Red blood cells (RBCs) are often subject to vibration during processing, transfusion, and transport. Further research is necessary to understand the effects of vibration on human RBCs and to reduce experimental deviations caused by device vibration.

Flow cytometry was used in this study to observe the cytokine expression of IgG and IgA and deformation of human red blood cells affected by the vibration of a vortex mixer with varying frequency (750 rpm and 1500 rpm), duration (5 min and 10 min), and container volume (96 well plate and 48 well plate).

The size of RBCs in duration of 10 min is obviously smaller than the duration of 5 min. The 10-minute duration led to visibly smaller RBC sizes compared to the 5-minute duration. There was little effect on the size of RBCs in the 10-minute groups from differences in frequency and container volume. However, decreased RBC size can be observed in the 5-minute groups, where frequency is increased or container volume is decreased. Echinocytes were present in photomicrographs of all 10-minute groups, but microstructure of the RBCs was not impacted by vortex mixer vibration. The elevated frequency or reduced container volume results in an increased cytokine expression of IgG within the 5-minute groupings.

It can be inferred that vibration must not be overlooked due to its potential impact on the shape and cytokine expression of RBCs. Hence, the inclusion of vibration must be taken into consideration in experiments and devices pertaining to RBCs.

# Introduction

Hemolysis is known to occur in red blood cells (RBCs) when exposed to intense vibration. A peak acceleration of 300 km/s2 for 30 min can result in 76.7% damage to RBCs [1]. To attain favorable deformation properties, RBCs can recover their double concave shape after being subjected to a shear force field oscillating at 2 Hz [2]. Although certain working conditions (such as karate, conga drum playing and hammering) can cause vibrations that affect RBCs, other types of vibration have been observed to impact them as well. During blood transfusion, blood is exposed to vibrations from devices such as extracorporeal membrane oxygenation and ventricular assist devices [3]. Additionally, mechanical vibrations frequently occur in experimental apparatus and can sometimes be a beneficial tool. This type of vibration is typically of low frequency, small amplitude, and continuous. It is unclear whether these vibrations can have an effect on the blood.

Red blood cells (RBCs) are the predominant component of blood and represent its physiological functions. RBCs are commonly selected for research as they can be collected and stored conveniently [4], with general physiological characteristics. They possess good deformation abilities, flowing effortlessly through capillaries [5]. The shape of RBCs can be influenced by various factors including temperature [6], pH, and certain diseases [7,8], which is a pivotal index for detecting abnormalities.

The vortex mixer is a frequently employed apparatus for vibrating and mixing materials in laboratory experiments. The vibration frequency ranges from 0 to 40 Hz, and the amplitude is typically several millimetres. This paper examines the impact of the vortex mixer's vibration on RBCs. This paper examines the deformation, microstructure, and surface immune factors of RBCs that were exposed to a vortex mixer at 750 rpm and 1500 rpm for 5 and 10 min, respectively. Forward scatter (FSC), side scatter (SSC), fluorescein isothiocyanate (FITC), and P-phycoerythrin (PE) data from the flow cytometer were analyzed.

# Materials and Methods

## Equipment

The vortex mixer (XH-C, Yuexin Corporation, Changzhou, China) is capable of generating vibrations in the frequency range of 0–50 Hz and an amplitude of 3 mm, with adjustable duration. The sample container is securely positioned on top of the mixer. The parameters of the processed red blood cells are measured by a flow cytometer (NovoCyte 2060R, ACEA Biosciences Inc., Hangzhou, China), and analyzed using the NovoExpress software for data acquisition, statistical analysis, and reporting. When passing through the laser beams in flow cytometry, larger cells yield a higher FSC value that is detected by the forward detector. The SSC value, detected by side detectors, increases with the cell's irregularity, number of protrusions on the cell surface, and the presence of organelles or granular substances. A specific fluorescein-conjugated antibody to the surface immune factor excitedly fluoresces in the fluorescence channel of the flow cytometer.

## Preparation of sample of RBCs

An anticoagulated whole blood sample was obtained from a healthy youth donor at the blood bank using a BD Vacutainer (product code: 68784) with 3.2% Heparin Anticoagulant. The sample was then washed twice using phosphate buffered saline (PBS), diluted accordingly, and centrifuged for 10 min at 1500 rpm in a cryogenic high-speed centrifuge (TGL-16D, Zhongjie Corporation, Changzhou, China). The supernatant was then aspirated and PBS was added to the washed red blood cells (RBCs) to create a resuspended RBC solution. The blood requirement was kept to a minimum (<100 ml), with all experiments being performed on the same sample.

