Characterization of shear stress preventing red blood cells aggregation at the individual cell level: The temperature dependence

1 Introduction

The red blood cell (RBC) reversible aggregation is one of the intrinsic properties of the blood that significantly affects the blood microcirculation [2, 8]. It is currently intensively studied for the perspective clinical application the optical-based techniques were widely used in these and relevant studies [3, 22]. Recently a new parameter was introduced to characterize the RBC aggregation [23]. The critical shear stress (CSS) or threshold shear stress (TSS) is defined as the minimum shear stress to prevent the RBC aggregation. Clinical studies have shown that this parameter is more sensitive compared to other characteristics of the RBC aggregation or ESR (erythrocyte sedimentation rate) [11]. Currently there are few methods for measuring CSS based on different shearing geometries: rotating cone-plate [21] or cup-and-bob [9] and microfluidic capillary flow [23]. Depending on the method used, the measured parameters differ by 2–3 times. The observed difference is assumed to be due to different geometries of shearing affect the aggregation process in different ways [5, 17]. The experiments aimed to directly confirm this assumption were performed using the optical tweezers at the individual cells level and found to be consistent [15].

However, a significant difference between the methods is also observed for the temperature dependence of the discussed parameters [17]. All methods show an increased aggregation at lower temperatures (4°C), which decreases by few times as the temperature raises until 20°C. At higher temperatures the behaviour of the parameter is different: for the rotating cone-plate method it keeps decreasing by ∼1.5 fold until 38°C; the cup-and-bob method yields a ∼1.5 fold decrease until 30°C and slight increase by ∼1.2 fold at the 38°C; the microfluidic capillary flow method shows an almost constant value throughout the whole range from 20 to 38°C. These observations could mean that the effect is dependent on the shearing geometry which makes challenging to assess the temperature effect itself. Determining the temperature dependent changes of the RBC aggregation is also crucial for understanding the effect of hypothermia on the blood flow [6, 18]. Hypothermia effect measured in vivo and ex vivo was found to impair the blood microcirculation by changing parameters of the RBC aggregation and deformability, functional capillary density, adherent/rolling leukocytes, while rewarming restored the parameters [7, 10]. In this work, we aim to figure out how the temperature affects the RBC aggregation using the direct measurements at the single cell level.

2 Materials and methods

3 Results

The measurements were carried out using the protocol introduced in our previous work [14]. We aimed to measure the minimum force required to hold two RBCs from spontaneously overlapping each other (i.e., aggregating force – F_A). The definition of this parameter is very similar to the definition of the CSS or TSS, which is the minimum shear stress required for holding the RBCs from aggregating. A set of frames demonstrating the measurement process is shown in Fig. 2(a): (1) Two individual RBCs are trapped by two independent optical traps; (2) The cells are brought to a small area contact and held to prevent the aggregation (overlapping each other). (3) F _T is decreased until one cell escapes from OT, as F _T becomes slightly weaker or equal to F _A . At this moment F _A is considered to be matching F _T . The single cell critical shear stress (SCSS) applied from the OT to the cells is obtained as the ratio of F _A and the cells interacting surface area S. S was calculated assuming the cells are interacting with their entire surfaces within the given linear overlap distance using Cassini‘s model. This assumption is supported by the data obtained with the scanning electron microscopy by Chien et al. [4], who showed that the interacting cells stick very close to each other and tend to overlap with their maximum surface. F_A values were measured on more than 100 pairs of the cells and recalculated to the SCSS.

Measurement results are shown in the Fig. 2(b). The obtained SCSS values are average within each temperature (50 cells per each temperature) and were 256±112 mPa (22°C) and 222±36 mPa (38°C). Within the measurement error intervals the SCSS values have almost no difference at the 22°C and at 38°C which agrees with the measurements made using the microfluidic capillary flow method.

4 Discussion

The direct measurements made at the single cells level using OT excludes the effect of the shearing geometry. The measurement results show that the RBC aggregation values are consistent with the microfluidic capillary flow method and the temperature dependent change of the RBC aggregation is nearly absent for the temperatures of 20°C and 38°C. We conclude that the temperature dependences observed for both cup-and-bob and cone-plate methods are caused by the peculiarities of the shearing geometry, which makes it very challenging to obtain the characteristics of RBC aggregation considering that the temperature dependent effect has also a non-linear character. In contrast, the CSS value measured with microfluidic capillary flow method is reflecting the process of RBC aggregation correctly.

Though in vitro measurements should be carefully transferred to in vivo case, our results indicate that temperature dependent changes in the blood microcirculation may be independent of the RBC aggregation, while more attention should be paid to other factors that could affect it [10, 12]. These findings should be considered in further studies of RBC aggregation.