Factors affecting the features of platelet-rich plasma in patients with knee osteoarthritis

Abstract

Objective:

The aim of this study was to present an analysis of platelet-rich plasma obtained from patients with knee osteoarthritis and reveal the factors affecting its features.

Methods:

A total of 62 patients (mean age: 56.68 ± 7.13 years) with symptomatic knee osteoarthritis were included in this study. Age (years), gender, height (m), weight (kg), body mass index (kg/m²), duration of symptoms, smoking status, smoking index, general health status, and physical activity scores were recorded. Whole blood and platelet-rich plasma cell counts were performed with a hematology analyzer. White blood cell, red blood cell, and platelet counts were recorded. According to the dose of injected platelets, efficiency of the procedure, purity of platelet-rich plasma, and activation classification, dose of platelets, efficiency of the procedure (platelet recovery rate, %), and purity of the obtained platelet-rich plasma product (relative composition in platelets, %) were calculated. Correlation analysis between the features of platelet-rich plasma and the patient-related variables, including age, gender, body mass index, smoking status, smoking index, presence of other health conditions, physical activity scores, duration of symptoms, and pain levels, was performed.

Results:

Dose of injected platelets, efficiency of the procedure, purity of platelet-rich plasma, and activation analysis showed that the dose of injected platelets was 3.25 billion, the efficiency of the process was 77%, and the purity rate of the platelet-rich plasma was 98.4%. Platelet-rich plasma platelet count was correlated with whole blood platelet count (r = 0.81, P < .001), whole blood white blood cell count (r = 0.39, P = .002), smoking status (r = 0.56, P = .03), smoking index (r = −0.63, P = .002), and the presence of hypertension (r = −0.31, P = .04). Platelet-rich plasma white blood cell and purity of platelet-rich plasma were correlated with the smoking status of the patients (r = 0.52, P = .01; r = 0.64, P = .003, respectively).

Conclusion:

This study has demonstrated that high dose and very pure platelet-rich plasma with medium efficiency was yielded with this platelet-rich plasma preparation procedure; whole blood platelet count, the presence of hypertension, and the smoking status of patients affect the features of the obtained platelet-rich plasma.

Level of Evidence: Level IV, Diagnostic Study.

Highlights

• Platelet-rich plasma (PRP) is an autologous blood product with a higher platelet concentration than whole blood which contains growth factors with highly anabolic properties. PRP has become widely used for knee osteoarthritis (OA). This study aimed to analyse of obtained PRP product and reveal the factors affecting the features of PRP in patients with knee OA.

• The results showed a significant and excellent relationship between platelet level within PRP and whole blood platelet count. Moreover, PRP platelet count was also correlated with whole blood WBC count, smoking status of the patients, smoking index, and the presence of hypertension.

• The results from this study indicate whole blood platelet count, the presence of hypertension, and the smoking status of the patients can affect the features of the yielded PRP in patients with OA. Considering these factors in the clinical decision of whether PRP injection should be considered as an option in the management of patients with osteoarthritis and in patient selection may be a guide in terms of preventing unnecessary injections and costs.

Introduction

Knee osteoarthritis (OA) is one of the leading sources of global disability, which affects approximately 300 million adults worldwide.^1,2 It is anticipated that the burden of knee OA will increase with the aging of the population and increasing obesity rates.^1,3 Multimodal therapies, including pharmacological and nonpharmacological approaches, are emphasized to address pain and functional limitations.^3-5 Although the pharmacological treatment options vary, they generally have small to moderate effects in the short term and can have several adverse effects.^6-8 On the other hand, exercise, which is a prominent nonpharmacological treatment among current options, can be safe and effective.^6,7 However, it is often challenging to maintain regular exercise, resulting in a lack of long-term benefits. Thus, it is obvious that there is a clear need for safe and efficient treatment techniques supported by evidence.

