Factors influencing platelet isolation: a prospective multicenter study from Western China

Abstract Tumor-educated platelets (TEPs) have been widely reported to have promising application potential; nonetheless, platelet isolation from peripheral blood is an important but neglected step in TEPs research for platelet-based liquid biopsy. In this article, we discussed some common influence factors for platelet isolation. To investigate the factors involved in platelet isolation, a prospective multicenter study was conducted on healthy Han Chinese adults (18 to 79 years of age). A total of 208 individuals were included in the final statistical analysis out of the 226 healthy volunteers who were prospectively enrolled from four hospitals. The primary study metric was the platelet recovery rate (PRR). The similar pattern was observed in the four hospitals, The PRR at room temperature (23°C±2°C) was slightly higher than the PRR at cold temperature (4°C±2°C). Moreover, the PRR gradually decreased as the storage time increased. The PRR for samples within 2 hours of storage is significantly higher than for samples beyond 2 hours (p < .05). Additionally, PRR was also affected by the equipment used in different centers. This study confirmed several factors that influence platelet isolation. In our study, we indicated that platelet isolation should be performed within two hours of peripheral blood draw and held at room temperature until isolation, and that centrifuge models should be fixed during the extraction process, which will further improve the research progress of platelet-based liquid biopsy in cancer. Plain Language Summary What is the context? Globally, cancer is one of the leading cause of premature death. Early screening is important for cancer diagnosis and treatment and can even significantly lower cancer mortality Globally, cancer is one of the leading cause of premature death. Early screening is important for cancer diagnosis and treatment and can even significantly lower cancer mortality For the liquid biopsy, isolation is an important step. Early studies have explored the influencing factors of exosome, circulating tumor cells (CTCs), and other components extraction in liquid biopsy. Despite platelet also being an excellent source of liquid biopsy, few studies have explored the factors that influence platelet isolation.Considering the importance of platelet isolation in tumor-based platelet liquid biopsy, our aim is to optimize platelet isolation conditions as much as possible to obtain a high platelet recovery rate. What is new? In this study, we conducted a prospective multicenter study ofhealthy adults from four centers, combining whole blood with platelet-richplasma to investigate factors influencing platelet recovery rate (PRR) during platelet isolation. In our study, we indicated that platelet isolation should be performed within two hours at room temperature, and that centrifuge models should be fixed during the extraction process, which will further improve the research progress of platelet-based liquid biopsy in cancer. What is the impact? In future platelet-related studies, we should fix the sample storage temperature, storage time and centrifuge model in the process of platelet extraction, so as to reduce the variables affecting platelet extraction as much as possible and ensure the stable recovery rate of platelet extraction.

• For the liquid biopsy, isolation is an important step.Early studies have explored the influencing factors of exosome, circulating tumor cells (CTCs), and other components extraction in liquid biopsy.• Despite platelet also being an excellent source of liquid biopsy, few studies have explored the factors that influence platelet isolation.Considering the importance of platelet isolation in tumor-based platelet liquid biopsy, our aim is to optimize platelet isolation conditions as much as possible to obtain a high platelet recovery rate.
What is new?
• In this study, we conducted a prospective multicenter study ofhealthy adults from four centers, combining whole blood with platelet-richplasma to investigate factors influencing platelet recovery rate (PRR) during platelet isolation.• In our study, we indicated that platelet isolation should be performed within two hours at room temperature, and that centrifuge models should be fixed during the extraction process, which will further improve the research progress of platelet-based liquid biopsy in cancer.

