Concentration Of Platelets In Plasma Research Articles

The effect of near-infrared low-level light on the in vitro quality of platelets during storage.

Near-infrared (NIR) light has been successfully applied to improve the quality of mouse platelets during storage. Because it is suspected that the mitochondria contain the primary photon acceptor, we hypothesized that human platelets for transfusion may be affected similarly and could benefit from NIR light treatment. The optimal light dose was determined using portions of platelet concentrates (PCs) in PAS-E. A pool-and-split design was used to prepare PCs in PAS-E or plasma (n = 6). On day 1, one unit of both pairs was illuminated with 830 nm light (light-emitting diodes, 15 J/cm2). PCs were stored at 22°C and sampled regularly for analysis. Data were compared with their corresponding controls with a paired two-sided t-test. Illuminated platelets in PAS-E were less activated with significantly lower CD62P expression (day 8: 10.8 ± 1.8 vs. 12.2 ± 2.6, p < 0.05) and lower Annexin A5 binding (day 8: 11.8 ± 1.9 vs. 13.1 ± 2.4, ns). They produced significantly less lactate resulting in a higher pH (days 6-10). ATP content and mitochondrial membrane potential were not affected. Although these trends were also observed for PCs in plasma, the differences did not reach statistical significance as compared with the control group. Our study demonstrates that the glycolysis rate of human platelets can be modulated through the use of NIR, possibly through mitochondrial aerobic metabolism, but this requires confirmation. If NIR illumination can be further optimized, it may potentially become a useful tool in situations in which glycolysis and platelet activation are exacerbated.

Predicting Platelet Age Using Artificial Intelligence Techniques

Background: Platelets are small, enucleated cells with their primary physiological role to repair vascular damage (hemostasis) and initiate thrombus formation in response to vascular injury. Low platelet count (thrombocytopenia) is associated with increased bleeding risk and is caused by decreased platelet production and/or increased platelet clearance. Platelet transfusion is a routinely used lifesaving procedure to control or prevent bleeding in patients with thrombocytopenia or platelet dysfunction. During storage platelet function deteriorates significantly over time and regulation of platelet granular content is essential in maintaining a healthy platelet function. Currently no in vitro tests are available to predict platelet age, which may be used to diagnose the underlying cause of thrombocytopenia, or to determine storage time of platelet concentrates (PC). Using confocal and super-resolution microscopy we could quantify loss of platelet granule content during aging. In this study we used Artificial intelligence techniques (AIT) to derive exact platelet age from imaging data. Aim: To develop AIT-based algorithms that can determine platelet age from images of platelets stored in plasma-rich-plasma (PRP) and in platelets concentrates (PC) measured by confocal microscopy. Methods: Platelets were stored in PRP for 8 hours at room temperature. Platelets were isolated and stained with antibodies for alpha-tubulin, von Willebrand Factor (VWF), SPARC or alpha-tubulin and imaged at T=0, 4 and 8 hours using confocal microscopy. Additionally 3 single donor PC in plasma were stored for 10 days at standard blood bank conditions. Platelet samples were pooled and analyzed at day 1,2,3,6,7,8,9 and 10. We trained a neural network consisting of multiple convolutional layers to classify age (time after donation) of platelets that were stored in either PRP or PC. Results: During storage in PRP as well as of PC we used for each time point 1334 62µm x 62µm x 4µm 3D images, containing 50 to 150 platelets per image, for training. When staining with markers VWF, SPARC or alpha-tubulin alone as training set we could already classify mean platelet age up to 78%, 85%, 88% respectively. When combining all three markers a maximum accuracy of 95% for platelets stored in PRP up to 8 hours was reached (Figure 1). The additional effect of combining these three markers highlights that the model used information in the images synergistically. For PC, an accuracy of 90% was reached for classifying mean platelet age of platelets stored up to 8 days. The model could not be trained to discriminate between days 9 and 10 resulting in an overfitted model. However the model was able to distinguish day between day 1, 3, 6 and 10 (Figure 2). As negative control a model trained on a randomized sample was made. This control model was not able to assign a category correctly, indicating an actual difference in platelet characteristics during storage was detected. By using visualization of the weight-gradient (e.g. where in the images the model assigns the most weight) we additionally confirmed that the model used separate individual platelets to characterize the age of the complete platelet sample. Conclusion: Combining confocal platelet images with a convolutional neural network, we trained a model that could synergistically integrate different stainings and information of individual platelets to predict with >95% accuracy platelet age in vitro as well as in PC up to 10 days of storage. Additionally our model could distinguish "young" platelets (stored 1, 3 or 6 days) from "old" platelets (stored 10 days). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Removal of platelets from blood plasma to improve the quality of extracellular vesicle research

