm high, 4.0 mm wide, and 40 mm long. The whole-blood sample (of 0.5 ml) is sheared in the microchannel under continuously decreasing pressure differentials. As the pressure differential exponentially decreases over time, the RBC aggregates tend to break down and disperse at high shear flows and the corresponding backscattered light (BSL) increases. When the pressure differential further decreases, the dispersed RBCs tend to re-aggregate and the BSL starts to decrease. Thus, the maximum value of BSL over the running index for time, t, indicates the termination of RBC disaggregation and the commencement of RBC aggregation. The time and shear stress values that correspond to the maximum value of the BSL intensity are defined as the critical time (tc) and the critical shear stress (tc), respectively. Since both aggregometers cannot operate below room temperature, they were placed in a temperature-controlled chamber and therefore, all measurements were conducted in the chamber. The thermostatic control of the measurement chamber was achieved by a fan-mounted heat exchanger that was connected to a water bath; the chamber temperature was maintained by a thermostat. The temperature was also checked continuously by thermocouples in the instruments. After the specified temperature was reached, 30 min was allowed for full thermal equilibrium between the measurement chamber and the instruments. As stated earlier, in order to correct the effect of the temperature on RCA, the plasma viscosity is to be measured at each temperature. Furthermore, it is also necessary to measure the whole-blood viscosity in a range of shear rates for determining the threshold shear stress. Therefore, the plasma and whole-blood viscosities were measured at 4, 10, 20, 30, and 37 1C using a pressure-scanning capillary viscometer (Shin et al., 2004) and a rheometer (AR2000, TA Instruments, USA). At each temperature, the dimensions of the glass capillary were measured with a digital microscope (Sometech, Seoul, Korea) and the instrument was calibrated using standard oil (Brookfield Corporation) of known viscosity. The whole-blood viscosity was measured over a range of shear rates (1–1000 s 1). All
Fig. 1. Schematics of two aggregometers (LORCA and a microfluidic aggregometer).
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rheological measurements were performed within four hours after the withdrawal of blood. In addition, the threshold shear stress (TSS, tthr) was determined by the product of the threshold shear rate (TSR, gthr) and the corresponding whole-blood viscosity (Zthr). Thus, the TSR was measured with LORCA and the whole-blood viscosity was measured with a pressure-scanning capillary viscometer. Then, the TSS was obtained by multiplying the TSR with the corresponding blood viscosity. The TSS represents the minimal shear stress for keeping RCAs dispersed. This parameter yields a quantitative measure of the aggregative strength of RCAs in a dimensional unit (Pa). A similar parameter, the critical shear stress (CSS, tcr), was introduced recently (Shin et al., 2009). The CSS is defined as the threshold shear stress for keeping RCAs dispersed in a channel flow. The CSS can be determined through a simple microfluidic aggregometer with simultaneous measurements of the pressure and the optical intensity. The detailed principle of CSS measurement can be found elsewhere (Shin et al., 2009).
increased. Due to the strong temperature dependence of the SA index, the corrected SA (SA m) still showed the same trend as the SA index.
3. Results The plasma viscosity exhibited a strong temperature dependence, as seen in Fig. 2. The plasma viscosity decreased monotonically as the temperature increased. As the temperature increased from 4 to 37 1C, the plasma viscosity decreased from 3.2 to 1.7 mPa s (a reduction of 48%). In addition, the whole-blood viscosity decreased as the temperature increased. For instance, at a shear rate of 10 s 1, the whole-blood viscosity decreased from 21.4 to 13.1 mPa s (a reduction of 40%) when the temperature increased from 4 to 37 1C. The aggregation index (AI) exhibited a strong temperature dependence, as shown in Fig. 3. AI increased monotonically as the temperature increased. As the temperature decreased from 37 to 4 1C, AI decreased from 65 to 41 (a reduction of 32%). However, the corrected AI (AI m) was quite different from the original AI. There was no significant change in the value of AI m over the whole range of the temperature. This phenomenon was due primarily to the decrease in the plasma viscosity as the temperature increased. Depending on the temperature dependence of the plasma viscosity, the corrected AI could decrease as the temperature increased (Neumann et al., 1987). The surface area (SA) also exhibited a strong dependence on the temperature, as shown in Fig. 4. There was a doubling of the SA index as the temperature increased from 4 to 30 1C. However, above 30 1C, the SA index decreased slightly as the temperature
Fig. 3. Variation of AI and the corrected AI with the temperature using LORCA.
Fig. 4. Variation of SA and the corrected SA with the temperature using LORCA.
Fig. 2. Variation of the viscosity of plasma and whole blood with the temperature at three different shear rates (measured with a pressure-scanning capillary viscometer) (Shin et al., 2004).
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of the b index, the corrected b index (b m) still showed the same trend as the b index. In contrast to the above indices, the threshold shear rate (TSR, gthr) significantly decreased as the temperature increased from 4 to 20 1C. Beyond 20 1C, the TSR reached an asymptotic value and became temperature independent (Fig. 6). The results on the TSR are quite the opposite to those of either AI or b. In addition, the threshold shear stress (tthr), which is the product of the TSR and the corresponding blood viscosity (gthr Zthr), showed similar results to the TSR (Fig. 7). The threshold shear stress (TSS) decreased monotonically as the temperature increased from 4 to 30 1C. Above 30 1C, the TSS slightly increased as the temperature increased. 4. Discussion Fig. 5. Variation of b and the corrected b with the temperature using LORCA.