Platelet-rich plasma (PRP) is an autologous blood product with a higher platelet concentration than whole blood and contains various growth factors with highly anabolic properties.^9,10 It is believed that PRP has a supra-physiologic effect to promote natural tissue healing by modulating the joint environment and reducing inflammatory distress.^11-16 PRP has become very popular and has drawn great interest as a regenerative adjunct therapy among clinicians and patients. In recent years, the use of PRP for the management of OA has gradually increased.^17-19 The heterogeneity of procedures and the lack of biological characterization of the obtained product are still the main limitations in the existing PRP literature, despite the fact that the number of studies has rapidly increased. Several classification systems have been suggested to address these problems and report the features of obtained PRP clearly.^20-24 DEPA (dose of injected platelets, procedure efficiency, purity of PRP, and activation) classification is one of these classification systems in the literature.²⁰

The primary goal of PRP injections is to deliver a higher concentration of platelets to the target area than blood circulation does. Thus, the platelet count is the most important factor that determines the features of PRP. The features and content of the injected substance are vital when evaluating the effectiveness of PRP.^16-20 It is well recognized that individual variations can have an impact on the hematopoietic system.^25-28 We hypothesize that features of PRP can be influenced by a variety of factors, such as age, gender, lifestyle, and habits, even when the same procedure and equipment are used for preparation. The question behind this study is: “Which factors should be considered before using PRP injections in the treatment of patients with osteoarthritis?” Therefore, the aim of this study is to present a prospective analysis of obtained PRP product and reveal the factors affecting the features of PRP in patients with knee OA.

Materials and Methods

Study design

This study is a prospective, observational substudy which is a part of multicenter randomized controlled clinical trial. It was coordinated in the Faculty of Health Science, Istanbul University-Cerrahpaşa and Faculty of Medicine, Istanbul University and conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Research Ethics Committee of Cerrahpaşa Faculty of Medicine, Istanbul University-Cerrahpaşa (IRB Study Protocol: 59491012-604.01.02). Volunteers provided written informed consent prior to the participation.

Patients

Potential volunteers between 40 and 70 years of age diagnosed with symptomatic knee OA were screened. Patients were included if they had knee pain greater than 3 points on an 11-point numerical pain rating scale and were graded as II or III on the Kellgren–Lawrence classification. Exclusion criteria were (i) lower hemoglobin count than 15 g/dL, (ii) ongoing anticoagulation therapy, (iii) using nonsteroidal anti-inflammatory drugs in the previous 3 weeks before the participation, (iv) patients with a history of previous operation in the affected knee, (v) active infection, malignancy, or systemic disorders limiting functional abilities, and (vi) impaired cognition that impacts the ability to give informed consent.

Sample size

A correlation sample size estimation was performed using a sample size calculator (http://www.sample-size.net/correlation-sample-size/). The following parameters were used for calculation: 80% power with a 2-sided alpha error of 0.05 and an expected correlation coefficient of 0.4. These parameters suggested a minimum of 47 patients as the total sample size. With an anticipated drop rate of 30%, a total of 62 patients were enrolled in the study.

Outcome measures

A patient information form was used to assess the sociodemographic characteristics of the participants, including age (years), gender, height (m), weight (kg), body mass index (BMI, kg/m²), duration of symptoms, smoking status, and general health status (presence of other diseases, medication, and previous treatments). The smoking status of the patients was assessed by the smoking status classification²⁹ and the smoking index.³⁰ The smoking status classification was performed based on the smoking status of the patients, and they were classified as current smokers, former smokers, or nonsmokers.²⁹ Current smokers are those who smoke on either a daily or an occasional basis. Former smokers are those who used to smoke on a regular or infrequent basis but are no longer smokers. A nonsmoker is defined as a person who has never smoked. For the current smokers, the smoking index was calculated using this formula: the number of cigarettes smoked daily × the number of years spent smoking.³⁰

The severity of pain was determined using an 11-point numeric pain rating scale, with “0” identifying no pain and “10” identifying the most severe pain imaginable. Using this scale, patients were asked to rate the severity of their knee pain from 0 to 10. The physical activity levels of the patients were assessed with the Turkish version of the International Physical Activity Questionnaire (IPAQ), which is valid and reliable.³¹ The MET-min (metabolic equivalent) score is used to assess the outcomes. It consists of 4 parts that question the duration and types of physical activities including sitting (1.5 MET-min), walking (3.3 MET-min), moderate physical activity (4 MET-min), and vigorous physical activity (8 MET-min). The total score is calculated and then categorized as low (lower than 600 MET-min/week), moderate (600-3000 MET-min/week), and high (above 3000). Data was collected the same clinician (12 years of clinical experience).

