Determination of whole blood and plasma viscosity in term neonates by flow curve analysis with the LS300 viscometer 1

Abstract

Determination of shear stresses at given shear rates allow approximation of flow curves by mathematical models and to calculate viscosities of non-Newtonian fluids. In term neonates, the mean arterial blood pressure (MAP) is markedly below that of adults, therefore rheological properties of blood play an important role in maintaining perfusion. Whole blood viscosity was measured in umbilical cord blood taken from 62 term neonates using the LS 300 viscometer. Individual parameters that influence the viscosity of whole blood were measured: red blood cell (RBC) aggregation, plasma viscosity, hematocrit, and RBC deformability. The flow curve of whole blood of neonates was approximated by the method of Ostwald with the highest quality whereas in adults the best approximation was found by the method of Casson. With hematocrits of 0.40, the viscosity of whole blood in newborns approximated by Ostwald (9.84 ± 5.12 mPa·s) was significantly lower than that of adults (15.34 ± 3.01 mPa·s). The aggregation index of the blood of newborns was markedly lower (2.98 ± 2.12) than in adults (14.63 ± 3.50) whereas RBC deformability was higher in neonates. The viscosity of plasma determined by Ostwald revealed a lower exponent (n) in neonates (0.94 ± 022) compared to adults (1.01 ± 0.12) and the viscosity determined by Newton was lower in neonates (1.04 ± 0.16 mPa·s) than in adults (1.19 ± 0.07 mPa·s). The flow curve of neonatal blood which is best approximated by the model of Ostwald emphasizes its important viscous properties necessary for conditions with physiologically low blood pressure.

Keywords

Ostwald Casson newborn viscosity flow curve yield shear stress

1 Introduction

Birth is followed by extensive changes in the cardiovascular system, such as a sudden interruption of placental blood flow and redistribution of cardiac output through the pulmonary arteries. Especially due to the low mean blood pressure in the newborn, the rheological properties of blood are very important for the maintenance of blood flow and microcirculation [17, 28]. The rheological properties of whole blood in newborns differ markedly from those in adults [2 , 44].

Investigations in adults have shown that high blood viscosity is a biological parameter which may be causally related to all significant cardiovascular risks, such as hypertension, high LDL cholesterol, low HDL, type-II diabetes, metabolic syndrome, obesity, smoking, age, and the male gender [16, 53]. Newborns are also subject to sudden rheological changes which might exert harmful effects on their microcirculation. Delayed severance of the umbilical cord or feto-fetal transfusion may increase hematocrit levels by as much as 50%, and thus increase the viscosity of whole blood. The resulting hyperviscosity may, in turn, impair blood flow to several organs like the kidney, brain or intestines, and thus endanger the oxygenation of tissue [9 , 47].

Perinatal hypoxia and limited oxygen supply to the placenta for a longer period of time, due to smoking, pre-eclampsia or placental insufficiency, stimulate erythropoiesis and raise hematocrit as well as fibrinogen values. This increases red blood cell (RBC) aggregation in the fetus and the newborn. A rise in whole blood viscosity and impaired microcirculation may also ensue as a result thereof [31]. Due to the child’s immature immune system, newborns and especially preterm infants are very susceptible to infection, which may rapidly develop into sepsis and septic shock. In fact, signs of impaired microcirculation are an early marker of sepsis in neonates. Several pathological rheological changes such as a rise in fibrinogen values, increased RBC aggregation, and reduced RBC deformability may be associated with this phenomenon [28]. Newborns with sepsis, anemia or thrombocytopenia frequently require transfusions with blood components derived from adult donors. A higher rate of cerebral hemorrhage, more frequent occurrence of necrotizing enterocolitis (NEC) and thrombosis have been reported after blood transfusions or the administration of immunoglobulins [13, 57]. A causal association between these conditions and rheological changes cannot be ruled out [35, 46]. Thus, it would be very interesting to expand our knowledge of the flow properties of blood - a non-Newtonian fluid - in newborns, under the impact of various shear forces [3].

The viscosity of whole blood provides very valuable information in this regard because it describes the interaction of blood components and flow resistance, and can be calculated from the flow curve of whole blood. Whole blood viscosity is especially dependent on hematocrit, plasma viscosity, the shape and deformability of red blood cells, leukocyte count, RBC aggregation, the interaction of blood particles with plasma, and the resulting shear forces acting on blood cells [8 , 46].

The viscosity of whole blood was determined with the LS300 viscometer in 62 term neonates. The software of LS300 permits the investigator to calculate the shear forces (shear stress τ) of a fluid at arbitrarily selected shear rates ( $\dot{γ}$ ). The flow curve thus obtained permits calculation of viscosity even in a non-Newtonian fluid like whole blood [48]. Thus, shear forces can be determined at any desired shear rate, and a viscosity profile can be created [23].

