Methods
This is a multicenter, cross-sectional study. From July 1, 2022 to September 30, 2022, plasmapheresis stations in Linwu (Hunan Province) and Jiange (Sichuan Province) recruited plasma donors as research subjects (the specific process of including and excluding subjects can be found in the Fig. S1 ). In order to minimize the interference of both primary and secondary osteoporosis risk factors as confounding factors, we used the International Osteoporosis Foundation (IOF) Osteoporosis Risk 1-Minute Test Questions for donor screening to ensure that no donor already exists the risk of osteoporosis. This questionnaire is recommended as a commonly used tool for osteoporosis risk assessment in the Guidelines for the Diagnosis and Management of Primary Osteoporosis 2017 ( Xia et al., 2019 ).

Inclusion criteria: (1) Registered qualified plasmapheresis donors (repeat donors,we named this group donors) who have donated more than 50 times and have continued to donate in the past six months, as well as new donors (we named this group controls) who have no history of plasmapheresis donation; (2) Male donors are over 50 years old, and female donors are postmenopausal; (3) Complete “the International Osteoporosis Foundation (IOF) Osteoporosis Risk 1-Minute Test Questions”, all answers are no; (4) Obtain the consent of the plasmapheresis donor and sign the “Informed Consent Form of the Plasmapheresis Donor”. Exclusion criteria: (1) Donors who may be affected by X-rays are excluded; (2) Taking steroid hormones or drugs that affect BMD and bone metabolism in the past 3 months should be excluded.

This study passed the review of the Ethics Committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences (approval number: NO: 2022029), and had each subject sign an informed consent form.

This article uses a cross-sectional study sample size calculation formula for estimation ( Wang & Cheng, 2020 ), and the formula is as follows where p represents the expected prevalence of osteoporosis in the donor population. Since there is no survey on the prevalence of osteoporosis in the donor population, and the average age of the population surveyed in this survey is over 50 years old. Therefore, using the results of the 2018 China Osteoporosis Epidemiological Survey in people over 50 years old  ( National Health Commission of the People’s Republic of China, 2018 ), it can be seen that p is 19.2%. q = 1 − p , d is the allowable error, and z is the significance test statistic. Set α = 0.05, d = 0.2 × p , z = 1.96. According to the formula, the sample size needs to be at least 421 people. n = p q d / z α 2 = z α 2 × p q d 2 .

A 3 ml venous blood was collected from each donor in a vacuum blood collection tube with a separating gel before donating plasma. Centrifugation (3,000r/min, 10min) was used to separate the serum, and the serum was stored at low temperature in a −20 °C freezer, and finally detected in the same lab. Blood test items include: serum total calcium, albumin (ALB), 25OHD, PTH, P1NP, β -CTX, SF levels.

Indicators of plasmapheresis donation behaviors including the total number of plasmapheresis donations, the recent plasma donation frequency ( i.e., the number of plasmapheresis donations in the last 12 months) and the interval from the last plasma donation were retrieved from the information system of the plasmapheresis station. Self-made questionnaires were used to collect background information such as age, BMI, annual household income, meat intake, calcium supplementation, and vitamin D supplementation. The International Physical Activity Questionnaire (IPAQ) was used to collect the physical activity of plasmapheresis donors.

P1NP was detected by total type I collagen amino-terminal elongated peptide detection kit (batch number: 653011, Roche, Basel, Switzerland), and the detection instrument was Roche E602. PTH was detected using a parathyroid hormone assay kit (batch number: 220901, Mindray, Shenzhen) and the testing instrument was Mindray CL-6000i. β -CTX was detected with β -collagen special sequence detection kit (batch number: 635899, Roche, Basel, Switzerland), and the detection instrument was Roche E602. Albumin was detected using an albumin detection kit (batch number: AUZ1125, Beckman, Brea, CA, USA) and the detection instrument was Beckman 5800. Serum calcium was detected by calcium assay kit (batch number: AUZ1199, Beckman, Brea, CA, USA), and the detection instrument was Beckman 5800. 25OHD was detected by total 25-hydroxyvitamin D assay kit (batch number: 230501, Mindray, Shenzhen), and the detection instrument was Mindray CL-6000i. SF was detected using chemiluminescence method using a ferritin assay kit (batch number: 47177UDOO, Abbott, Chicago, IL, USA) and the detection instrument was Abbott Alinity.

