Assessment of Quality of Platelet-Rich Plasma Produced With Desktop Centrifuge and Comparison With Standardized Commercially Available Platelet-rich plasma.

AIMS: The aim of this study was to assess the quality of platelet-rich plasma (PRP) produced by in-house desktop centrifuge method and compare it with that of standardized commercial PRP.
MATERIALS AND METHODS: REMI desktop centrifuge was used to prepare PRP and to compare with standardized commercial PRP by calculating the quantity of platelets using Beckman Coulter cell counter in 10 PRP samples and assessing the morphological quality of platelets using JEOL JEM transmission electron microscope (TEM). STATISTICAL ANALYSIS USED: The t test for platelet count in desktop PRP with the test value of therapeutic PRP was 12.618. The P value was <0.001, which was significant statistically. The data followed a normal distribution in normal Q-Q plot for platelet count in desktop centrifuge. So the test samples were not much deviated.
RESULTS: The platelet count was lesser than that of standardized commercial PRP. When viewed under JEOL JEM transmission electron microscope, the α granules in platelets were intact and the morphological quality of the PRP was good.
CONCLUSIONS: With this study, we have determined that the morphological quality of PRP produced by the in-house desktop centrifuge method is comparable to that of standardized commercial PRP. Though the quantity of platelets was less than 1 million cells/μL, the clinical results were good with desired bone formation, thereby providing good avenue for further research. Copyright:

INTRODUCTION

Platelet-rich plasma (PRP) was introduced by Whitman and Berry in 1997.[1] According to Marx et al.[2] in 1998, mixing PRP with autogenous bone resulted in the release of growth factors, producing denser bone regeneration and radiographic maturation within a short time. Since then quality and efficacy of various PRP preparations have been analyzed by various authors using different methodologies.[234]

The α granules of the platelets contain growth factors[5] such as vascular endothelial growth factor, platelet-derived growth factors, and transforming growth factor-β.[6]

Since 2002, we have developed a technique based on vacutainer system as described by Sonnleitner et al.[7] in 2000, to make PRP and have used it successfully in many cases. The standard techniques for evaluating the quality of PRP are by depicting the growth factor levels in PRP[56] and quantity by assessing the platelet concentration[2] and platelet count using cell counters.[8]

MATERIALS AND METHODS

This study was aimed to evaluate the quality of desktop PRP by calculating the amount of platelets present in the collected plasma and assessing the morphological quality of α granules in the platelets. We have used JEOL JEM 100 SX (JEOL, Akishima, Tokyo, Japan) Transmission Electron Microscope (TEM) and Beckman Coulter cell counter Ac-T 8 (Beckman Coulter, Pasadena, CA) series for the analysis.

Blood samples were taken from 10 patients who were healthy and need PRP gel[9] or PRP sheets to fill the bony cavities as in case of cyst, multiple impacted teeth orthognathic, surgeries etc.[101112131415] PRP samples were also taken from 10 volunteers. The PRP obtained by this procedure is analyzed for quality and quantity.

Harvesting platelet-rich plasma

Autogenous venous blood was centrifuged using desktop centrifuge [Figure 1] at 1200rpm for 10min [Figure 2], which resulted in three layers: the blood cell concentrate, buffy coat, and supernatant layer of platelet-poor plasma (PPP) [Figure 3]. The buffy coat or PRP[16] extends 6–8 mm into blood cell component layer. Using Eppendorf micro pipette, the supernatant layer and buffy coat including its extensions into the blood cell component layer were pipetted into another test tube [Figure 4] and centrifuged again. The PPP was pipetted out and the PRP was isolated [Figure 5]. The PRP was mixed with calcium chloride resulting in the formation of PRP sheet [Figure 6] and PRP gel [Figure 7]. PRP gel was then mixed with autologous bone graft and placed into the surgical defect.[9] The cell counter used for counting platelets is Beckman Coulter cell counter Ac-T 8 series. The plasma samples were then processed and the ultrathin sections obtained were viewed at 60 or 80kV using JEOL JEM 100 SX TEM.

Figure 1

REMI desktop centrifuge used to obtain platelet-rich plasma

Figure 2

The collected blood was centrifuged at 1200rpm for 10 min

Figure 3

The centrifuge resulted in three layers—the blood cell concentrate, buffy coat, and supernatant layer of platelet-poor plasma

Figure 4

The buffy coat was pipetted into another test tube and centrifuged again

Figure 5

Platelet-rich plasma obtained from REMI desktop centrifuge

Figure 6

Calcium chloride was added to platelet-rich plasma in concentration of 1:10 to form platelet-rich plasma sheet

Figure 7

Platelet-rich plasma gel

RESULTS

The transmission electron microscopic study showed that the α granules in platelets of desktop PRP obtained with REMI table top centrifuge using Sonnleitner et al.[7] technique were intact without loss of growth factors.