Individual factors that influence viscosity were also determined: aggregation index (aggregometer), plasma viscosity (LS 300, dynamic viscosity; capillary tube viscometer, kinematic viscosity), hematocrit (microcentrifuge) and RBC deformability (Rheodyn).

2 Material and methods

Samples of umbilical cord blood were taken by slow aspiration immediately post-partum from 62 term neonates (37 + 0 to 41 + 6 weeks of gestation (w. gest.), mean: 38+2 w. gest.; 3261 g birth weight; 32 male; 30 female). Standard test tubes coated with ethyleneediaminetetraacetic acid (EDTA) (1.5 mg/ml) were filled with the blood [48]. In all samples, whole blood viscosity at the original hematocrit and at a set hematocrit of 0.40, as well as plasma viscosity was determined. In 17 blood samples, whole blood viscosity was determined additionally after setting hematocrit to the original hematocrit by centrifugation. To set hematocrit, the blood samples were centrifuged at 2000 g for 10 minutes (Haereus Labofuge 400, Haereus Holding GmbH, Hanau, Germany). Plasma and the buffy coat were then pipetted carefully as usual. The desired hematocrit of 0.40 or the original hematocrit of the sample was set by adding autologous plasma. Hematocrit was assessed in all samples using the microhematocrit method, by performing centrifugation of a small sample (60 μl) at 15000 g for 10 minutes (Biofuge A, Heraeus Sepatech GmbH, Germany) [2]. All preparations for the measurements were performed at the ambient temperature of 21°C. Viscosity, aggregation and RBC deformability were measured at 37°C. For this purpose the aggregometer and Rheodyn SSD were placed in an incubator set to 37°C (Inkubator 7510, Drägerwerk, Lübeck, Germany). All measurements were performed within four hours after blood sampling [4, 38].

For comparison, blood samples of 66 healthy non-smoking adults (33 male, 33 female; age 33 ± 8 years) had been investigated. The outcome of these measurements had been described in detail previously [49].

The present study was performed in accordance with the Declaration of Helsinki and had been approved by the ethics committee of the University of Heidelberg.

2.1 Measuring principle of LS300

The measurement procedure has been described earlier [48, 49].

2.2 Measurement of whole blood and plasma viscosity (LS300)

The samples (0.9 ml) were investigated by geometric distribution of ten measuring points in a logarithmic shear velocity range from 0.5 s^–1 to 50 s^–1 within 120 s (0.5, 0.76, 1.1551, 1.7556, 2.6683, 4.0557, 6.1642, 9.3691, 14.2402, 21.6438, 32.8967, 50 1/s) (Fig. 1). The flow curves can be approximated according to rheological models (Newton, Ostwald, Bingham or Casson) [49]: $Newton : τ = η_{N} \cdot \dot{γ}$ (1) $Ostwald : τ = η_{Ost} \cdot {\dot{γ}}^{n}$ (2) $Bingham : τ = τ_{0} + η_{BH} \cdot \dot{γ}$ (3)

$\begin{matrix} Casson : \sqrt{τ} & = & \sqrt{τ_{0}} + \sqrt{η_{Ca} \cdot \dot{γ}} \\ τ & = & τ_{0} + 2 \sqrt{τ_{0} \cdot η_{Ca} \cdot \dot{γ}} + η_{Ca} \cdot \dot{γ} \end{matrix}$ (4) where: η_N, viscosity by Newton; η_Ost, viscosity by Ostwald; n, exponent by Ostwald; η_BH, viscosity by Bingham; τ₀, yield shear stress by Bingham; η_Ca, viscosity by Casson; τ₀, yield shear stress by Casson; $\dot{γ}$ , shear rate.

The agreement between the determined curve and the measuring points is calculated by the LS300 software. Ostwald’s model includes mention of the exponent in addition to viscosity while Bingham’s and Casson’s model includes mention of the yield point (point of intersection of the flow curve and the y-axis).

The impact of the RBC sedimentation rate on whole blood viscosity during the measuring cycle, or the formation of a surface film at the air-fluid interface of plasma could be ruled out [49].

2.3 Measurement of plasma viscosity by Capillary tube viscometer (KPSV4)

Plasma viscosity was additionally determined with the capillary tube viscometer (KSPV 4, Rheomed GmbH, Aachen, Germany) [24] and compared to LS300.