Fig. 6. Variation of the threshold shear rate with the temperature using LORCA.
Fig. 7. Comparison of the threshold shear stress (TSS) and the critical shear stress (CSS) at different temperatures. The TSS was determined through the LORCA aggregometer and a blood viscometer, whereas the CSS was measured directly through a microfluidic aggregometer. (The results on the TSS reported in Neumann et al. (1987), are also plotted for the sake of comparison.)
Similarly, the b index (the rate constant of RCA) increased monotonically as the temperature increased from 4 to 30 1C (Fig. 5). The b index approximately tripled when the temperature increased from 4 to 30 1C. In fact, the b index exhibited the strongest temperature dependence among the measured aggregation indices. Similar to the SA index, the corrected b index (b m) still showed the same trend as the b index. Above 30 1C, the slope of the b index curve significantly changed and there was a slight increase in the b index. In addition, the slope of the corrected b index (b m) became opposite to that of the b index above 30 1C. However, due to the strong temperature dependence
Conventional aggregation indices, such as the AI, SA, and b, increase as the temperature increases, which are opposite trends to that of the blood viscosity. As mentioned earlier, since RBC aggregation is the main determinant of low-shear blood viscosity, the temperature dependence of aggregation indices seem to be contradictory to that of the blood viscosity. These results were caused by the temperature-dependent viscosity of the suspending medium, viz., the plasma. Neumann et al. (1987) reported that the opposite trend of temperature-dependent aggregation indices could be corrected by multiplying the aggregation indices with the plasma viscosity. In the present study, however, the corrected indices could not yield straightforward interpretations for the temperature dependence of the blood viscosity, since the temperature dependence of aggregation indices showed mixed patterns of variation even after the plasma viscosity was corrected for. These different results between the two studies were due to the degree of temperature dependence of the plasma viscosity. In other words, the plasma viscosity in the present study was slightly less temperature-dependent than that in the previous study (Neumann et al., 1987). In the present study, the threshold shear rate and shear stress of RBC aggregates decreased as the temperature increased, which showed good agreement with the results on the blood viscosity. Thus, the temperature dependence of whole-blood viscosity may be interpreted through the threshold shear rate and shear stress. However, since the threshold shear rate (TSR) is a strong function of the hematocrit (Shin et al., 2009), it is necessary to adjust the hematocrit for comparing TSR with other results. Furthermore, the determination of the threshold shear stress requires measurements of both the threshold shear rate (gthr) and the threshold blood viscosity (Zthr), even though the threshold shear stress is hematocrit independent (Eldwood et al., 1993; Shin et al., 2009). However, the critical shear stress (CSS), which has the same physical definition as the TSS, can be measured directly through a transient microfluidic aggregometer (Shin et al., 2009). In Fig. 7, the CSS (tcr) is compared with the TSS (tthr). Additionally, the TSS obtained in a prior study (Neumann et al., 1987) is plotted in Fig. 7 for the purpose of comparison. Both threshold shear stresses were at least twice the CSS. This difference is attributed to the lower effectiveness of the shear stress, the direction of which is parallel to the rouleaux interface if there is no alignment with the rouleaux interface (Chien et al., 1977). The difference between the two threshold shear stresses may be caused by the measurements of the whole-blood viscosity. However, the CSS showed the same trend as the TSS when the temperature varied between 4 and 30 1C. In addition, CSS and TSS were found to be hematocrit independent; hence, there is no need to adjust the hematocrit value for comparing CSS with TSS (Razavian et al., 1992; Shin et al., 2009). Thus, the CSS may provide a direct and intuitive understanding of the hemorheological characteristics of whole blood.
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The threshold (or critical) shear stress significantly increased as the temperature decreased. This fact indicates that increasingly high shear stresses are required for dispersing RCAs at low temperatures. Hence, under hypothermic conditions, the mechanical stability of RCAs appears to be increased, which may cause a significant increase in the blood-flow resistance, especially in microcirculation. The significant increase of the threshold shear stress may be correlated with the seasonal rise of circulatory diseases in winter. In general, the seasonal variation of the rate of occurrence of ischemic circulatory diseases is strongly correlated with elevated blood pressure, lipids, and other factors related to thrombosis and hemostasis (Eldwood et al., 1993). Since red-cell aggregation is also closely related to thrombosis and hemostasis, it is worth investigating the possible correlation between seasonal ischemic heart disease and red-cell aggregation.
5. Conclusion In summary, various aggregation indices were examined for their temperature dependencies on the basis of the interpretation of the variation of the blood viscosity with the temperature. Even though the aggregation indices were corrected with reference to the temperature-dependent plasma viscosity, they did not show the same trend with regard to the temperature dependence of the blood viscosity. The threshold shear rate and the threshold shear stress yielded the same trends as that of the whole-blood viscosity with regard to temperature variations. The threshold shear rate requires the adjustment of the hematocrit, whereas the threshold shear stress that is measured in a Couette-flow-type aggregometer requires the additional measurement of the viscosity. However, the critical shear stress, which can be directly measured in a microchannel flow, yielded the same trend as for the wholeblood viscosity with temperature variations. As the temperature decreases, RBC aggregates become more resistant to hydrodynamic dispersion and the corresponding threshold (or critical) shear stress becomes large and the blood becomes viscous. Thus, the threshold (or critical) shear stress may help to better clarify the mechanics of RBC aggregation under both physiological and pathological conditions.
Conflict of interest None.
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