Hielscher Ultrasound Technology

Ultrasonic Preparation of Platelet Rich Serum

  • Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets, which is used for medical and estethic treatments.
  • Ultrasonic lysis and conditioning is an very effective method to prepare platelet-rich plasma.
  • Hielscher Ultrasonics offers various ultrasonic systems matching the requirements of the blood treatment (e.g. for inside and outside preparation of a clean room).

Platelet Rich Serum

Sonication is a reliable and easy method to lyse the platelets in autologous blood serum in order to release the growth factors. The platelet-derived fractions – also known as autologous conditioned serum (ACS) – are used for tissue repair or aesthetical treatments. To obtain optimal results, the PRP is enriched prior to injection.
In order to prepare enriched plasma with cell growth factors, sonication is a very effective and efficient method to lyse the cell membranes and to release almost the complete cell growth factors from the platelets. By ultrasonic lysis and conditioning, even anti-coagulated blood can be used.
Conditioning of PRP via sonication is a convenient and cost-effective way to yield in concentrated growth factors.

Ultrasonic preparation of platelet-rich plasma (PRP) © Fortunato, T. M. et al. (2016)

Ultrasonic lysis of platelet-rich plasma (PRP)

Ultrasonic Lysis Effects

Ultrasonic treatment of blood serum results in a homogeneous lysis of the platelets in order to release the entrapped growth factors (growth factor AB (PDGF-AB), growth factor AA (PDGF-AA), growth factor BB (PDGF-BB), vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), epidermal growth factor (EGF), insulin-like growth factor (IGF), epithelial cells growth factor ECGF and fibroblastic growth factor (FGF)). The platelets and their alpha-granules are disrupted by sonication, using the phenomenon of acoustic cavitation. Using ultrasonic cavitation for platelet lysis diminishes the possible negative impact on the growth factor activity of the repeated freeze-thaw method.

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Protocol for Ultrasonic PRP Preparation

Probe-type insonifier UP200St for lysisApprox. 16mL of fresh collected autologous blood sample is gently inverted 8-10 times in a tube so that the blood and anticoagulants are well mixed. Afterwards, the sample is centrifuged at 1800 g for 20 min at room temperature. After the centrifugation, the platelets and mononuclear cells build a whitish layer at the bottom of the plasma layer, which is known as platelet-rich plasma (PRP). The upper layer is known as platelet-poor plasma (PPP). The mononuclear cells can be removed or remain in the sample. Often, the mononuclear cells remain in the PRP sample to avoid additional steps, which bear the risk of contamination.
Note: The ultrasonic treatment of autologous PRP must be carried out under sterile conditions.
PRP and PPP are separated and collected. To the PPP, 1.5mL 10% calcium chloride solution is added, whilst the to PRP 0.5mL 5% ethanol is added. Both suspensions are gently agitated and allowed to stand for 5 min at room temperature. Finally, 1mL PPP is added to the PRP suspension and is allowed to stand until a fibrin clot forms.
Ultrasonic treatment: As the formed platelet gel acts as a reservoir for the released growth factors, the fibrin gel must be disrupted in order to release the available growth factors. The parameters for the ultrasonic lysis and growth factor release are the following: Sonicate with an UP200Ht or UP200St (200W, 26kHz) with sonotrode S26d2 the sample in the tube. To avoid overheating of the blood sample, sonication can be performed in pulsing cycles (e.g. 10 sec. on, 30 sec. off). The sample should be placed on ice for cooling so that the temperature is optimally kept between 2 – 5°C. After sonication, the sample is centrifuged at 16000 g for 5 min. at room temperature. The supernatant is collected and can be stored at -70°C.
The platelet concentration in the PRP fraction was enriched 1.7 – 4.5 fold in the sonicated method when compared to the literature average platelet count in whole blood.

PRP has been combined with adipose-derived stem cell therapy (ultrasonically released autologous stem cells from adipose tissue, mixed with PRP, and injected back into the patient’s tissue for repair).