2.4 Aggregation index

The aggregation index was determined with the Myrenne Aggregometer MA1 (Myrenne GmbH, Roetgen, Germany). Twenty microliters of a sample of whole blood was pipetted into the shear opening and sheared at 600 s^–1 for 10 seconds, assuming that all pre-existing RBC aggregates are disaggregated by the process. Rotation is stopped for 10 seconds and aggregation is measured by the passage of light (M0). The aggregation index thus obtained is proportional to the area under the light transmission curve, which reflects the degree of aggregation attained at the end of the 10-second period [7].

2.5 RBC deformability

In accordance with previous studies [7], RBC deformability was investigated by RBC elongation, using the shear stress diffractometer Rheodyn SSD (Myrenne Gmbh, Roetgen, Germany). Various shear rates (0.3, 0.6, 1.2, 3.0, 6.0, 12.0, 30.0, 60.0 Pa) are employed and the elongation index is mentioned in percentage. The whole blood sample is diluted with dextran. The viscosity of dextran (Dextran Lsg. FP 60, Article no. 8072921, 100 ml, Burg-Apotheke (pharmacy), Königstein, Germany) as a shear medium was tested with LS300; it was 0.24 mPa·s at room temperature and 0.11 mPa·s at the measuring temperature of 37°C.

2.6 Statistical analysis

Statistical analysis was performed to determine differences between the groups of term neonates and adults, the influence of hematocrit and of centrifugation on rheological parameters as whole blood viscosity and aggregation. Means and standard deviations were determined for all data. Mann-Whitney Rank Sum Test was used to compare two groups. To investigate differences between more than two groups, the null hypothesis was discarded and variance analysis was performed by Kruskal-Wallis One-Way Analysis of Variance on Ranks. Multiple mean value comparisons were performed by an all pairwise multiple comparison procedure (Tukey Test) [21].

3 Results

The determination of the shear forces of whole blood or plasma at given shear rates allows to create a flow curve. Thus, at every shear rate the corresponding viscosity can be calculated (Fig. 1). The highest quality of approximation of these flow curves is achieved by various mathematical models. In term neonates the best quality of approximation for the flow curve and viscosity was achieved by the model of Ostwald (99.65 ± 0.22; p < 0.001) (Table 1, Fig. 2). For a better visible discrimination mainly of the lower shear rate range we used the square root function of the approximation of the flow curve by Ostwald and Casson (Fig. 2). In contrast, the best quality of approximation for the whole blood of adults was achieved by the model of Casson (99.71 ± 0.13) (Table 1) [49]. The viscosity of whole blood in mature newborns showed an essential lower yield point τ₀ in the flow curve as compared to that of adults (approximated by the model of Casson). At similar hematocrits (calculated), whole blood of newborns revealed a lower viscosity than whole blood of adults either by approximation by Ostwald or Casson (Table 1).

In concurrence with the data obtained for adults, the best quality of plasma viscosity measurement with LS300 was achieved by the model of Ostwald (p < 0.001) [49]. The flow curves of plasma of healthy newborns were only slightly different by the methods of Ostwald or Newton (Fig. 3). However, best quality was achieved by the approximation of Ostwald. The plasma viscosity of newborns measured with LS300 approximated by Ostwald (exponent n) and Newton, as well as the measurement of dynamic viscosity determined with the capillary tube viscometer, was significantly lower (p < 0.001) than that of adults (Tables 1, 2, Fig. 3).

By correlating hematocrit with viscosity by Ostwald (y = – 28.87 + (96.77·x); r = 0.71; p < 0.001) and hematocrit with the exponent by Ostwald (y = 1.10 – (0.86·x); r = 0.59; p < 0.001) as well as hematocrit with viscosity by Casson (y = 1.21 + (5.52·x)) and yield point τ₀ by Casson (y = – 11.87 + (37.87·x)), the impact of hematocrit could be calculated by linear regression (Fig. 4). Thus, viscosity according to Ostwald and Casson could be corrected to an arbitrarily set hematocrit within a range of 0.32– 0.65 (Table 3), without the need to alter the blood sample by centrifugation for setting hematocrit and thus falsifying viscosity. In order to obtain a standardized comparison of adult data with those of newborns, the viscosity was calculated with an arbitrary hematocrit set to 0.40. The data for adults were obtained earlier [49], linear regression for Casson η_Ca y = – 0.50 + (10.98·x), τ₀ y = – 9.98 + (36.09·x) and for Ostwald η_Ost y = – 17.70 + (82.59·x); exponent (n) y = 0.81 – (0.35·x).