Introduction
Currently, cancer is one of the most common cause of premature death in most countries [1].Early screening is important for cancer diagnosis and treatment and can even significantly lower cancer mortality [2].Liquid biopsy refers to the detection technology that reflects the molecular characteristics and dynamic changes of tumors by collecting human body fluids (blood, urine, saliva, etc.) as test samples [3].Liquid biopsy has gained increasing interest in recent years due to its noninvasive nature, frequent multiple detection capabilities, and quick reaction capabilities [3].Currently, the primary components of liquid biopsy used in clinical trials, include circulating tumor cells (CTCs) [4], circulating tumor DNA (ctDNA) [5], exosomes [6], TEPs [7], circulating tumor RNA (ctRNA), etc [8,9].For the liquid biopsy, isolation is an important step [10].For the exosome extraction from whole blood, Yang et al found that the efficiency of separation of exosomes by differential centrifugation depends on factors such as acceleration, rotor type and sample viscosity [11].Additionally, the organic solvent precipitation method is affected by many factors, including temperature, time, pH value and ionic strength, etc [11].Yirui C et al found that exosomes stored at 4°C had the highest concentration, and 3 h was suitable for cell uptake of exosomes.Longer incubation time will increase the difficulty of exosome aggregation and counting [12].For the CTC extraction process, the CTC sorting and enrichment will directly affect the subsequent detection (counting, immunofluorescence, gene amplification, gene sequencing, etc.) [13].Studies have found that it is difficult to maintain the integrity and high cell activity of CTCs during enrichment [14].Nasiri have proposed that many factors can limits the application of CTCs in clinical practice [15].
Despite platelets also being an excellent source of liquid biopsy, few studies have explored the factors that influence platelet isolation.Hechler B et al found that the citrated plateletrich plasma is stable for no more than 2 hours [16].Previous research has pointed out that the platelet isolation method is critical for functional platelet and Extracellular Vesicles studies, and for the use of platelets for diagnostic purposes in particular [17].Isolated platelets were incubated with different agonists for 30 min at 37°C with continuous agitation.After stimulation, activation was stopped by addition of 10 mM EDTA, and platelets were bolled by two-step centrifugation (750 g, 15 min).The supernatant was recovered and hyper centrifuged at 100 000 g for 2 h at 10°C to collect PEV [17].Moreover, in our previous study, we found that PRR was generally low by general platelet isolation method [18].
Considering the importance of platelet isolation in tumor-based platelet liquid biopsy, a systematic and comprehensive investigation of the factors that influence the platelet extraction process under the conditions of large sample sizes and multi-center studies is necessary.Our aim is to optimize platelet isolation conditions as much as possible to obtain a high PRR for liquid biopsy diagnostics.

Experimental arrangement
The Department of clinical laboratory of Sichuan Cancer Hospital & Institute arranged a multi-center collaborative study to examine the influence of several factors such as blood sample storage temperature, blood sample storage time, and research center on platelet recovery rate.Four hospitals were chosen to take part in this study: the People's Hospital of Pengzhou City, the People's Hospital of Fushun County, the People's Hospital of Guangyuan City, and the first People's Hospital of Shuangliu.Each of these hospitals was in charge of choosing healthy local participants, conducting tests, and gathering samples, while the department of clinical laboratory at the Sichuan Cancer Hospital & Institute was in charge of project quality assurance.

Inclusion and exclusion of subjects
The prospective study was carried out between January 2021 and June 2021 by recruiting 226 healthy ethnic Han candidates between 18 and 79 years of age (average age 41.2 years old) from four cities in southwest China.
Inclusion criteria:1.Age is between 18 and 79 years old.2. Fasting time>8 hours.3. The availability of the necessary data.4. Body mass index (BMI) between 18.5 kg/m2 and 24.9 kg/m 2 .5. The Han ethnic group.6. Nutritional condition is good in the absence of acute or chronic illnesses.7. The coagulation is normal.8. Do not take antiplatelet drugs.
Exclusion criteria were as follows: 1. Existence of any existing health issues (comorbidities) 2. Being pregnant or having previously used or still using alcohol, cigarettes, or oral contraceptives; 3. Aberrant clinical laboratory values, such as platelet count<100 × 10 9 /L or > 300 × 10 9 /L; 4. Any surgery within 6 months of enrollment; 5. Blood donation or transfusion within 4 months of enrollment; 8. Body mass index (BMI) <18.5 kg/m 2 or (BMI) >24.9 kg/m 2 .After applying the inclusion and exclusion criteria to a total of 226 patients from four centers, 208 healthy subjects were included in the final analysis (Table I).

Ethics declaration
Clinical study protocol for the investigation was authorized by the medical ethical committee of Sichuan Cancer Hospital (SCCHEC-02-2021-037).All individuals provided written informed consent in accordance with the Declaration of Helsinki prior to participating in the study.Monitoring was done by the Clinical Research Office on a regular basis.During the process of collecting, storing and processing of data, all information was anonymized.