The platelet count in the PRP produced using REMI desktop centrifuge, counted using Beckman Coulter cell counter Ac- T 8 series, was less with a mean value of 0.45 million cells/µL.

DISCUSSION

The healing of both soft and hard tissues was accelerated by PRP.[17] However, the clinical efficacy of PRP varies depending on the preparation method and the present methods to evaluate efficacy of PRP systems have several limitations.[181920]

According to Marx,[4] platelet membrane integrity is of paramount importance because fragmented platelets may spill more growth factors and that their tertiary structure is altered resulting in decreased clinical efficiency.

The morphological quality assessment of the PRP produced by our method was carried out using TEM, which showed that the platelets were intact and α granules were also intact [Figure 8]. So the PRP produced by desktop centrifuge method was of good quality.

Figure 8

Assessment of the platelet-rich plasma was performed using transmission electron microscope, which showed that the platelets were intact and α granules were also intact

According to Marx,[4] therapeutic PRP should contain 1 million platelet cells/µL or 400% of the peripheral blood platelet count as measured with Beckman Coulter cell counter.[4]

The platelet count in 10 PRP samples counted using Beckman Coulter cell counter showed the PRP count was lesser than therapeutic range [Graph 1]. The statistical analysis showed that the t test [Table 1] for platelet count in desktop PRP with the test value of therapeutic PRP is 12.618. The P value was significant statistically (< 0.001) [Table 2]. In the normal Q-Q plot for platelet count in desktop centrifuge, the data followed a normal distribution and the test samples were not much deviated [Graph 2].

Graph 1

The platelet count in desktop platelet-rich plasma was lesser than therapeutic range

Table 1

The t test for platelet count in desktop PRP with the test value of therapeutic PRP was 12.618; the P value was < 0.001, which was statistically significant

T. TEST
One-sample statistics
	N	Mean	Std deviation		Std.Error mean
Platelet count in desktop PRP	10	440400.00	140242.013		44348.418
One-sample test
		Test value = 1000000
	T	DF	Sig (2-tailed)	Mean difference	95% Confidence interval of the difference
					Lower	Upper
Platelet count in desktop PRP	12.618	9	.000	-559600.00	-659923.09	-459276.91

Table 2

In the normal Q-Q plot for platelet count in desktop centrifuge, the data followed a normal distribution. So the test samples were not much deviated

Explore descriptives
			Statistic	Std. Error
Platelet count in desktop PRP	Mean		440400.00	44348.418
	95% Confidence interval for mean	Lower bound	340076.91
		Upper bound	540723.09
	5% Trimmed mean		439888.89
	Median		420000.00
	Variance		19667822222.222
	Std. Deviation		140242.013
	Minimum		264000
	Maximum		626000
	Range		362000
	Interquartile range		274500.00
	Skewness		.063	.687
	Kurtosis		-1784	1.334

Graph 2

In the normal Q-Q plot for platelet count in desktop centrifuge, the data followed a normal distribution and the test samples were not much deviated

The platelet count was less when compared with the commercial PRP [Graph 3] produced with Smart-Prep, PCCS,Curasan[22] or Placon type of centrifuge as given by Marx,[4] Weibrich et al.,[222324] and Eby.[25]

Graph 3

The platelet count of desktop platelet-rich plasma (PRP) was less when compared with the commercial PRP

The reason for this may be the cell counter what we used was not as effective as that used in counting commercial PRP. We have used Beckman Coulter counter Ac-T 8 series instead of Cell-dyn 3700 where clumps of platelets may have been counted as individual platelet. But the morphological quality was good when viewed under JEOL JEM TEM. The platelets and α granules were intact, which showed that there was no spillage of growth hormone during the preparation, which may reduce the effectiveness of PRP. Tsay.C.R (2005) shows PRP prepared with thrombin results in a large, immediate release of growth factors, which could be lost into the interstitium in vivo. While thrombin receptor activator peptide(TRAP) and bone substitutes are more efficacious in sustaining growth factors and bone formation.²¹

CONCLUSION

Tsay.C.R (2005) shows PRP prepared with thrombin results in a large, immediate release of growth factors, which could be lost into the interstitium in vivo. While thrombin receptor activator peptide(TRAP) and bone substitutes are more efficacious in sustaining growth factors and bone formation.[²¹] With this study, we have determined that the quality of PRP produced by the in-house desktop centrifuge method is comparable to that of standardized commercial PRP. Though the quantity of platelets as assessed by the Cell Dyne 3700 is more accurate than the Beckman Coulter cell counter Ac-T 8 series, the clinical results were good, providing good avenue for further research. The morphological quality of the alpha granules assessed using JEOL JEM TEM is a good method for evaluation and standardization of in-house PRP as it is important to preserve the platelet membrane integrity which relates to its ability to release growth factors. Fragmented platelets may spill more growth factors into solution and therefore their effectiveness are lessened.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.