Quality Analysis of Autologous Platelet-Rich Plasmapheresis

Abstract

To summarized the technology of autologous platelet-rich plasmapheresis and analyzed the product quality, in order to provide safe and effective product guarantee service for clinical treatment. Technical parameters were set according to patient age, weight, height, and preoperative routine blood indices. Autologous platelet-rich plasma (PRP) was collected, and the product quality and adverse reactions of patients were statistically analyzed. Autologous PRP had platelet (PLT), white blood cell (WBC), and red blood cell (RBC) counts of (1250.26 ± 435.88) × 10⁹/L, (1.19 ± 1.95) × 10⁹/L, and (0.05 ± 0.04) × 10¹²/L, respectively. The PLT enrichment ratio in PRP was 5.66 ± 1.66. There was no significant difference in PLT, RBC, WBC, or hematocrit before and after apheresis (P > 0.05). The incidence of adverse reactions was 8%, and all were mild. When clinical patients use PRP in the treatment of diseases, autologous platelet-rich plasmapheresis technology was used to apheresis PRP, which has good product quality and few adverse reactions, and thus can be adopted more widely.

Introduction

Autologous platelet-rich plasma (PRP) is a protein concentrate isolated from autologous peripheral blood [1]. PRP is prepared using manual centrifugation or an automatic blood component separator. At present, the most common method is manual separation; e.g., test tube centrifugation, blood bag centrifugation, and preparation kits [2–5]. However, autologous platelet-rich plasmapheresis is rarely reported. Platelet-rich plasmapheresis refers to the use of an automatic blood cell component separator, which collects whole blood, returns red blood cells (RBCs), white blood cells (WBCs), and plasma back to the body, and concentrates platelets (PLTs). The apheresis component of this technology is quite mature [6]; improving the process of collecting autologous PRP could provide high-purity and high-PLT-count PRP. Here, we report the application of this technology.

Materials and Methods

Clinical Data

From June 2020 to March 2021, 50 patients were admitted to our hospital, including 36 females and 14 males with a mean age of 44.8 ± 14.3 years (range 20–75 years) (Table 1). There were 29 cases of bone and joint diseases, 4 of facial rejuvenation, 1 of alopecia, 11 of facial rosacea, and 5 of local ulcer (Table 1).

Table 1.

General information of patients

Factors	n (%)
Gender
Male	14 (28)
Female	36 (72)
Age (years)
20–30	10 (20)
31–50	22 (44)
51–60	12 (24)
61–75	6 (12)
Diseases
Bone and joint diseases	29 (58)
Facial rejuvenation	4 (8)
Alopecia	1 (2)
Facial rosacea	11 (22)
Local ulcer	5 (10)

Inclusion Criteria

Patients were included in the study if they had no obvious cardiopulmonary dysfunction, maintained a cardiopulmonary circulation of 400–500 mL of blood for 5–10 min, and had a preoperative PLT count ≥ 100 × 10⁹/L. Patients were asked to abstain from eating high-fat and high-protein meals before apheresis. Peripheral vascular access is important to evaluate the extracorporeal circulation, ensure high product quality and not increase the pain of the patient.

Signed Informed Consent

This study was approved by the institutional subcommittee and ethics committee of Hospital. The IRB approval and informed patient consent were obtained.

Exclusion Criteria

Patients were excluded if they had moderate to severe anemia and coagulation dysfunction, and/or acute systemic infection or tumor. Patients prescribed drugs that affect PLT function and the coagulation system were instructed to refrain from taking them during the week before apheresis. Female patients were excluded 3 days before and after their menstrual period.

Instruments and Materials

Blood samples were collected and separated using single-use butterfly needles, calcium carbonate, a fully automatic blood cell component separator (NGL-XCF-3000; Sichuan Nigale Biomedical Co., Ltd., Chengdu, China), single-use kits for blood cell separation (P-2000 IE; Sichuan Nigale Biomedical Co., Ltd., Chengdu, China), anticoagulant ACD-A (Sichuan Nigale Biomedical Co., Ltd.), a fully automatic blood analyzer (XS-900i; Sysmex, Kobe, Japan), and a sterile feeder machine (TSCD-II; Terumo, Tokyo, Japan). Samples were stored in transfer bags in an ultralow-temperature freezer.

Environmental Requirements

Class II environmental requirements were met by providing sterile air and disinfecting the operating table with a wet paper towel.

