“More is not necessarily better as too high of a platelet concentration  can be detrimental to the healing process.”

– Fadadu et al. (2019)

What is the optimal composition of PRP? I have been asked this question countless times and my answer is always the same. It depends. It depends on the injury. Is it acute or chronic? Is the wound clean or contaminated? Is the structure a joint or a tendon? It should be obvious to anyone with medical training that there is no single formulation of PRP that will work best for every condition. In the coming weeks, I will review the scientific literature and summarize what is known about the various cellular components of PRP, namely: leukocytes, platelets, and erythrocytes. For part two I will be discussing platelets.

PRP vs PPP

Platelet RICH plasma simply means that a given sample of plasma has platelets concentrated more than whole blood. When a plasma sample has a lower concentration than whole blood, it is referred to as platelet POOR plasma (PPP). PRP has been compared to PPP in a wide variety of studies (e.g., reducing oxidative stress, reducing apoptosis, increasing angiogenesis, antimicrobial activity, nerve regeneration, adjunct to spinal fusion, and carpal tunnel surgeries). Nearly universally, PRP outperforms PPP, so clearly platelets are a good thing.

Focusing then within the realm of PRP, the next question is what concentration of platelets is optimal? Humans tend to think that more is better. Following that logic, we would expect the literature to clearly show that higher concentrations of platelets work best. Alas, this is not the case.

Dr James Corey Orava, DVM working in the lab

In Vitro Evidence

Beginning with in vitro studies, there are a variety of published papers looking at cell lines derived from pigs, dogs, horses, and humans. Yoshida et al. performed a study with porcine cruciate ligament fibroblasts. They compared three different concentrations of PRP (low, medium, high). They found the low concentration outperformed the high concentration under all conditions. Choi et al. investigated the effects of seven different concentrations of PRP on the viability and proliferation of canine bone cells.

Their conclusion: “alveolar bone cells are suppressed by high PRP concentrations but are stimulated by low PRP concentrations.” Boswell et al., from Cornell University, evaluated equine tendon explants cocultured in three different PRP formulations. They concluded “too high of a platelet concentration is detrimental to tendon healing.”

Lastly, Mazzocca et al. utilized three commercially available PRP systems (i.e., made three differing concentrations) on three different human cell lines: osteoblasts, tenocytes and myocytes. Similar to other studies, they reported: “a ‘more is better’ theory for the use of higher platelet concentrations cannot be supported.”

In Vivo Evidence

Quite a few in vivo studies have also evaluated the effect of varying platelet concentrations. Beginning in 2011, researchers from Martha Murray’s lab at Harvard (Mastrangelo 2011) utilized two platelet concentrations, 3X and 5X, in conjunction with a collagen scaffold to repair cruciate ligaments in adolescent pigs. While both groups responded well, they “found no evidence that reducing the platelet concentration from 5X to 3X compromises mechanical outcome.”

Continuing this work, researchers from the same lab (Fleming 2015) performed the same basic experiment, this time with three PRP concentrations (1X, 3X, and 5X) and using older pigs. First, they noted that all the PRP groups had better (and equivalent) cartilage scores than the traditional surgical repair. However, they also reported that “ACL reconstruction procedure using the 1X preparation was significantly greater than traditional reconstruction while the 3X and 5X preparations were not.” They noted the lack of response in the higher concentration groups was likely due to the younger animals used in the first study, which are known to repair CCLs better. Another reason for the disparity may be because the 3X and 5X groups used leuko-poor PRP and a recent meta-analysis has shown that PRP with leukocytes works better for tendons.

Filardo et al. published a study comparing a single-spin PRP (1.5X) with a double-spin PRP (4.7X) for people with knee osteoarthritis. They noted both groups showed statistically significant improvement in pain and function at all time points, but the high concentration
group “produced more pain and swelling reaction.” It should be noted that the 4.7X group was also leuko-rich, while the 1.5X was leuko-poor, and a recent systematic review demonstrated that leuko-poor PRP works better for osteoarthritis, so increased platelets may not be the sole responsible component.

Researchers from Colorado (LaPrade et al.) transected the medial collateral ligaments in rabbits and then injected the joint, at the level of injury, with either saline, PPP, 2X PRP, or 4X PRP (all were leuko-poor). They observed that compared to other treatments, the high dose “PRP demonstrated a significant negative effect on ligament strength as well as collagen orientation.”

Also from Turkey in 2020, Aydin et al. saturated Vicryl suture with two concentrations of PRP, 2.7X and 5.1X, and used this for colonic anastomosis. They concluded “platelet concentration of PRP seems to have a significant impact on the outcome with superior efficacy of [low concentration] over [high] in terms of bursting pressures and collagen concentration at the anastomotic site.”

It is important to note that not every study gives the nod to lower concentrations of PRP. In 2020, Yaradilmis et al. from Turkey compared two concentration of PRP with HA in patients with knee arthritis. They reported “PRP injections produced superior results than HA” and the higher concentration “seems to be the most effective.” However, it must be noted that even their high concentration was only 4.7X.

Systematic Reviews/Meta-Analyses

In 2017, in the journal Arthroscopy, Dai et al. published a meta-analysis that evaluated 10 randomized controlled trials involving 1,069 patients. While they concluded that PRP, in general, was more effective than HA in terms of pain relief and functional improvement, when they evaluated the “subgroup of platelet concentrations >5x” they noted HA performed better.