Dose of injected platelets, efficiency of the procedure, purity of PRP, and activation classification was published by Magalon et al²⁰ to introduce a standardized classification based on biological parameters. “Dose” identifies the injected platelet dose, which is determined by multiplying the volume of PRP by the platelet concentration in the injected substance. It is categorized as A (very high dose of injected platelets >5 billion), B (high dose of injected platelets, 3-5 billions), C (medium dose of injected platelets, 1-3 billions), and D (low dose of injected platelets, <1 billion). “Efficiency” identifies the recovery rate in platelets and is calculated by the percentage of platelets recovered in the PRP from the blood. It is classified as follows: A, high efficiency if the platelet recovery rate is greater than 90%; B, medium efficiency if the recovery rate is between 70% and 90%; C, low efficiency if the recovery rate is between 30% and 70%; and D, poor efficiency if the recovery rate is less than 30%. “Purity” identifies the relative composition of platelets, white blood cells (WBCs), and red blood cells (RBCs) in the obtained PRP. Categorization is as follows: A, very pure if the percentage of platelets compared with RBC and WBC is >90%; B, pure if the percentage of platelets compared with RBC and WBC is from 70% to 90%; C, heterogeneous if percentage of platelets compared with RBC and WBC is from 30% to 70%; D, whole blood PRP if percentage of platelets compared with RBC and WBC is <30%. Finally, “activation” defines whether an exogenous clotting factor adds to the activation of platelets or not. This action depends on the treatment indications as well as the physician’s decision.^20,32

Platelet-rich plasma preparation procedure

Platelet-rich plasma was prepared using a commercially available kit (Figure 1, T Lab, T-Biotechnology Laboratory, Istanbul, Turkey), and the whole preparation procedure was managed by the same experienced orthopedic surgeon (18 years of clinical experience). To perform complete cell counts for both whole blood and PRP, 20 mL of the whole blood was drawn from the ante cubital vein of the patients under sterile conditions. Tubes contain 3.20% concentrated 0.1 M of sodium citrate to block the coagulation. After phlebotomy, the tubes were placed swing-out rotor centrifuging device and were centrifuged at 830 g for 4 minutes. Following the single centrifugation, 6 mL of the obtained PRP was used for platelet count analysis. Platelet-rich plasma and the whole blood samples were held at room temperature and analyzed as soon as possible.³³

Whole blood and PRP cell counts were performed with a hematology analyzer, and WBC, RBC, and platelet counts were recorded. According to DEPA classification, dose of platelets, efficiency of the procedure (platelet recovery rate, %), and purity of the obtained PRP product (relative composition in platelets, %) were calculated.²⁰

Statistical analysis

Statistical analysis was conducted using the Statistical Package for the Social Sciences version 21.0 (IBM SPSS Corp.; Armonk, NY USA) statistic program for Windows. Shapiro–Wilk test was used to check the distribution of the data. It was normally distributed; thus, parametric tests were preferred for analysis. Pearson’s correlation coefficient, point biserial correlation, and paired sample t-test were performed. Data were summarized as means, SDs, frequency, and percentages, and the alpha level was set at 0.05 in all analyses. It was considered satisfactory for correlation coefficients to reach values of 0.4 or higher (r = 0.81-1.0, excellent; 0.61-0.80, very good; 0.41-0.60, good; 0.21-0.40, fair; and 0.00-0.20, poor).³⁴

Results

A total of 62 patients who satisfied the eligibility criteria and agreed to participate (mean age: 56.68 ± 7.13 years) were included in the analysis. Details of the patient characteristics are presented in Table 1. Cell counts (platelets, leukocyte, erythrocytes, and platelet distribution width) within whole blood and obtained PRP are demonstrated in Table 2. Whole blood platelet counts ranged from 169 to 452 × 10⁹/L and PRP platelet counts ranged from 316 to 794 × 10⁹/L. The mean WBC count was 1.45 ± 0.99, and mean RBC count was 0.20 ± 0.03 within PRP. As expected, the WBC and RBC counts within PRP were lower than the levels of whole blood. Dose of injected platelets, procedure efficiency, purity of PRP, and activation analysis showed that the dose of injected platelets was 3.25 billion, the efficiency of the process was 77%, and the purity rate of the PRP was 98.4%. These results indicated “high dose (B),” “medium platelet recovery rate (B),” and “very pure (A)” PRP with a final DEPA score as “B-B-A” (Table 3).