Quality control
We developed a strict quality control process for factors that affect platelet recovery in multicenter clinical trials, including the specimen collection process, the data collection process, the experimental process, and the data analysis process, in order to minimize results bias.
During the specimen collection process, specimens were collected in strict accordance with ethical requirements, subjects' blood samples were collected in severe accordance with inclusion and exclusion criteria, and the collection criteria were strictly followed throughout the entire process.In order to collect data effectively, it is necessary to establish a process for training information collectors and to ensure that they are trained sternly.During the experimental process, each research center monitored the temperature in the operating environment to ensure that the sample storage temperature was within the specified range.All procedures must be gentle to prevent platelet activation and aggregation.For each machine test in the same research center, the same type of hematometer and centrifuge should be used to avoid experimental error.The storage conditions for sample grouping and the time point of on-machine detection are strictly followed, and the whole blood centrifugation for PRP is severely followed according to protocol's instructions.Data analysis should be conducted sternly in accordance with the statistical analysis process, and data should not be modified to ensure the validity of both the data and the results of the analysis.

Measurement equipment
Following is a list of routine blood testing instruments used in each research center: The BC-6800 hematology analyzer from Mindray Medical Electronics Co., Shenzhen, China, was used in the Pengzhou center to measure platelet count (PLT) and other platelet-related parameters, the Mindray BC-6800PLUS hematology analyzer and The Mindray BC-6900 hematology analyzer are used by Guangyuan and Shuangliu centers respectively, while the XN-1000 hematology analyzers from Sysmex Corp., Kobe, Japan, were used in Fushun.XN-1000 and Mindray series blood cell analyzers routinely detect platelets using the electrical impedance principle, also known as Coulter's principle.In this method, electrical resistance that is produced by passing a particle through an aperture is plotted as a volume histogram.A blood cell or particle fitting the platelet size profile of the platelet distribution curve is counted as a platelet [19].
The hematology analyzer instruments of the four research centers have passed the provincial ventricular quality assessment in 2020.Each research center utilizes centrifuges as follows:

Specimens processing and hematological analysis
Following are the steps taken to process the blood samples: Blood samples were divided into 8 1.5 mL Eppendorf (EP) tubes, each of which contained 1 mL, and then the 8 EP tubes were categorized into A and B groups, numbered a1, a2, a3, a4 and b1, b2, b3, and b4 (corresponding to 2 h, 4 h, 6 h and 8 h samples, respectively).
The samples of group A were stored in cold temperatures (4°C ±2°C) refrigerator, and the samples of group B were directly stored at room temperature.
The whole blood samples were tested by a blood cell analyzer in duplicate; average values of platelet parameters were recorded for the impedance channel and optical channel parameters.After detection, the remaining whole blood samples were transferred to a new EP tube and centrifuged for 20 min at 120*g at room temperature.Platelet-rich plasma (PRP) was obtained from the upper portion of the tube following centrifugation.This plasma contains a considerable number of platelets.PRP separation method used by our research group was derived from Myron G. Best et al [22,23].The supernatant was then absorbed into a new EP tube and tested again.The mean values of plateletrelated parameters in PRP were recorded.
Other samples stored according to the conditions were removed from cold storage and room temperature storage at the set time points of 2 h, 4 h, 6 h, and 8 h.The same procedures were used to process the samples as described above, and the platelet parameters in whole blood and PRP were recorded at the specified times.In batches at different storage temperatures and times, platelets were isolated from blood samples without break during the centrifugation process.Following the collection of qualified samples, the data was sorted out and rechecked.After checking for errors, the data was registered in the data table and sent to the director of Sichuan Cancer Hospital.
The research units sorted out the paper records into electronic files, extracted one-tenth of the data for verification, and sent it to the leading unit after verification.For a summary of the experimental design and protocol, see (Figure 1).

Platelet activation
Citrated-anticoagulated peripheral blood was collected from 6 healthy adults.By centrifuging the citrated peripheral blood samples at 120 × g for 20 minutes, platelet-rich plasma (PRP) samples were prepared.In the antigen assay tube, two 5 μL fluorescentlylabeled monoclonal antibodies (e.g.CD62P PE and CD41a APC) were added, and in the negative control tube, two 5 μL fluorescently-labeled MIgGs (e.g.MIgG PE and MIgG APC).Add 5 µl citrated-anticoagulated PRP to the antigenic tubes and negative control tube respectively.Incubate at room temperature for 20 minutes without exposure to light.After adding 1.5 ml of phosphate buffered saline (PBS)buffer, mixing blood samples, and centrifuging at 300 × g for five minutes, the supernatant was discarded.It was fixed at 4-8°C for 15 minutes, then detected by flow cytometry (FCM) after 1 ml of 1% paraformaldehyde was precooled and mixed with eddy current.The platelets were resuspended and washed with PBS to minimize nonspecific binding to fluorescent antibodies, as was reported previously in FCM [24].