Technical Parameters

Technical parameters were adjusted according to patient height, weight, gender, and preoperative hematocrit (HCT) and PLT counts. Pre-apheresis settings were as follows: apheresis speed, 50–80 mL/min; input speed, 50–80 mL/min, ratio of anticoagulant to whole blood, 1:10–12 mL; PLT apheresis, 75–80 (based on the preoperative PLT count); platelet apheresis volume equal to the apheresis volume; and no use of plasma apheresis function.

Measurements

We conducted routine pre- and postoperative blood tests (PLT, RBC, WBC and HCT counts) in patients. We assessed the safety of PRP apheresis by evaluating patients postoperatively for blood circulation interruptions, numbness in the mouth, dizziness, congestion, hematoma and other adverse reactions at the puncture site.

Statistical Analysis

All statistical analyses were conducted using SPSS Statistics for Windows (version 25.0; IBM Corp., Armonk, NY, USA). Count and measurement data were analyzed using the χ² tests and t-test, respectively. P values < 0.05 were considered statistically significant.

Results

The Characteristics of Gender, Age and Disease Spectrum of Patients

Most of the patients treated with PRP were female (72%), mainly aged 31–60 years old (68%), and the disease spectrum was mainly bone and joint disease and facial rosacea (80%) (Table 1).

Routine Blood Test Indices and PRP Quality of Patients Before and After Apheresis

Indices from routine blood tests (i.e., PLT, WBC, RBC and HCT) did not statistically significant before and after apheresis (P > 0.05) (Table 2). The PLT concentration in the PRP product was (1250.26 ± 435.88) × 10⁹/L, which met the requirements for clinical treatment. RBC and WBC counts were (0.05 ± 0.04) × 10¹²/L and (1.19 ± 1.95) × 10⁹/L, respectively, which were relatively low (Table 2).

Table 2.

Patient test indicators and product quality of platelet-rich plasmapheresis ($\overline{\mathrm{x}}$±s)

Group	n	PLT × 109/L	WBC × 109/L	RBC × 1012/L	HCT (%)
Before apheresis	50	226.56 ± 66.25	6.25 ± 1.93	4.41 ± 0.46	38.86 ± 4.58
After apheresis	50	230.00 ± 66.13#	4.93 ± 1.39#	3.98 ± 0.30#	33.67 ± 2.91#
PRP product	50	1250.26 ± 435.88	1.19 ± 1.95	0.05 ± 0.04	–
P value		0.818	0.619	0.301	0.167

^#Compared to before apheresis

Operating Data and Adverse Reactions

Collecting one cycle versus two cycles, the apheresis time and the product volume, increased for the latter (P < 0.05), but product quality remained unchanged and the adverse reaction rate was not statistically significant (Table 3).

Table 3.

Actual operating data and adverse reaction ratio ($\overline{\mathrm{x}}$±s)

Group	n	Circulating volume (mL)	Apheresis time (min)	Product Capacity (mL)	Product content of PLT (× 109/L)	Anticoagulant (mL)	Adverse reaction classification			Adverse reaction rate (%)
							Oral numb	Brief dizzy	Hematoma at site of puncture
One circulating blood volumes	39	447.38 ± 59.06	18.51 ± 5.42#	41.84 ± 7.82#	1268.85 ± 448.67	37.92 ± 7.64	0	2	1	7.7#
Two circulating blood volumes	11	941.36 ± 85.63	46.72 ± 21.44	89.72 ± 19.24	1184.36 ± 399.82	83.27 ± 12.41	1	0	0	9.1
P value			0.00	0.003						0.620

^#Compared to two circulating blood volumes

The PLT Enrichment Ratio in PRP Products Relative to Whole Blood

The PLT enrichment ratio in PRP products was 5.66 ± 1.66 relative to whole blood; 6.0%, 50.0%, and 44.0% of PRP products had enrichment ratios of < 3, 3–6, and > 6, respectively (Fig. 1).

Fig. 1.

The PLT enrichment ratio in PRP products relative to whole blood (5.66 ± 1.66, n = 50)

Discussion

Autologous PRP contains several times more PLTs than the physiological concentration of the body. It is widely used for tissue regeneration, wound repair, facial rejuvenation, and functional reconstruction because of its low-trauma acquisition process and therapeutic potential [7–10]. This study included 50 patients treated for bone and joint diseases, facial rejuvenation, alopecia, local ulcer, and other conditions. The age distribution of the patients was wide (20–75 years), demonstrating the broad range of clinical application of autologous platelet-rich plasmapheresis. PLT counts in PRP are generally reported as 3–6 times those in whole blood. PLT counts > 1000 × 10⁹/L achieve the best cell proliferation effects. The mean PLT count of our products was (1250.26 ± 435.88) × 10⁹/L, indicating adequate therapeutic concentrations. Furthermore, PLT enrichment was 5.66 ± 1.66 times higher on average relative to whole blood (Fig. 1).