Table 1.

Patient characteristics

n = 62	Mean	SD	Minimum	Maximum
Age (years)	56.68	7.13	48	70
Height (m)	1.64	0.08	1.50	1.90
Weight (kg)	75.63	10.91	54	102
BMI (kg/m2)	27.81	3.53	22.31	35.16
Duration of symptoms (months)	37.24	47.81	6	240
Pain level (0-10/NRS)	6.69	1.16	4	8
Smoking Index (for current smokers)	64.81	32.23	15	150
Physical activity levels (total IPAQ score)	992.41	425,.60	328	2146
n = 62	n		Frequency (%)
Gender
Female	40		64.5
Male	22		35.5
BMI categories
Underweight	0		0
Normal weight	13		21.0
Overweight	32		51.6
Obesity class I	14		22.6
Obesity class II	3		4.8
Obesity class III	0		0
OA severity
Grade I	0		0
Grade II	27		43.6
Grade III	35		56.5
Grade IV	0		0
Smoking status
Yes
Current smokers	26		41.9
No
Former smokers	31		50
Nonsmokers	5		8.1
Other diseases
Yes
Hypertension	13		21
Diabetes Mellitus	6		9.7
Other	5		8.0
No	38		61.3
Physical activity categories
Low	19		30.6
Moderate	43		69.4
High	0		0

BMI, body mass index; IPAQ, the International Physical Activity Questionnaire; NRS, numeric rating scale; OA, osteoarthritis.

Table 2.

Cell counts within PRP and whole blood

n = 62	Whole blood				PRP
	Platelets (×109/L)	Leukocytes (×109/L)	Erythrocytes (×1012/L)	Pdw	Platelets (×109/L)	Leukocytes (×109/L)	Erythrocytes (×1012/L)	Pdw
Mean	267.64	6.93	5.17	16.66	542.40	1.45	0.20	17.36
SD	56.85	2.35	1.52	0.41	85.63	0.99	0.03	0.35
Maximum	452.0	15.30	12.90	16.10	794.0	2.70	0.30	18.40
Minimum	169.0	4.20	3.95	17.40	316.0	0.70	0.01	16.40
Range	283	11.10	8.95	0.70	478	2.0	0.29	2.0

Pdw, platelet distribution width; PRP, platelet-rich plasma.

Table 3.

Analysis of PRP based on the DEPA classification

	Dose of injected platelets (billions)	Efficiency of the process (platelet recovery rate, %)	Purity of the PRP (relative composition in platelets, %)	Activation
n = 30	A: >5 very high dose	A: >90 high	A: >90 very pure PRP
	B: 3-5 high dose	B: 70-90 medium	B: 70-90 pure PRP
	C: 1-3 medium dose	C: 30-70 low	C: 30-70 heterogeneous PRP
	D: <1 low dose	D: <30 poor	D: <30 whole blood PRP
	3.25	77%	98.4%	Endogenous activation
Final DEPA score	B High dose	B Medium efficiency	A Very pure	–

DEPA, dose of injected platelets, efficiency of the procedure, purity of PRP, and activation; PRP, platelet-rich plasma.

Results of the correlation analysis of the patient-related variables, including age, gender, BMIs, smoking status, smoking index, presence of other diseases, physical activity scores, duration of symptoms, and pain levels with features of PRP based on DEPA classification, are summarized in Table 4. A significant and excellent relationship was found between platelet level within PRP and whole blood platelet count (r = 0.81, P < .001). Platelet-rich plasma platelet count was also correlated with whole blood WBC count (r = 0.39, P = .002), smoking status of the patients (r = 0.56, P = .03), smoking index (r = 0.63, P = .002), and the presence of hypertension (r = −0.31, P = .04). Dose of the injected platelets is calculated using platelet count within PRP; therefore, the same correlations were also found for “dose of injected platelets” (Table 3). Platelet-rich plasma white blood cell and purity of PRP were correlated with the smoking status of the patients (r = 0.52, P = .01; r = 0.64, P = .003, respectively). In addition, the smoking index score of the current smokers was showed a good correlation with PRP-WBC (r = 0.57, P = .02) and a fair correlation with the purity of PRP (r = 0.35, P = .04).

