Introduction
Whole Blood and Platelets
Platelet-rich plasma (PRP) was first used in cardiac surgery by Ferrari and colleagues in 1987 as an autologous blood transfusion after open heart procedures to avoid homologous blood product transfusions. The use of PRP to treat musculoskeletal disorders (MSK-D) was noted in the mid-1990s for tendinosis and tendinitis. Currently PRP is being utilized by providers in multiple specialties, including maxillofacial surgery, spinal disorders, orthopedics, chronic wounds, and more. During the physiologic cascades of soft tissue regeneration, a variety of cellular, hormonal, and growth factors play pivotal roles and are stored in the alpha and dense granules of platelets.
Platelets are formed from megakaryocytes in the bone marrow by pinching off from their progenitor cells before being released into the peripheral circulation. Platelets are small, anucleate, discoid blood cells (1 to 3 μm), with an in vivo half-life of 7 days, and the average platelet count in adults ranges from 150 to 350 × 10 6 /mL of circulating blood.
Since platelets are an integrated part of whole blood, mimicking and accelerating physiologic tissue healing and regeneration, harvesting a calculated amount of fresh blood is mandatory to acquire platelets and eventually other whole blood constituents.
Medical and US Food and Drug Administration (FDA)-approved point-of-care devices can fractionate a unit of autologous whole blood into various components and formulations, consisting of platelet-poor plasma, platelets, leukocytes, and red blood cells (RBCs).
Phlebotomy Procedure
Administrative duties, proper patient identification, and informed consent precede a blood harvesting procedure, with proper labeling of syringes. Patients can be placed in a comfortable seated or recumbent position. Sterile and single-use needles and syringes are used, and blood is generally drawn by phlebotomy with a venous catheter ranging from 18 to 21 gauge in adults. The practitioner responsible for harvesting blood should maintain proper local guidelines and use personal protective equipment (PPE). Several PRP manufacturers produce complete PRP kits that include processing devices, syringes ( Fig. 6.1 ) and in most kits an anticoagulant and a phlebotomy kit (including tourniquet, butterfly assembly, gauzes, alcohol pad, labels) ( Fig. 6.2 ).
The whole blood collection syringe should contain an anticoagulant to avoid blood clotting during blood harvesting and PRP preparation, thus preventing platelet activation prior to its use. Manufacturer instructions provide guidelines on loading sufficient anticoagulant in the harvesting syringe.
Vein Assessment
Assessment of the patient prior to phlebotomy to locate an adequate vein is advised. In most instances the median antecubital vein is preferred by practitioners, but care should be taken in this area due to the close proximity of the median nerve. For the blood draw, it is important to select a large and stable vein in the antecubital fossa, with the cephalic and basilic vein connected by the median antecubital vein ( Fig. 6.3 ) . Another common option is the base of the cephalic vein, located over the wrist joint proximal to the thumb (note that the superficial radial nerve is close to this area). In muscular patients, the cephalic vein is more superficial near the elbow in the antecubital fossa, presenting as the median antebrachial vein. Veins at the back of the hand ( Fig. 6.4 ) are more peripheral veins and are more suitable for intravenous access to inject medication than for blood harvest. Supportive technology such as ultrasound devices or vein visualization systems can be used to map and access veins when not clearly visible/palpable on visual inspection.
Tourniquet and Aseptic Technique
A tourniquet is placed to fill the patient’s venous vascular system. Prior to venipuncture, aseptic techniques are used for skin preparation. Single stick draws are preferred to decrease chances of activation and patient discomfort. PRP systems that minimize exposure of blood and cellular components to open air, allowing for minimal manipulation of tissue, are mandatory as per FDA guidance.
Venipuncture
Before performing the venipuncture, flush the butterfly assembly with the anticoagulant in the syringe to avoid clotting. Pull the skin taut to anchor the vein, and insert the needle bevel up, entering the skin at a shallow angle of 5 to 15 degrees. Direct the needle into the vein from its top or side surface ( Fig. 6.5 ). A slight resistance is felt as the needle passes through the dense skin tissue and vein wall. A slight ease in resistance is noted when the needle enters the lumen of the vein. If the blood does not flow, reposition the needle; if the blood is flowing slowly, adjust the angle of the needle, as it may be sitting against the wall of the vein.