Measurement of platelet RNA yield
The PRP was isolated from the whole blood of six healthy adults that had been anticoagulated with EDTA, and then centrifuged at 360 g for 20 minutes.The precipitated platelets were what we want.Total RNA was then extracted using BioTeke's Total RNA isolation kit.Then we ran the NanoDrop 2000 machine according to the Standard Operating Procedures (SOP) [25] to test RNA samples (concentration and purity).

Statistical analysis
Statistical analysis was conducted with SPSS 26.0 software (SPSS Inc., Chicago, IL) and R (http://www.R-project.org)[26].Shapiro-Wilk tests were used to assess the normality of the distribution.The final data display consisted of the mean and standard deviation (SD).Wilcoxon test was used to test for non-normal distributions.Finally, the data were presented as a median, interquartile range (IQR), and the 25th and 75th percentile.The ggplot2 package was used for data visualization [27].p-values<0.05 were considered statistically significant.

Results
Effect of blood storage temperature on PRR Initially, we analyzed the impact of blood storage duration and temperature on PRR.In this situation, the temperature of the blood storage facility is the most important indicator.In general, the PRR obtained at room temperature(23°C±2°C) is higher than that obtained at cold temperature(4°C±2°C) (Figure 2).Our findings indicate that storage of blood at room temperature for up to 8 hours is suitable for platelet separation.

Effect of blood storage time on PRR
Following that, we assessed the effect of blood storage time on PRR at room temperature within two hours from specimen collection and beyond two hours.In 3/4 of the enrolled hospitals, PRR within two hours was significantly higher than PRR after two hours (p < .05)(Figure 3).According to our study, storing platelets for less than two hours was more beneficial for platelet isolation and recovery.
To verify this conclusion, we recruited 6 healthy volunteers from Sichuan Cancer Hospital, and the results were consistent with the results of the multicenter study (Figure 5a).

Effect of the research center's instrument on PRR
Finally, we examined the impact of several research institutes on PRR.In this study, Pengzhou, Guangyuan and Shuangliu center all used Mindray series blood cell analyzer, while Fushun used XN-1000 blood cell analyzer.Among them, Pengzhou and Guangyuan used desktop low-speed refrigerated centrifuges, while Fushun and Shuangliu used desktop low-speed automatic balancing centrifuges.There was no significant difference in PRR between Pengzhou center and Guangyuan Center, no significant difference in PRR between Fushun Center and Shuangliu Center, and there were significant differences among the other centers in PRR (all P < .5; Figure 4).The following conclusions can be Furthermore, we analyzed EDTA-anticoagulated peripheral blood samples in 6 healthy adults using the XN-1000 and Mindray BC6800 as supplementary.In the hematological analysis, both platelets and Mean Platelet Volume (MPV) were examined.The PRR at room temperature (23°C±2°C) was slightly higher than the PRR at cold temperature (4°C±2°C).As the storage time increased, the PRR gradually decreased.Compared to the PRR values stored for 4 hours, the PRR values for 0 hours were significantly higher (p < .05)(Figure 5a).MPV values measured by the XN-1000 were significantly higher than those measured by Mindray (p < .05)(Figure 5b).

Effect of platelet extraction factors on platelet activation
We then analyzed CD62P expression in platelets by gating all CD41a-positive events (whole gating).The CD62P/CD41a (%) values of samples stored for 4 hours were significantly higher than those stored for 0 hours (p < .05),but the increase was more evident for samples stored at room temperature (Figures 5d, 6).

Effects of platelet extraction factors on RNA concentration
As well, we used a Nanodrop 2000 spectrophotometer to quantify the total RNA concentration.RNA concentrations measured at room temperature for 4 hours were higher than those measured at 0 hours according to our results.At low temperatures, RNA concentrations at 4 hours were significantly lower than those measured at 0 hours (p < .05)(Figure 5c).