Table 4.

Correlation analysis of the patient-related variables with PRP quality based on DEPA classification

	Wb_Plt	Wb_WBC	Wb_RBC	Wb_Pdw	Gender	Age	BMI	Physical activity score	smoking status	Smoking index	Other diseases: HT	Other diseases: DM	Duration of symptoms	Pain level
PRP_Plt	0.81	0.39	0.14	0.02	−0.15	0.11	−0.10	0.15	0.56	−0.63	−0.31	0.05	0.12	−0.18
PRP_WBC	0.09	−0.04	0.31	−0.18	−0.25	−0.17	0.23	0.03	0.52	−0.57	−0.17	−0.08	−0.38	−0.14
PRP_RBC	−0.06	−0.13	−0.06	0.16	−0.09	−0.19	0.18	−0.11	−0.12	0.25	0.21	−0.23	−0.16	−0.03
PRP_Pdw	0.12	0.01	0.16	0.05	−0.21	0.24	0.22	0.14	0.15	−0.29	0.12	−0.07	−0.10	0.13
Dose of injected platelets	0.81	0.39	0.14	0.02	−0.15	0.11	−0.10	0.15	0.56	−0.63	−0.31	0.05	0.12	−0.18
Efficiency of the process	−0.30	0.07	0.19	0.25	−0.23	0.07	0.25	0.07	0.13	−0.25	−0.08	0.01	−0.21	0.16
Purity of the PRP	−0.12	−0.03	0.20	−0.20	−0.20	−0.29	0.30	−0.01	0.64	−0.35	−0.09	−0.11	−0.23	−0.19

BMI, body mass index; DM, diabetes mellitus; HT, hypertension; PRP, platelet-rich plasma; PRP_Pdw, platelet-rich plasma platelet distribution width; PRP_Plt, platelet-rich plasma platelet count; PRP_RBC, platelet-rich plasma red blood cell count; PRP_WBC, platelet-rich plasma white blood cell count; Wb_Pdw, whole blood platelet distribution width; Wb_Plt, whole blood platelet count; Wb_RBC, whole blood red blood cell count; Wb_WBC, whole blood white blood cell count.

*The bold p values indicate statistical significance (p<0.05).

Discussion

Platelet-rich plasma injections have gained increasing popularity in knee OA management as a novel minimally invasive therapy. However, features of injected PRP are crucial for the promising effects of concentrated platelets in the tissue healing process. This is the first study that presents a prospective analysis of PRP procedure and reports the factors affecting the features of PRP in patients with knee OA. The results of the current study indicated that this automated PRP preparation procedure produces medium-dose and very pure PRP with an increased platelet concentration over the platelet count of baseline whole blood. On the other hand, the correlations between PRP cell counts and patient-related factors that may affect the quality of PRP were analyzed in this study. Whole blood platelet count was highly correlated with PRP dose and platelet count as expected. There were high degree correlations between PRP-WBC and purity with smoking status, yet there was no correlation between any other factors including age, BMI, physical activity level, OA severity, duration of symptoms, and pain levels in patients with knee OA.

Variations in PRP preparation procedures, as well as a lack of ­characterization of injected PRP, limit the ability to draw an ­evidence-based conclusion regarding the efficacy of PRP. Dose of injected platelets, procedure efficiency, purity of PRP, and activation, which is one of the reported classification systems addressing this gap, is used to assess the features of injected PRP in this study.²⁰ The results of this study revealed that “high dose (B),” “medium platelet recovery rate (B),” and “very pure (A)” PRP. Magalon et al²⁰ presented a retrospective analysis of 20 different PRP procedures, and they reported the dose of injected platelets in the range of 0.21 billion and 5.43 billion.^20,35 In the same study, the authors found that previously published recovery rates ranged from 13.1% to 79.3%, and the global purity of the PRP products ranged from 6.0% to 99.7%.^20,35,36 The procedure in the present study furnished 3.41 billion platelets, 76% recovery rate, and 99.2 % global purity in accordance with the literature.