To compare the blood of newborns with that of adults, the viscosity of whole blood was calculated for a corrected hematocrit of 0.40 and approximated in both groups according to the two models of Ostwald and Casson. The viscosity of newborns according to Casson was 3.42 ± 0.42 mPa·s; yield point 2.59 ± 2.42 mPa, and quality 99.17 ± 0.19. The viscosity of adults according to Ostwald was 15.34 ± 3.01 mPa·s, exponent 0.67 ± 0.03 and quality 99.21 ± 0.13. For the selected hematocrit of 0.40, the viscosity of whole blood in newborns (9.84 ± 5.12 mPa·s, exponent 0.76 ± 0.06) was significantly lower (p < 0.001) than that of adults by Ostwald as well as Casson (viscosity 3.64 ± 0.32 mPa·s, yield point 4.42 ± 1.63 mPa) (Table 1). When the hematocrit was arbitrarily set to 0.48 the whole blood viscosity was also lower for neonates than for adults (p < 0.001).

Centrifugation of the blood samples of newborns for adjusting hematocrit to 0.40 and to the original hematocrit of the blood samples influenced whole blood viscosity as well as RBC aggregation. While the viscosity of whole blood and the yield point according to Ostwald were significantly lower after centrifugation (p < 0.05), RBC aggregation was markedly increased (p < 0.001) (Table 3). RBC deformability, in contrast, remained unchanged. However, whole blood viscosity of term neonates either adjusted to a hematocrit of 0.40/0.48 or calculated by linear regression to a hematocrit of 0.40/0.48 did not reveal significant differences.

RBC aggregation of newborns (native hct: 2.98 ± 2.12; hematocrit 0.40: 5.30 ± 3.01) compared to that of adults (native hct: 14.63 ± 3.50; hematocrit 0.40: 16.51 ± 4.40) revealed significantly lower values in newborns in the original blood samples and also when set to hematocrit 0.40 (Table 2).

Deformability of RBC was significantly higher from RBC from newborns than from adults (Table 2).

4 Discussion

The viscosity of whole blood and plasma [18 , 55] plays a significant role in several cardiovascular diseases [22, 56] and hyperviscosity syndromes (M. Waldenström) in adults [20, 54]. Even in newborns, many diseases like polycythemia of the newborn [9 , 47], feto-fetal transfusion, sepsis or asphyxia [28, 46] are associated with serious changes in flow conditions in the circulatory system and in microcirculation. Especially due to low shear rates in the hypotensive circulatory system of fetuses and neonates, the rheological properties of blood and pathological changes in these are of special significance.

LS300 permits the investigator to determine the viscosity of Newtonian and non-Newtonian fluids by creating a flow curve. According to the measured shear stresses of the investigated fluid, the resulting flow curve can be approximated by the models of Newton, Ostwald, Bingham or Casson, which are incorporated in the software of the device [49]. The quality of the chosen approximation is given as well. The flow curves could be produced by even more precise mathematical models such as those of Quemada or Ree-Eyring [32, 39], but with the disadvantage of having to define a large number of arbitrary parameters.

With regard to the whole blood viscosity of adults, the best agreement was obtained by Casson’s method compared to the other three models [49]. This may be due to the distinct yield point. With regard to the whole blood viscosity of mature newborns, on the other hand, the best quality of approximation was achieved with the method of Ostwald. The relevant low yield point in newborns may be due to the lower aggregation behavior of children’s red blood cells [14 , 44]. Lower fibrinogen levels in the blood of newborns, fetal fibrinogen variants with less ability to interact with the RBC surface, and lower immunoglobulin levels in newborns, are major causes of lower aggregation [37 , 44].

Since aggregation is much lower in the blood of newborns than in that of adults, the yield point may be also much lower. Therefore, the method of Ostwald provides better approximation of whole blood viscosity in newborns. Erythrocytes of adults suspended in PBS also revealed an almost imperceptible yield point, non-measurable aggregation, and therefore much better approximation by Ostwald than by Casson [49]. Especially at low shear rates and therefore in venous blood flow, aggregation and yield point exert a very significant impact on the rheological properties of whole blood [41]. Baskurt et al. mention that the magnitude of RBC aggregates in whole blood is inversely proportional to the magnitude of active shear forces [5, 11]. Pre-existing RBC aggregates (such as rouleaux) are cleaved under increasing shear forces, while new aggregates are formed under sluggish flow conditions or stasis [34, 45]. Low aggregation and the absence of an internal structure, possibly represented by a relevantly low yield point are decisive factors in the circulation of newborns, which is marked by low blood pressure [17] and low vascular resistance [27]. Conversely, diseases like septicemia, tissue injury, necrosis or ischemia [5 , 58], which increase aggregation, also raise the yield point in newborns and thus exert a negative impact on flow behavior. This aspect needs furtherinvestigation.