BackgroundBlood plasma is commonly used for biomarker research of extracellular vesicles (EVs). Removing all cells prior to analysis of EVs is essential. ObjectivesWe therefore studied the efficacy of the most commonly used centrifugation protocol to prepare cell‐free plasma. MethodsPlasma was prepared according to the double centrifugation protocol of the International Society on Thrombosis and Haemostasis (ISTH) in three independent studies. The concentrations of platelets, platelet‐derived EVs, and erythrocyte‐derived EVs were measured by calibrated flow cytometry. ResultsThe mean platelet concentration ranged from 5.1 × 105/ml to 2.8 × 107/ml and differed 55‐fold between studies. Thus, the ISTH centrifugation protocol does not remove all platelets and results in variation between studies. As the concentration of platelet‐derived EVs and platelets correlates linearly (R2 = .56), and the volume fraction of EVs and platelets in plasma are similar, the presence of platelets affects downstream analysis. To remove platelets a 0.8‐μm polycarbonate filter was used to lower the platelet concentration 146‐fold (p = .0013), without affecting the concentration of platelet‐derived and erythrocyte‐derived EVs (p = .982, p = .742). ConclusionsTo improve the quality of EV research, we recommend (1) measuring and reporting the platelet concentration in plasma used for EV research, or (2) removing platelets by centrifugation followed by filtration.

Platelet-Rich Plasma: Applications in Cosmetic Dermatology

Platelet-rich plasma (PRP) or autologous platelet gel, plasma-rich growth factors, and platelet-concentrated plasma are blood component that is rich in thrombocytes on a small volume of plasma. There were seven primary growth factors that actively secreted by thrombocytes including platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor, connective tissue growth factor, and insulin-like growth factor-1. PRP application in cosmetic dermatology includes tissue regeneration, wound healing, repairing scar tissue, skin rejuvenation, and being used in alopecia patients. PRP could accelerate soft-tissue formation in the subdermal, activate fibroblast formation and new collagen deposition, and formation of blood vessels and fat cells. PRP is also considered a potential therapy for accelerating hair growth for alopecia.

Platelet-rich plasma treatment in patients with refractory thin endometrium and recurrent implantation failure: A comprehensive review.

Refractory thin endometrium and recurrent implantation failure are among the most challenging infertility-related factors hindering successful pregnancy. Several adjuvant therapies have been investigated to increase endometrial thickness and the pregnancy rate, but the treatment effect is still minimal, and for many patients, these treatment methods can be quite costly and difficult to approach. Platelet-rich plasma (PRP) is an autologous concentration of platelets in plasma and has recently been elucidated as a better treatment option for these patients. PRP is rich in cytokines and growth factors, which are suggested to exert a regenerative effect at the level of the injured tissue. Another advantage of PRP is that it is easily obtained from the patient’s own blood. We aimed to review the recent findings of PRP therapy used for patients with refractory thin endometrium and recurrent implantation failure.

The blood cells count in leukocyte and leukocyte-poor platelet-concentrated plasma in patients with musculoskeletal disorders

The blood cells count in leukocyte and leukocyte-poor platelet-concentrated plasma in patients with musculoskeletal disorders

Platelet-Rich Plasma: The Journey so Far !

Platelet-rich plasma (PRP) is an autologous blood product, obtained after blood centrifugation. PRP is rich in growth factors which promote tissue-healing, alter angiogenesis, and possess versatile immunomodulatory effects, in the relative absence of any significant demonstrable adverse effects. Consequently, PRP has found application in multiple specialities in recent years, including dermatology. A literature search was performed on PubMed, Medline database, and Google Scholar, using keywords like platelet-rich plasma (PRP), platelet-concentrated plasma, platelet-rich growth factors, autologous platelet concentrate. Relevant studies were selected, and data was analyzed following extraction. Studies show that PRP has not only been used as an adjunctive modality but has been employed as a stand-alone therapy as well. Multiple authors have reported PRP to be efficacious in disparate dermatological conditions, like alopecia, skin rejuvenation, healing of refractory cutaneous ulcers, and even acne scar management. The strongest evidence so far has been demonstrated in androgenetic alopecia and facial skin rejuvenation. However, routine use in dermatological conditions is hampered by the relative paucity of high-quality evidence and large randomized studies. Furthermore, PRP composition and preparation methods are not yet standardized and even the treatment regimens proposed too vary widely. The present review provides a bird's eye view of the evidence available so far regarding the use of PRP in dermatology. The review focusses more on recent prospective studies, including randomized trials and tries to summarize the evidence in a brief, but comprehensive manner.

Hypersensitivity transfusion reactions to platelet concentrate: a retrospective analysis of the French hemovigilance network.

Among labile blood products, platelet concentrates (PCs) are the leading cause of hypersensitivity transfusion reactions (HTRs). These reactions often lead to interruption of PC transfusion and can result in a prolonged transfusion process leading to significant morbidity and use of premedication and close monitoring for patients with a history of allergic transfusion reactions. The French hemovigilance database is one of the largest standardized databases providing information on HTRs following administration of labile blood products. In this study, we analyzed this database to assess the relative risk of HTR for each type of PC. HTRs following PC transfusion were retrospectively extracted from the e-Fit Hemovigilance database of the French National Agency for Medicines and Health Products Safety (ANSM). Frequencies were calculated using the number of specific PCs transfused. Between 2008 and 2014, the overall estimated incidence of HTRs following PC administration was calculated at 232 HTRs per 100,000 PCs transfused. The rate of HTRs was significantly higher with apheresis PC (337/100,000) than with buffy-coat PC (94/100,000). Platelets in additive solutions (PAS) were associated with a significantly lower frequency of HTRs when compared with PCs in native plasma. Amotosalen/UVA- PCs (APCs and BCPCs) which are always in PAS in France, exhibited the lowest frequency of HTRs when compared with their corresponding PCs in native plasma or in PAS (p < 10-7 in all comparisons). Our results showed that the type of PC and its processing may have an impact on the risk of HTR.