The PRP products also had very low RBC counts. Low RBC counts, and thus low oxygen consumption, can reduce the probability of hemolysis, which is beneficial for storage and freezing. Small amounts of WBCs can provide resistance against infections and clear necrotic tissue, which is helpful for local, difficult-to-heal wounds [11–13]. However, inflammatory factors, such as IL-1β, TNF-α, IL-6 and IL-8, released by leukocytes may inhibit the repair of some tissues or certain periods of the repair process [14–16]. It is controversial as to whether WBCs in PRP products are beneficial for tissue repair, so further prospective clinical studies are needed. However, WBC filtration can be conducted if WBCs need to be completely removed.

Autologous platelet-rich plasmapheresis is clinically safe. Pre- and postoperative blood parameters did not differ significantly among the 50 patients in our study. With two compared to one circulating blood volume, product volume increased but product quality and concentration were unchanged. The four cases of adverse reactions were mild, and the adverse reaction rate of one cycle was 7.7% (Table 3). One case showed congestion after back-transfusion, which has several plausible explanations. First, platelet-rich plasmapheresis requires patient blood vessels to be in good condition. Older patients, and those with longer hospital stays treated with certain drugs, may have weakened blood vessels. Additionally, medical staff may have accidentally punctured the blood vessel walls. Patients may also not have been in the position required for platelet-rich plasmapheresis, leading to needles being misplaced. We took several steps in response to this incident. We evaluated patient blood vessels and avoided venipuncture on both elbows 2 days before apheresis. We also improved the puncture techniques of the staff. Finally, we increased the number of patient observations to promote safety. If a hematoma appeared during apheresis, the procedure was stopped immediately, another blood vessel was selected, and the puncture site was treated accordingly.

Transient dizziness occurred in two patients. Our department uses an automatic blood cell component separator, which has the advantage of simple PRP apheresis from blood donors via a puncture needle. Also, only one tube is used for blood apheresis and blood doping, and used in this way is single channel single centrifugal cup type apheresis, a apheresis the extracorporeal circulation blood volume required for 447.38 ± 59.06 mL, to strengthen attaches great importance to the prevention. A low blood volume reaction can slow down the blood collection process, in turn reducing patient tolerance. With two circulating blood volumes, the apheresis adverse reaction rate was 9.1%. Only one patient had slight mouth numbness, possibly because calcium was not administered quickly. Patients are given solid oral calcium supplements 0.5 h prior to using anticoagulant ACD-A to prevent adverse reactions to low calcium, such as a sensation of pins and needles in the mouth and convulsions. If lip numbness or other phenomena occur, the rate of reinfusion should be slowed. The patient should also be closely observed and given calcium carbonate orally or, if necessary, intravenously via slow injection of 10% calcium gluconate to alleviate the hypo-calcium reaction.

Apheresis duration, circulation volume, and anticoagulant dose were determined based on the PRP volume, vascular condition, and age of the patient. Patients with good vascular condition require a lower PRP volume, shorter apheresis duration, less whole-blood processing, and lower anticoagulant dose. Platelet-rich plasmapheresis is advantageous because it is fast and can be conducted based on the specific amount of PRP required. The collected PRP was typically divided into 3 bags using an aseptic interchange machine. One bag was used on the same day, and the rest were cryopreserved in our department, which reduced the pain and expense caused by multiple punctures.

Conclusions

We demonstrated the clinical value and safety of PRP techniques, which merit wider use. When clinical patients need to use PRP for multiple times to treat diseases, autologous platelet-rich plasmapheresis technology was used to apheresis PRP, the volume of PRP is adjustable, the quality was good, and the PRP could be stored in different doses, which has a wide application demand.

Author contributions

BZ designed the research, interpreted the data, NS and ZY wrote the manuscript. NS and ZY performed the experiments, collected and interpreted the data. QR, CL and MS participated in manuscript preparation. All authors critically revised the manuscript and approved the final version.

Funding

The PLA Military Logistics Research Project Cultivation Special Project (NO.2021HL012).

Declarations

Conflict of interest

The authors have no conflicts of interest to declare.

Ethical approval

This study was approved by the institutional subcommittee and ethics committee of Hospital. The IRB approval and informed patient consent were obtained (KY2021114).