That same year, Milants et al. from Belgium, published a very thorough technical analysis that investigated the use of PRP for osteoarthritis. They reviewed 19 randomized controlled trials involving 1,946 patients. Rather than simply evaluating significant changes in validated outcomes, they utilized a more clinically relevant measure known as Minimal Clinically Important Improvement (MCII). This sets a standard for when an observed difference is clinically relevant to the patient. Further, they set the goal for success as greater than double the MCII.

These were known as the Very Good Responders Group (VGRG), while those studies that did not meet MCII were classified as the Bad Responders Group (BRG). Results have been summarized in Figure 1. The data appear to show that PRP concentrations for osteoarthritis have a sweet spot between 1.5X and 4X. The authors concluded that for osteoarthritis a PRP concentration <5X should be preferred.

CONCLUSION

Despite the widespread utilization of platelet rich plasma, much debate remains. What volume should I inject? How often should I inject? What is the optimal composition of my PRP? While not completely answering this final question, this review has hopefully cleared some of the smoke. In medicine, you can have too much oxygen; you can have too much water; and, it appears, you can have too many platelets. As we continue to gather information, undoubtedly we will fine tune the manner in which we administer PRP. Until then, the current consensus is well summarized by researchers from the Mayo Clinic, and other institutes, who concluded:

Figure 1. Platelet concentrations between various randomized controlled trials. Blue columns were categorized by Milants et al. as Very Good Responders, while Red columns represent Bad Responders. This data appears to represent a concentration ‘sweet spot’ in the 1.5X to 4X platelet range. Note: the study by Napolitano, classified as a Bad Responder, had an average platelet concentration of 2.3X but the actual values varied greatly from 0.17X to 5.23X (dotted bar)

Table 1. Summary of PRP studies that evaluated varying platelet concentrations and their general outcomes.

References

References 

Aydin MA et al. Comparison of Platelet-Rich Plasma-Impregnated Suture Material with Low and High Platelet Concentration to Improve Colonic  Anastomotic Wound Healing in Rats. Gastroent Res Pract 2020, Article ID 7386285. 

Boswell SG et al. Increasing Platelet Concentrations in Leukocyte-Reduced Platelet-Rich Plasma Decrease Collagen Gene Synthesis in Tendons.  Am J Sports Med 2014, 42, 42. 

Cetinkaya RA et al. Platelet-rich plasma as an additional therapeutic option for infected wounds with multi-drug resistant bacteria: in vitro  antibacterial activity study. Eur J Trauma Emerg Surg 2019; 45(3): 555-65. 

Choi BH et al. Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. Int J  Oral Maxillofac Surg 2005; 34: 420-4. 

Dai WL et al. Efficacy of Platelet-Rich Plasma in the Treatment of Knee Osteoarthritis: A Meta-analysis of Randomized Controlled Trials.  Arthroscopy 2017; 33, 3: 659-70. 

Fadadu, PP et al. Review of concentration yields in commercially available platelet-rich plasma (PRP) systems: a call for PRP standardization. Reg  Anesth Pain Med 2019;44:652–659. 

Filardo, G et al. Platelet-rich plasma intra-articular injections for cartilage degeneration and osteoarthritis: single- versus double-spinning approach.  Knee Surg Sports Traumatol Arthrosc 2011; 20(10): 2082-91. 

Fleming, BC et al. Increased Platelet Concentration does not Improve Functional Graft Healing in Bio-Enhanced ACL Reconstruction. Knee Surg  Sports Traumatol Arthrosc 2015; 23(4): 1161-70. 

LaPrade, RF et al. Use of Platelet-Rich Plasma Immediately After an Injury Did Not Improve Ligament Healing, and Increasing Platelet  Concentrations Was Detrimental in an In Vivo Animal Model. Am J Sports Med 2018; 46(3): 702-12. 

Liao JC. Positive effect on spinal fusion by the combination of platelet-rich plasma and collagen-mineral scaffold using lumbar posterolateral fusion  model in rats. J Orthop Surg Res 2019; 14: 39. 

Mastrangelo, AN et al. Reduced platelet concentration does not harm PRP effectiveness for ACL repair in a porcine in vivo model. J Orthop Res.  2011; 29(7): 1002–7. 

Mazzocca, AD et al. The positive effects of different platelet-rich plasma methods on human muscle, bone, and tendon cells. Am J Sports Med 2012;  40(8): 1742-9 

Milants, C et al. Responders to Platelet-Rich Plasma in Osteoarthritis: A Technical Analysis. BioMed Research International 2017, Article ID 7538604. 

Song D et al. Effect of platelet-rich and platelet-poor plasma on peri-implant innervation in dog mandibles. Int J Implant Dent 2019; 5: 40. 

Trull-Ahuir et al. Efficacy of platelet-rich plasma as an adjuvant to surgical carpal ligament release: a prospective, randomized controlled clinical  trial. Sci Rep 2020;10: 2085. 

Yang W et al. Platelet-rich plasma protects HUVECs against oX-LDL-induced injury. Open Med 2018; 13: 41-52. 

Yaradilmis YU et al. Comparison of two platelet rich plasma formulations with viscosupplementation in treatment of moderate grade gonarthrosis:  A prospective randomized controlled study. J Orthop 2020; Jan 28;20:240-246. 

Yoshida, R et al. Increasing Platelet Concentration in Platelet-Rich Plasma Inhibits Anterior Cruciate Ligament Cell Function in Three Dimensional Culture. J Orthop Res 2014; 32(2): 291-5