Discussion
So far, few studies have been conducted on the influencing factors of platelet isolation.In this study, we systematically explored variables deemed to require optimization in a multicenter, large sample, including blood storage temperature, storage time, and instruments Figure 3.The boxplot of PRR stratified by the amount of time that blood is stored before and after 2 hours.Our boxplot is not a standardized approach and illustrates the distribution of data based on the three numbers summary: quartiles, the median, and the third quartile.
used at each research center, since these variables may effect platelet production, purity, and platelet extraction recovery.
Here, we conducted a prospective multicenter study of healthy adults from four centers, using PRP separated from whole blood specimens to investigate factors influencing PRR during platelet isolation.PRR, is a crucial indicator of the platelet isolation process.There were no statistically significant difference in PRR between blood samples stored at room temperature and cold temperatures, although PRR from room temperature sample storage was marginally superior to cold storage.These results are consistent with previous studies which have observed in vitro hypothermia enhances platelet αIIbβ3 activation and P-selectin expression induced by platelet agonists [28].Platelet activity in fresh blood, especially when stored at 4°C, quickly decreased, with about 40% activity within 6 h and only 20% activity within 12 h [28].
A supplementary experiment examining the effect of temperature on platelet activation was conducted in six samples, and the results were consistent with those found in previous studies [29,30].The presence of P-selectin in the plasma membrane of platelets, or the increase in its positive percentage, is considered a specific marker of platelet activation [31].In our study, unstimulated platelets showed high P-selectin, possibly due to limited immediate access to flow cytometry in clinical Settings, lack of rapid sample processing, and standardization of time intervals.The main obstacle to using flow cytometry to analyze platelet activation is sensitivity to changes in sample collection and processing in the laboratory, especially during the critical time period between blood collection and sample testing.Sample fixation is used to maintain platelet aggregation at the time of phlebotomy and minimize platelet activation in vitro [28].Shattil et al demonstrated that fixing the blood immediately after blood collection with 1% paraformaldehyde (PFA) prevents spontaneous platelet activation and stabilizes the sample, which is particularly beneficial in clinical Settings [32].Cho et al demonstrated that the proliferative effect of platelets was reduced by fixing platelets  Factors influencing platelet isolation 7 with PFA [33].Schmidt et al have shown that platelet fixation with PFA can significantly reduce CD 62P expression after stimulation [34].Without stimulation, platelet activation in the PFA fixed samples was limited to 0.8 ± 0.2% CD62P-positive cells [34].The purpose of our study was to investigate the influence trend of sample storage temperature and storage time on platelet activation during platelet extraction.There was no fixed blood after sample collection, resulting in spontaneous activation of unstimulated platelets and expression of high P-selectin.It is recommended to store blood samples at room temperature for up to 8 hours, ideally testing within 2 hours after collection, because cold temperatures can activate platelets, promote their adhesion to red blood cells and white blood cells, thus resulting in a decrease in the amount of platelets subsiding in the centrifugation process [35].When the platelets are centrifuged, the larger and aggregated platelets are precipitated, which results in the consumption of platelets, while the smaller platelets stay in the plasma.
We then analyzed how the recovery rate of platelets for blood stored at room temperature changes with storage time.Compared to platelet recovery beyond 2 hours, platelet recovery within 2 hours was significantly higher.Previous research has indicated that too long storage of platelets can result in low platelet counts.
Blood samples were left at room temperature for 6 hours with significantly reduced platelet counts [36].As blood is stored for a longer period of time, more activated platelets are produced [30].Our study found that platelet extraction should be completed within two hours of blood storage, and that storage time in excess of two hours would reduce platelet recovery.This conclusion indicates that blood storage time has an impact on platelet isolation, and platelet isolation should be conducted within 2 hours as much as possible to minimize the impact of storage time on platelet isolation.Furthermore, we investigated the effect of different research centers on platelet recovery during 2 hours of platelet extraction at room temperature, and our study found that, there were differences in PRR among the various research centers, but neither the differences between Pengzhou and Guangyuan nor the differences between Fushun and Shuangliu were statistically significant.Here, we consider the following reasons:The blood routine analyzer used in Pengzhou center and Guangyuan center is Mindray 6800 series, so there is no significantly statistical difference between the two centers; The blood routine analyzer used by Fushun is XN-1000 series, while Shuangliu uses Mindray 6900 series, but the two results are not significantly different.According to quality control assessments every day and comparisons from different types of equipments every-week, the performance of the blood routine analyzer in the four centers were acceptable.External Quality Assessment results of the laboratory was 91 to 100 points in 2020.Therefore, we prefer the difference of results due to different centrifuges.By checking the official website of each centrifuge, it can be seen that Fushun and Shuangliu use desktop low-speed refrigerated centrifuges, while Pengzhou and Guangyuan use desktop low-speed automatic balancing centrifuges, which may be the reason for the above results.
In future platelet-related studies, we should fix the blood sample storage temperature, storage time and centrifuge model in the process of platelet extraction, so as to reduce the variables affecting platelet extraction as much as possible and ensure the stable recovery rate of platelet extraction.By studying the influencing factors of platelet isolation, this project will facilitate a more comprehensive study of PRR's relationship to clinical diseases, as well as establish a reference value range for PRR and include it in the blood routine report, so that some clinical diseases may be diagnosed and prognosed using a new test basis.
Nevertheless, there are still some limitations to the study of platelets in liquid biopsy.Presently, most studies related to platelets are conducted in single centers, and it is hoped that future studies will include larger samples and be conducted in multiple centers.In the future, we should standardize platelet extraction procedures in order to maximize the benefits of liquid biopsy using platelets.