Ultrasonic Equipment

The ultrasonic device VialTweeter allows for simultaneous sample preparation of up to 10 vials under same process conditions. (Click to enlarge!)Hielscher Ultrasonics is expert for sophisticated and reliable ultrasonic technology. Our ultrasonic devices are used worldwide for lysis, extraction and sample preparation – by direct and indirect. Autoclavable as well as disposable accessories ensure a contamination-free treatment of autologous blood samples. Most of our systems can be used for ultrasonic lysis of stem cells and PRP, which makes our ultrasonic devices economically even more efficient.
This table below lists our ultrasonic devices, which are suitable for the preparation of platelet-rich plasma, and their recommended sample volume, respectively. Larger ultrasonicators and customized systems are available, too.

Device Power [W] Freq. [kHz] Type Volume [mL]
VialTweeter 200 26 stand-alone 0.5 1.5
UP50H 50 30 handheld or standmounted 0.01 250
UP100H 100 30 handheld or standmounted 0.01 500
UP200Ht 200 26 handheld or standmounted 0.1 1000
UP200St 200 26 standmounted 0.1 1000
UP400St 400 24 standmounted 5.0 2000
UP200St-SonoStep 200 26 stand-alone 30 500
GDmini2 200 26 contamination-free flow cell

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GDmini2 at UP200St-TD Transducer (200 watts) for contamination-free sonication

GDmini2 at UP200St-TD Transducer (200 watts)

Literature/References

  • Fortunato, T. M.; Beltrami, Ch.; Emanueli, C.; De Bank, P.A:; Pula, G. (2016): Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications. Sci. Rep. 6, 2016.
  • Hamid, M.S.A.; Yusof, Ashril; Ali, Mohamed Razif Mohamed (2014): Platelet-Rich Plasma (PRP) for Acute Muscle Injury: A Systematic Review. PLOS ONE Volume 9, Issue 2, 2014.


Facts Worth Knowing

Platelet Rich Plasma

Platelet-rich plasma contains growth factor AB (PDGF-AB), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor BB (PDGF-BB), vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), epidermal growth factor (EGF), insulin-like growth factor (IGF), epithelial cells growth factor ECGF and fibroblastic growth factor (FGF), which are important stimulants for cell growth and renewal.

Autologous Conditioned Serum (ACS)

Autologous Conditioned Plasma (ACP) is a platelet-rich plasma that is extracted from autologous blood which is used to support regeneration in a variety of orthopedic conditions and surgical procedures. Conditioned serum is prepared by separating the plasma from the other blood components (such as erythrocytes), and concentrate it. ACP is a platelet-rich plasma (PRP). PRP is a general term for a type of plasma that contains a significantly higher concentration of thrombocytes when compared with whole blood. It is extracted from whole blood. Main components of ACP include thrombocytes (platelets), and several growth factors that play an important role in the healing process. Unlike other platelet-rich plasma formulations, ACP is distinguished by a low concentration of white blood cells such as neutrophil granulocytes that can be detrimental to the healing process when present in high concentrations.

Platelet-Rich Plasma Injections

Platelet-rich plasma is used as injection in regenerative medicine as well as in cosmetic procedures. The application of PRP promises significant acceleration of muscle healing and muscle recovery. Furthermore, it is successfully used to treat inflammation, arthritis, pain, wounds, slow healing open wounds, and serious skin burns. The PRP contains a high level of growth factors, which stimulate and protect the healing cells ao that the damaged tissue is repaired.
For cosmetic treaments, PRP has been shown effective to stimulate hair growth, skin renewal (also known as vampire treatment).

Platelet-Derived Growth Factors (PDGF)

The term platelet-derived growth factor (PDGF) refers to the factors PDGF-A, PDGF-B, PDGF-C and PDGF-D. It is a signalling network of four ligands (PDGF-A, PDGF-B, PDGF-C and PDGF-D), and two receptors, PDGFR-alpha and PDGFR-beta.
Growth factors, or proteins, regulate cell growth and division. As the principal mitogen found in mammalian serum, which is released from platelets during clot formation, it plays a significant role in blood vessel formation (angiogenesis), the growth of blood vessels from already-existing blood vessel tissue.