Especially at shear forces above 10/s, the orientation and deformability of RBC‘s influences the resulting shear stress [5, 7]. In agreement with previous studies [27, 50], the RBC deformability of newborns was significant greater than that of adults.

Any disruption of the streamlined orientation of fluids through cellular elements like red blood cells is, under laminar flow conditions, one of the main reasons for higher viscosity values, such as those observed in whole blood compared to plasma [5, 10]. Depending on the concentration of cellular elements - in this case hematocrit - there is an exponential rise in whole blood viscosity [1, 8]. As mentioned in several publications [28], the hematocrit of newborns (0.48 ± 0.05) is markedly higher than that of adults (0.41 ± 0.034). A hematocrit range of 0.45– 0.55 l/l is reported to be normal in newborns [40]. Hematocrit may well increase to 0.65 in the first few hours after birth [52]. The exponential effect of hematocrit on whole blood viscosity becomes very evident above 0.65 l/l and therefore plays a decisive role in the microcirculatory effects of polycythemia of the newborn [12, 36].

To compare viscosity values in newborns with those of adults, it is common practice to set hematocrit to 0.40 l/l. However, since the centrifugation of blood has a marked effect on viscosity, the measurement of viscosity should be standardized and centrifugation avoided [49]. Even in other non-Newtonian fluids, stirring or centrifugation alters viscosity: cream turns stiff while honey becomes more fluid [19]. Using the algorithm described in the present study, the impact of hematocrit on whole blood viscosity can be measured within the range of 0.32– 0.65. Viscosity increases very rapidly when hematocrit exceeds 0.65 [28]; the present algorithm cannot be used in these conditions.

The flow curves of plasma by Ostwald and Newton were very similar for newborns. The exponent (n) by Ostwald was below 1 (0.94). Nevertheless, a significantly better quality was noted for Ostwald’s approximation of the flow curve (98.89%) compared to the Newtonian approximation. The exponent, an important additional parameter, reflects the course of the flow curves: increasing (>1) – shear thickening; decreasing (<1) – shear thinning. In adults the flow curves of plasma approximated by Ostwald and by Newton were nearly the same; better quality was noted for Ostwald’s approximation [49], too. Overall, Newtonian plasma viscosity and the dynamic viscosity of newborns, as described in previous studies [27, 44], were markedly below those of adults.

With hematocrit set to 0.40 and 0.48, the whole blood viscosity of newborns approximated by Ostwald (hct 0.40: 64%; hct 0.48: 80%) and Casson (hct 0.40: 93%; hct 0.48: 85%) was significantly lower than that of adults (100%). Especially in newborns, the impact of the viscosity of blood is very significant because of the physiological low blood pressure values of newborns. Lower viscosity values compared to adults are attributed to the sum of interactions of several factors. Lower plasma viscosity, lower fibrinogen content, and better RBC deformability are as important as the reduced aggregation ability of RBC’s and the Fahraeus-Lindqvist effect in narrow capillaries [59]. Despite previous investigations in blood samples of adults [23], the impact of individual parameters in newborns is still poorly understood and should be addressed in future studies.

5 Conclusions

Especially in newborns, whose blood pressure is much lower than that of adults (the mean arterial pressure of newborns is 40 mmHg as opposed to 90 mmHg in adults), the rheological properties of blood play a major role in maintaining blood perfusion. LS300 enables the investigator to determine the viscosity of fluid by the analysis of flow curves. Thus, shear forces can be calculated for predefined shear rates, and these can be extrapolated to the entire relevant physiological shear range. Whole blood viscosity should be determined according to standardized methods (EDTA as anticoagulant; avoiding centrifugation). Using the algorithm described here, the impact of hematocrit on the whole blood viscosity of newborns is taken into account within the range of 0.32– 0.65. Ostwald’s method yielded the best quality of blood viscosity calculation in newborns, while Casson’s method was best for adults. The flow curve of neonatal blood which is best approximated by the model of Ostwald emphasizes its important viscous properties necessary for conditions with physiologically low blood pressure. For the determination of the viscosity of plasma, the best agreement was obtained by Ostwald’s method in newborns as well as in adults. However, the flow curves approximated by Ostwald and by Newton were nearly similar.

The rheological parameters of newborns differ very markedly from those of adults. Whole blood viscosity, plasma viscosity and RBC aggregation are lower, and RBC deformability is higher than that of adults. These facts should be given special attention when administering blood products to sick newborns because the products may exert harmful effects on their blood viscosity as well as microcirculation.

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