Conclusion
In this paper, the common affecting elements of platelet recovery, such as blood storage temperature, blood storage time, and research center were discussed through multi-center investigation.The following are the study's findings: After blood samples are taken, whole blood samples intended to be tested using plateletbased liquid biopsy should be identified and stored at room temperature until platelets are isolated by separating platelet-rich plasma from the blood sample within two hours from specimen collection.This will greatly improve platelet recovery and make it easier to conduct subsequent liquid biopsy-related experiments using tumor-educated platelets.In addition, the utilization of various instruments in different research facilities may have an effect on platelet recovery.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 2 .
Figure 2. The line chart of PRR stratified by blood storage time and blood storage temperature.

Figure 4 .
Figure 4.The violin plot of PRR stratified by center.Our violin plot is not a standardized approach and illustrates the distribution of data based on the three numbers summary: quartiles, the median, and the third quartile.

Figure 5 .
Figure 5. Hematologic analysis and platelet activation.A: The violin plot of PRR stratified by blood storage time and blood storage temperature.B: The MPV values measured by XN-1000 and Mindray.C: The violin plot of RNA concentration stratified by blood storage time and blood storage temperature.D: The violin plot of CD62P/CD41a (%) stratified by blood storage time and blood storage temperature.

Figure 6 .
Figure 6.FCM analysis of platelet activation.A, C, E: gating of the platelets population separated from the larger red blood cells.B, D, F: The blood samples were double-stained with APC-conjugated human CD41a antibody as well as PE-conjugated human CD62P antibody.CD62P PE-A intensity is shown on the horizontal axis and CD41a APC-A intensity is shown on the vertical axis.The numbers in black in B/D/F represent percentage of CD62P positive platelets in CD41a positive platelets.A and B, C and D, E and F are RT 0 h, RT 4 h, and CT 4 h, respectively. .

Table I .
[20,21]haracteristics.Pengzhou center uses LDZ5-2 table low speed automatic balance centrifuge from Beijing Jingli Instrument Co. Ltd, Guangyuan center uses GENIUS 6K-C table low speed automatic balance centrifuge from Changsha XINAO Instrument Co. Ltd, Fushun center uses KDC-2046 Low speed refrigerated centrifuge from Anhui USTC Zonkia Scientific Instruments Co.Ltd, Shuangliu center uses LD-5 G low speed refrigerated centrifuge from Chengdu Shuke Instrument Co. Ltd.The instruments used in each research center are shown in (Table I).All instruments are regularly maintained and calibrated.This study recruited a total of 226 healthy, drug-free volunteers including 42 members of the Pengzhou group, 60 members of the Fushun group, 64 members of the Guangyuan group, and 60 members of the Shuangliu group.It was mandatory for all candidates to fast for at least eight to fourteen hours without solid food and beverages before sample collection.All specimens (10 mL/ person) were drawn into EDTA-K 2 tubes (Becton, Dickinson and Company)[20,21]using vacuum tube needles (Shangdong Chengwu Instrument Co. Ltd; 0.55 × 19 mm) for hematological analysis.