White blood cells in platelet rich plasma, the good, bad, and ugly?

From: P. A.M. Everts: Autologous platelet-leukocyte enriched gel. Basics and efficacy 1.
The literature is not always clear on definitions and effects of white blood cells in PRP products. Since the initial use of PRP in the 90's, the presence, and functions of leukocytes in PRP has been greatly underestimated. The literature still does not give a clear direction on their roles in the various healing processes. The PRP literature, addressing leukocytes, can be sometimes very confusing when pointing to certain PRP formulation for specific indications, like osteoarthritis. The scientific conclusions from these papers are contradicting. I mention this in regard to studies, by the Rizzoli Institute in Bologna Italy (Kon, Filardo, et. al.)2, by Yin et al3, and Dragoo and co-workers from of Stanford University4. This might also be partially the reason why a true, and generally accepted, PRP classification system is not yet in place, creating more confusion among practitioners. Most strikingly, in many papers it is even not clear what "kind" of PRP has been used.

Background on white blood cells.

Let me first clarify "leukocytes", a.k.a. white blood cells (WBC's). The word leukocyte origins from Greek leukos, white, and kytos, cell. WBC's cell develop from the haemopoietic cell line, mainly in the bone marrow, and mature in several differentiation pathways: myeloblast finally into granulocytes, lymphoblast into lymphocyte, and monoblast into monocyte5. Leukocytes are divided into granular (neutrophils, eosinophils, basophils) and a-granular cells (lymphocytes and monocytes). We don't care to much for the eosinophils and the basophils in the PRP, since they are very fragile and do not survive the centrifugation process.

Leukocytes have clear duties in healing processes.

The interactive healing process involves mediators, extracellular matrix components, resident cells, including platelets, and infiltrating leukocytic cells, who all participate in classic pathway of wound healing: hematoma, inflammation, tissue formation, and ultimately tissue remodeling. The neutrophils have a clear function in executing healing since they form a dense barrier against invading pathogens and counteract against infections6. Macrophages, originating from monocytes, secreting collagenase and transforming growth factor, therefore playing a key role in wound repair, and inflammatory control7. Lymphocytes produce insulin like growth factors, and they may contribute to tissue remodeling8. Furthermore, leukocytes are also well-known for their roles in immune and host-defense mechanism, this I will discuss at a later moment.

The main focus in the PRP literature was on platelets.

It is only since a few years that authors start to discuss the role of the WBC's in PRP. in particular in treating osteoarthritis (OA). During the first years, publications were addressing platelets, what is the ideal platelet concentration, how often should we inject, how much volume, where should we inject, do we need platelets at all? All very relevant questions, and the answers are still not conclusive. However, there is a tendency towards some kind of opinion that WBC's might also play a role to achieve a positive result. In particular, the neutrophils are thought to have a detrimental effect on cartilage and synoviocytes. This resulted in the creation of more undefined nomenclature. Neutrophils have been called bad, and for convenience purposes, in many peer-reviewed paper, all the leukocytes are bad, and new product definitions have been called for.

Literature starts to point to new terminology: L-PRP/ LR-PRP/LP-PRP/P-PRP.

The literature added more unclear definitions to the existing tables of nomenclature with regard to product definitions.  It is often unclear what these definitions really mean, since rarely any laboratory analysis's are performed stating the true content of any PRP formulations used. It should be strongly advised that, at least in research and patients outcome studies, the biological injection, or application, is supported by adequate laboratory analysis in order to objectively quantify the bio-cellular activity of the injectate or graft, and thus effecting outcomes.

Food for thought: Define proper PRP protocols.

If neutrophils are considered to be detrimental in any PRP indicated therapy, then use a PRP formulation that does not include neutrophils, while maintaining other, valuable, WBC's that clearly contribute to healing or tissue regeneration, like e.g. monocytes9.