DXA was used to measure the BMD of anterior and posterior lumbar spine (L1-L4), bilateral femoral neck and non-dominant radial BMD of plasmapheresis donors with GE LunarDPX Prodigy. In both Linwu and Jiange plasmapheresis stations, Haier Blood Technology XJ-II blood component apheresis machine was used for plasmapheresis donation.

P1NP was measured by electrochemiluminescence with normal reference values ranging from 16.3 to 78.2 ng/mL, and data were cited from the Guidelines for Clinical Application of Bone Turnover Biomarkers ( Li, Xia & Zhang, 2021 ). β -CTX was measured by electrochemiluminescence, and the normal reference value ranged from 0.114–0.628 ng/mL, and the data were quoted from the 2021 Guidelines for the Clinical Use of Biochemical Markers for Osteotransition. Albumin was determined by BCG, with normal reference values ranging from 40 to 55 g/L, and data were quoted from the Hong, Yusan & Ziyu (2014) . Serum calcium was determined by colorimetric method, and the normal reference value ranged from 2.2–2.7 mmol/L, and the data were quoted from the “National Clinical Laboratory Operating Regulations”. PTH was determined by chemiluminescence method, and the normal reference value ranged from 12-65 pg/mL, and the data were quoted from the “National Clinical Laboratory Operating Regulations”. 25OHD was determined by electrochemiluminescence, and vitamin D inadequacy was indicated at <20 ng/ml, as shown in the Guidelines for the Use of Vitamin D and Bone Health in Adults ( Liao et al., 2014 ). According to the WHO criteria for iron accumulation ( World Health Organization, (2020) ), male SF > 200 ug/L and female SF > 150 ug/L are classified as iron accumulation. BMD is presented as g/cm 2 and as T -score. Based on the DXA classification criteria for BMD, a T -value of ≥-1 is normal, a T -value of −2.5 < T <-1 is low bone mass, and a T -value of ≤−2.5 is osteoporosis, as shown in the Guidelines for the Diagnosis and Management of Primary Osteoporosis 2017 ( Xia et al., 2019 ). According to the guideline, a T -value ≤−2.5 at either site is diagnosed as osteoporosis.

Graphpad Prism 8.0 and SPSS 24.0 (Armonk, NY, USA) were used for data collation and statistical analysis. Firstly, the overall population was descriptively statistically analyzed, and the basic differences between the two groups of new plasmapheresis donors and repeat plasmapheresis donors were compared. For the continuous data, the data conforming to the normal distribution were expressed as mean and standard deviation, and one-way ANOVA was used. Data that do not conform to the normal distribution are expressed by median and interquartile ranges, and Wilcoxon rank-sum test is used. The frequency (composition ratio) was used to express the counting data, and the chi-square test was used. The overall population was divided into groups according to the type of donor, and whether there were statistically significant differences between groups in bone biomarkers. The statistically significant indexes were analyzed according to three different indicators of donation behaviors ( i.e., the total number of plasmapheresis donations, recent frequency, and the interval from the last plasma donation), and multiple linear regression analysis was used to further assess the association between donation behaviors and bone metabolism biomarkers. We use Restricted Cubic Splines (RCS) to better demonstrate the nonlinear trend of change between bone metabolism biomakers and plasmapheresis donation behaviors. The Bootstrap method was used to test the mediating effect of SF on bone metabolism markers in relation to plasma donation behaviors. Finally, analyze whether there are statistically significant differences in BMD at different locations among different types of plasmapheresis donors (new and repeated donors). When conducting multiple comparisons, the Bonferroni method is used to adjust the P -value.

The test level was α = 0.05, and p < 0.05 indicated that the difference was statistically significant.

In the information collection stage, a unified and standardized project operation manual and questionnaire were produced and the staffs involved in the project were trained through online meetings to ensure the standardization of data collection. In the data entry stage, two independent members entered separately and then cross-checked to ensure the accuracy of the data. In the statistical analysis stage, the detection value of serum total calcium was corrected by the correction formula [corrected calcium = measured calcium + (40-measured albumin) × 0.02] and included in the statistical analysis.