## Experimental procedure

Table 1
                         displays the processing conditions for the control group (Group C) and experimental groups. The resuspended RBC solution was added to the well plate with 0.5 ml per well for 10 wells in the 48 well plate and 0.25 ml per well for 10 wells in the 96 well plate. Subsequently, the well plate carrying the samples was securely placed on the vortex mixer and exposed to vibration in accordance with the processing conditions of the experimental groups, as detailed in Table 1. 0.2 ml of the processed sample was diluted with PBS. Following a 20 min incubation period, a photomicrograph of red blood cells (RBCs) was captured. Subsequently, 0.1 ml of the processed sample and 1 ml PBS were transferred to a flow tube using a transferpette. Next, the mixture was loaded into the flow cytometer for shape analysis and structural complexity analysis. 1 × 106–3 × 106 RBCs were aspirated from the sample and mixed slightly with 0.1 ml the PBS, 5 μl the Mouse Anti-human IgA/FITC (BioLegend 333512), and 5 μl Mouse Anti-human IgG/PE (BioLegend 409303). Incubate the mixture in the dark at room temperature for 20 min. After centrifuging at 1500 rpm for five minutes and discarding the supernatant, repeat the process once more. Then, mix with 0.5 milliliters of PBS and load onto a flow cytometer for the expression of surface immune factors. Each group was tested in triplicate and the experiment was conducted three times for each sample.

# Results

The photomicrographs in Fig. 1
                      display the results of RBCs from nine groups at a magnification of 600X. The groups of 5-minutes showed scarce presence of echinocytes, however, the deformation of RBCs could not be determined from the photomicrographs. Echinocytes were present in the photomicrographs of all 10-minute groups. The number of echinocytes counted under the microscope in Group 5 was around 34 echinocytes/100 RBCs, in Group 6 was around 23 echinocytes/100 RBCs, in Group 7 was approximately 30 echinocytes/100 RBCs, and in Group 8 was approximately 20 echinocytes/100 RBCs. Due to the random selection of sight and distribution of cells, the photomicrograph is unable to quantitatively investigate the deformation of RBCs.

The forward scatter (FSC) measurement is directly proportional to the size of the cell. The statistical analysis of FSC-H and FSC-A of the red blood cells (RBCs) loaded on the flow cytometer are illustrated in Fig. 2
                      and Fig. 3
                      respectively. Detailed information is provided in the supplementary material. The trend of FSC-H and FSC-A of all nine groups is identical. When compared to the FSC-H and FSC-A of the control group, all experimental groups exhibited different results. The RBC size over a duration of 10 min was substantially smaller than that over a duration of 5 min. There is negligible influence of frequency and container volume on the size of RBCs in the 10-minute groups. However, in the 5-minute groups, an increase in frequency or a decrease in container volume can result in a reduction in the size of RBCs.

The SSC can indicate the intricacy of cellular microstructure. Fig. 4
                      and Fig. 5
                      display the SSC-H and SSC-A statistics of RBCs that were measured via flow cytometry. There appears to be no significant difference between the nine curves in both figures, suggesting that the vortex mixer has limited impact on the microstructure of RBCs.

On the surface of RBCs, various immune factors can be expressed under certain conditions. The figures depicting cytokine expression of IgG and IgA of RBCs from nine groups are shown in Fig. 6
                      and Fig. 7
                     , respectively. The increase in frequency or decrease in container volume results in higher cytokine expression of IgG in the 5-minute groups. There is no discernible difference in cytokine expression of IgG between the 10-minute groups and the control group. Only group 7 demonstrates significant characteristics in comparison to the control group. The difference in cytokine expression of IgA between the experimental and control groups is not noticeable according to Fig. 7.

# Discussion

The threshold for mechanical injury of RBCs has been studied through various factors, such as peak acceleration, threshold laminar shear stress, and Reynolds shear stress [9–12]. These investigations primarily focused on hemolysis resulting from RBC injury and its measurement, often leading to a disregard for the condition of undamaged RBCs necessary for optimal deformation recovery performance. The red blood cells affected by the vortex mixer, which is a frequently employed instrument for processing the sample, ought to be investigated to mitigate the influence on the outcomes of subsequent experiments.