Blood Aspiration
Pull slowly back on the plunger of the collection syringe in a controlled fashion at a rate of approximately 1 mL/s to prevent the vein from collapsing and inducing too much negative pressure, which can cause cell damage. During aspiration, agitate the blood in the syringe with the anticoagulant twice (by moving an air bubble backward and forward) to assure proper mixing, as the whole blood is more dense than the anticoagulant and will not be properly anticoagulated otherwise during the blood draw, potentially initiating clotting within the central part of the syringe ( Fig. 6.6 ) .
When the required amount of blood is obtained the tourniquet can be released, the syringe is disconnected, and a sterile cap is placed. Remove the butterfly, or other needle assembly, and apply a folded gauze to the site as a pressure bandage. Invert the syringe clockwise-to-counterclockwise five times to mix the anticoagulant and blood more completely ( Fig. 6.7 ).
Contaminated Materials
Dispose of sharps and contaminated materials in designated medical waste containers.
Adverse Events and Complications Following Phlebotomy
Pain and Ecchymosis
Adverse events and complications following a phlebotomy are rare. However, a poorly executed phlebotomy can cause adverse effects for patients, ranging from pain or ecchymosis at the site of puncture, nerve damage, and hematoma. Transient localized pain and localized ecchymosis are common and improve with time. Occasionally, patients may experience a vaso-vagal reaction with associated light-headedness, hypotension, nausea, and sweating. This is treated with laying the patient back, oral fluids, and the passage of time with reassurance of the patient. Pulse and blood pressure should be checked until they normalize and then the patient should be slowly brought to a seated, and then upright, position. The more severe adverse events that have been documented in blood transfusion services usually involve poor venipuncture practice or the presence of anatomic variants resulting in hematoma and injury to anatomic structures in the vicinity of the needle entry.
Infection
Poor infection-control practices can lead to bacterial infection at the site where the needle was inserted into the skin. This is an extremely rare event. Such an infection could ascend more proximally in the venous vascular system and develop into a septic thrombophlebitis, which needs to be managed according to local protocols, potentially with culture-directed antibiotics. Furthermore, healthcare workers can be exposed to bloodborne pathogens from needlestick injuries.
Paresthesia
In rare cases post-venipuncture paresthesias are observed, mainly due to edema at the site of the needlestick rather than a nerve injury. This should resolve within 3 weeks. Patients with longer-lasting paresthesia can be referred to a neurologist or electromyographer for further assessment.
Arterial Puncture
Another potential complication is an arterial puncture. This would be apparent by the spontaneous appearance, or withdrawal, of bright red blood in the syringe. This is managed by quick and direct pressure on the area and monitoring for any local expansion and/or change in pulse distally.
Possible Factors Influencing the Quality of Platelet-Rich Plasma
Blood Draw Anatomic Site and Time
Waters and Roberts discussed several possible factors that have an effect on the quantity and quality of platelets in aspirated whole blood intended for PRP preparation. The authors concluded that the duration of the blood draw and manner in which the blood was drawn shows a relationship between the platelet count in the PRP prepared. Furthermore, the PRP platelet concentrations were also influenced by the whole blood harvesting site. Optimal platelet counts were obtained when blood was drawn from a peripheral vein as compared to an arterial collection site or a central venous line.
Shear Stress
The use of small-gauge harvesting needles, excessive pull force, and venipuncture of small blood vessels increase shear forces applied to blood components and will result in platelet activation and RBC lysis, resulting in plasma-free hemoglobin. Under normal operational procedures and avoiding high shear forces, PRP-platelet function is preserved ( Fig. 6.8A ) . Intact platelets maintain the alpha granules integrity, containing a variety of platelet-derived growth factors. Blood draw from small hand veins that have tendency to collapse during aspiration and the use of small needles lead to high shear forces at the point of the needle, resulting in premature platelet activation ( Fig. 6.8B ). The release of platelet contents, including the alpha granules platelet growth factors, result in the platelets losing functionality.