EmCyte PurePRP® protocols gives you the option to choose a neutrophil-poor protocol, at the completion of this preparation protocol in which 98% of pro-inflammatory neutrophilic granulocytes are eliminated. A neutrophil-rich protocol can be used, also low erythrocyte concentrations, but the PRP is enriched with neutrophilic granulocytes. This protocol can be used when a significant inflammatory reaction is desired, in order to facilitate a strong leukocytic chemotaxis, to induce a phagocytic response.

In intra-articular PRP injections for OA, clinicians should understand the effects of any PRP formulation affecting, pro-inflammatory mediator production10 (TNF?, IL-1?, IL-6, IL-15, IL-17, IL-18, IL-10, IL-13), cell death, and the role of the various platelet derived growth factors in potentially the prevention and preservation of the cartilaginous matrix11.

The leukocytes have a great impact on the intrinsic biology of PRP.

Although the presence or absence of leucocytes in PRP preparations was previously neglected, in the last years more attention has been paid to their role, and several studies have been conducted to define their immune and metabolic effects in PRP. I think it is fair to state the WBC's are crucial plasma components of (wound) healing processes, and we need to identify which WBC's we need in PRP, in which pathophysiological condition.  The role of leukocytes in PRP requires further investigations, but, first of all, the presence and mechanisms of these cells must not be neglected, or forgotten anymore in any application.  A similar, overlooked, phenomena of PRP, are the PRP antimicrobial-bactericidal properties.  I will address these performance characteristics in a later blog.

Leukocytes in PRP: "The good, the bad, and the ugly"? You tell me.



References

  1. Autologous platelet-leukocyte enriched gel. Basics and efficacy. A novel method to support soft tissue and bone healing. Everts, PAM. 2007: ISBN 10: 90-8590-016‐6.
  2. Leukocyte-Rich Platelet-Rich Plasma Injections Do Not Up-Modulate Intra-Articular Pro-Inflammatory Cytokines in the Osteoarthritic Knee. Erminia Mariani, Valentina Canella, Luca Cattini, Elizaveta Kon, Maurilio Marcacci, et al. PLOS. 2016; 11: 1-13.
  3. Advantages of Pure Platelet-Rich Plasma Compared with Leukocyte- and Platelet-Rich Plasma in Treating Rabbit Knee Osteoarthritis. Yin, Xu, Sheng, An, Guo, et al. Med Sci Monit. 2016 Apr 17;22: 1280-90.
  4. The Effect of Platelet-Rich Plasma Formulations and Blood Products on Human synoviocytes: Implications for Intra-Articular Injury and Therapy. Braun, H., Kim, H., Chu, C., Dragoo, J. L.; American J. Sports Med.2014: 42(5), 1204-1210.
  5. The Role of Leukocytes from L-PRP/L-PRF in Wound Healing and Immune Defense: New Perspectives. Bielecki T., Dohan Ehrenfest D., Everts PA, Wiczkowski A. Curr Pharm Biotechnol. 2012 Jun;13(7):1153-62.
  6. Active participation of endothelial cells in inflammation. Cook-Mills, J.M.; Deem, T.L. J. Leukoc. Biol., 2005, 77(4), 487-495.
  7. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Leibovich, S.J.; Ross, R. Am. J. Pathol., 1975, 78(1), 71-100.
  8. Immortalized B lymphocytes produce B-cell growth factor. Gordon, J.; Ley, S.C.; Melamed, M.D.; English, L.S.; Hughes-Jones, N.C. Nature, 1984, 310(5973), 145-147.
  9. Monocytes and macrophages in tissue repair: Implications for immune-regenerative biomaterial design. Ogle, M. E., Segar, C. E., Sridhar, S.,Botchwey, E. A. ; Experimental Biology and Medicine. 2016:241(10), 1084-1097.
  10. The Role of Inflammatory and Anti-Inflammatory Cytokines in the Pathogenesis of Osteoarthritis. Wojdasiewicz P,Poniatowski A, SzukiewiczD. Mediators Inflamm. 2014;56: 1459-1468.
  11. Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo. Davidson RK, Jupp O, de Ferrars R, et al. Arthritis Rheum, 2013; 65: 3130–40.

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