The rotation of the motor in the vortex mixer generates vibrations that induce a vortex effect for the well plate held on the platform. This effect produces a turbulent swirling flow that enhances mixing efficiency [13]. Several mechanical factors, including shear stress, turbulence, and impact, can affect the shape of RBCs and potentially cause their damage [14]. The free energy of red blood cells (RBCs) increases when subjected to a sufficient force field. The representative shapes of RBCs follow a sequence of stomatocyte-discocyte-echinocyte-rapture [15,16].

Vibration applied to the paper impacts the shape of red blood cells (RBCs), as shown in Fig. 1, Fig. 2, and Fig. 3. The 10-minute vibration groups are harmful to RBCs due to the presence of echinocytes in the sample. Although the photomicrograph of the 10-minute groups shows no cell debris, there may be free hemoglobin released from the RBCs. Compared to the 5-minute groups, the difference in FSC-A and FSC-H in the 10-minute group is minor. Consequently, the deformation of RBCs affected by the vibration applied in the paper is restricted, and the process takes time. The frequency, container volume, and duration all affect the deformation of RBCs. Typically, the shape of RBCs is capable of rapid recovery under normal conditions. It is advisable to allow at least 10 min between the RBC sample leaving the vortex mixer and its placement on the flow cytometer to maintain the stability of the RBC shape. Consequently, the RBC shape can be considered stable.

The vibration and swirling flow are the two primary factors that affect RBCs in the vortex mixer. The acceleration, which the paper reports is about 10 m/s2 for 750 rpm and 40 m/s2 for 1500 rpm, is significantly lower than the minimum acceleration required for RBC damage, which is 200 km/s2
                     [1]. Moreover, the volume of the well plate is too small for turbulent flow to extend. Therefore, the RBCs in the experiment remained unharmed, with no cell debris detected under the microscope or via flow cytometry.

The vortex mixer's vibration is directly applied to the well plate, causing the RBCs near the plate's wall to be impacted by the container wall, while other RBCs are influenced by the fluid force. As RBCs possess remarkable deformation characteristics and lack biochemical reactions, it is unlikely that the vibration of the vortex mixer will affect the microstructure of RBCs.

Immune factors of RBCs play an important role in the human immune system. While research often focuses on the impact of drug, antigen, and disease on surface immune factors [17–19], the effects of mechanical factors are seldom studied. If the expression rate of surface immune factors increases when there is no disease, it can lead to over-immunity. This can cause normal tissue and cells to be attacked, leading to bodily malfunction. Only the IgG in groups 1, 2, and 3 is higher than the control group. It appears that a certain frequency can induce cytokine expression of IgG on the surface of RBCs.

# Conclusions

To analyze the impact of vibration on RBCs, this study examined the photomicrograph, deformation, microstructure, and cytokine expression of IgG and IgA of RBCs influenced by the vortex mixer, a widely used experimental device. Tests were conducted at both 750 rpm and 1500 rpm, across 48 and 96 well plates, for a duration of 5 and 10 min. Results indicate that RBCs exposed for 10 min had greater deformation than those for 5 min, and echinocyte formation was present across all 10-minute groups. The vibration does not appear to have an effect on the microstructure of RBCs. However, groups subjected to vibration for 5 min exhibited elevated expression of cytokine IgG. To ensure proper processing of RBCs, it is recommended to use a vortex mixer with a rotation speed of less than 750 rpm and a loading time of less than 5 min. Vibration cannot be ignored as a factor that affects the shape and surface immune factor of RBCs during processing, transmission or transport, even potentially causing damage. To reduce the effects of vibration on RBC deformation, both frequency and duration should be decreased, and larger containers may also be used.

The RBC is a complex system with many components that can perform various functions. This paper examined only the effects of vibration on the deformation, microstructure, and cytokine expression of IgG and IgA of RBCs. However, important signaling molecules and physiological functions such as ATP [20] and oxygen-carrying capacity [21] were not investigated. Therefore, further research is necessary to comprehensively understand the impact of vibration on RBCs. The properties of RBCs vary between cells and individuals, with a general range of values. Our study collected RBC samples from healthy young donor, which were tested three times per group. We did not investigate the effects of age and gender on our results, and the sample size was relatively small, potentially leading to larger errors in our findings.

# Informed consent and patient details

The authors declare that this report does not contain any per sonal information that could lead to the identification of the donor(s).

# Funding

This research was supported by Education Department of Hunan Province, China (Grant No. 21C0525, 21C0523).

# CRediT authorship contribution statement

Chuang Xiang: Conceptualization, Methodology, Validation, Formal analysis, Writing – original draft, Funding acquisition, Supervision. Liang Wang: Data curation, Resources, Writing – review & editing.