The anabolic properties attributed to PRP are mainly derived from supra-physiologic counts of platelets in this product.^9,10 It is supposed that the increased platelet concentration promotes natural tissue healing by modulating the joint environment and reducing the inflammatory distress.¹⁶ As a consequence, the platelet count of PRP is the major factor affecting the quality of PRP procedure.³⁷ The procedure used in this study produced supra-physiologic platelet concentration over the baseline with an average 2-fold increase. It was reported in the literature that the ideal platelet concentration of the injected PRP is 2- to 10-fold over the baseline whole blood platelet count.^37,38 Higher platelet concentrations may not directly indicate a high-quality PRP alone.³⁹ On the contrary, over-concentrating platelets with increased growth factor concentrations do not enhance the regenerative properties of PRP and may delay the natural healing process by causing increased scar tissue formation.^40,41

Platelet distribution width, which is likely affected by preparation procedure or anticoagulant type, is an indicator of platelet activation and one of the quality-related factors of PRP. It was reported that keeping the platelet distribution width within the normal ranges indicates good viability of the platelets.³⁹ Analysis showed that the platelet distribution width was within the physiological reference ranges, and the PRP procedure in this study did not cause preactivation of the platelets. Verma et al³⁹ emphasized that the platelet distribution width analysis may serve as a measure of quality for PRP activation.

Results of correlation analysis revealed that whole blood platelet count was highly correlated with PRP dose and platelet count. This is an expected result and well-documented in previous literature; however, the magnitude of this correlation may be an indicator for the platelet recovery rate from the baseline whole blood.^39,42,43 Platelet recovery rate represents the efficiency and quality of the PRP preparation process.²⁰ Significant correlations between platelet counts of whole blood and PRP were reported by Verma et al³⁹ (r = 0.62) and Weibrich et al⁴² (r = 0.73), previously. In this study, the results indicated a positive strong correlation (r = 0.81) and 77% platelet recovery rate. Besides, good correlations were found between PRP-WBC and purity with smoking status of the participants. It is known that smoking can cause structural changes in the hematopoietic system and increase inflammation as well as WBCs.^44,45 Pedersen et al⁴⁶ revealed that smoking raises WBCs by around 17% in current smokers and 0.6% to 15% in former smokers. The presence of hypertension was negatively correlated with PRP platelet count and dose of injected platelets. We did not find any significant correlation between features of PRP with the other patient-related factors including age, BMI, physical activity score, duration of symptoms, and pain levels. To the best of our knowledge, there is no study that analyzes the affecting factors for the features of PRP in patients with OA. Besides, similar to our results, it was reported in the literature that the gender and age of the patients do not alter the platelet count of PRP.^39,42,47

This study has several limitations. First, we did not analyze the quantification of growth factor content of the PRP product as a quality and viability control parameter. Second, the effects of other preparation procedures and devices did not compare. Furthermore, the final pH of PRP and using of different anticoagulants which have potential to affect the quality were not evaluated. The other limitations are the relatively small sample size and the absence of a control group. Although the concentration of platelets is crucial, it does not necessarily indicate the quality of the PRP product alone. To confirm the quality and content of the PRP delivering to patients with OA, future studies should consider other factors such as pH, platelet swirling, and growth factors.

Platelet-rich plasma has drawn great interest as a regenerative adjunct therapy, and it is increasingly used in the management of knee OA. Whole blood platelet count, the presence of hypertension, and the smoking status of the patients can affect the features of the yielded PRP in patients with OA. Considering these factors into account in the clinical decision is important for determining whether PRP is an option in the management of patients with knee osteoarthritis. These results may serve as a guide in terms of patient selection and avoiding unnecessary injections and their associated costs. The variations in PRP preparation procedures and lack of characterization are still the major challenging factors in the PRP literature. To make a global conclusion in effectiveness and quality of PRP, further studies should clearly report quality control measures of the procedure.

Figure 1.

PRP kit and obtained product after procedure