Platelet-rich plasma for androgenetic alopecia: A review of the literature and proposed treatment protocol.

Androgenetic alopecia (AGA) is a common hair loss disorder caused by genetic and hormonal factors that are characterized by androgen-related progressive thinning of scalp hair in a defined pattern. By the age of 60 years, 45% of men and 35% of women develop AGA. Currently, U.S. Food and Drug Administration-approved treatments for AGA include oral finasteride and topical minoxidil. Due to the limited number of effective therapies for AGA, platelet-rich plasma (PRP) has become an effective alternative treatment. PRP is an autologous concentration of platelets in plasma with numerous growth factors that contribute to hair regeneration. The growth factors contained within the alpha granules of platelets act on stem cells in the bulge area of the hair follicles and stimulate the development of new follicles along with neovascularization. PRP has become a promising treatment modality for AGA. Although there have been several studies previously reported, a standard practice for PRP preparation and administration as well as a method to evaluate results have not been established. This literature review was conducted to evaluate the effectiveness of PRP for AGA and discuss the various treatment protocols that have been proposed.

Administration of platelet concentrates suspended in bicarbonated Ringer's solution in children who had platelet transfusion reactions.

Adverse reactions to platelet transfusions are a problem. Children with primary haematological and malignant diseases may experience allergic transfusion reactions (ATRs) to platelet concentrates (PCs), which can be prevented by giving washed PCs. A new platelet additive solution, using bicarbonated Ringer's solution and acid-citrate-dextrose formula A (BRS-A), may be better for platelet washing and storage, but clinical data are scarce. A retrospective cohort study for consecutive cases was performed between 2013 and 2017. For 24months, we transfused washed PCs containing BRS-A to children with primary haematological and malignant diseases and previous adverse reactions. Patients transfused with conventional PCs (containing residual plasma) were assigned as controls, and results were compared in terms of frequency of ATRs, corrected count increment (CCI) and occurrence of bleeding. We also studied children transfused with PCs washed by a different system as historical controls. Thirty-two patients received 377 conventional PC transfusions. ATRs occurred in 12 (37·5%) patients from transfused with 18 (4·8%) bags. Thirteen patients, who experienced reactions to regular PCs in plasma, then received 119 transfusion bags of washed PCs containing BRS-A, and none had ATRs to washed PCs containing BRS-A. Before study period, six patients transfused 137 classical washed PCs with different platelet additive solution, under same indication, ATRs occurred in one (16·7%) patient from transfused with one (0·7%) bags. CCIs (24h) in were lower with classical washed PCs (1·26±0·54) compared to regular PCs in plasma (2·07±0·76) (P<0·001), but there was no difference between washed PCs containing BRS-A (2·14±0·77) and regular PCs (2·21±0·79) (P=0·769), and we saw no post-transfusion bleeding. Washed PCs containing BRS-A appear to prevent ATRs without loss of transfusion efficacy in children with primary haematological and malignant diseases. Their efficacy should be further evaluated through larger prospective clinical trials.

Platelet-rich plasma stimulates angiogenesis in mice which may promote hair growth

BackgroundPlatelet-rich plasma (PRP) is an autologous concentration of human platelets in plasma. In this paper, we aimed to investigate the effect of PRP on hair growth.MethodsPlatelet-rich plasma and platelet-poor plasma were prepared by sterile centrifugation and injected into shaved dorsal skin of mice (n = 10). Saline injection was used in the control group. The length of randomly plucked hairs was measured at 8, 13, 18 days after PRP injection. Histological examination was preformed to observe the histologic changes of skins. The immunohistochemistry analysis of CD31 was performed to detect the changes of hair length and formation of new vessels.ResultsAt 13 and 18 days after the last injection, the hair length of mice in PRP group (4.24 ± 0.60 and 8.29 ± 0.48 mm, respectively) was significantly longer compared with the control group (3.70 ± 0.52 and 7.21 ± 0.64 mm, p < 0.05). No significant difference in the hair length was found between the PPP group and the control (p > 0.05). In addition, the number of CD31-positive vessel in the PRP group (9.90 ± 0.60) was more than that in the control group (8.60 ± 2.34, p < 0.05).ConclusionPlatelet-rich plasma might promote hair length growth and increase the number of hair follicles by inducing angiogenesis.

Pulsed xenon flash treatment inactivates bacteria in apheresis platelet concentrates while preserving in vitro quality and functionality.

Pulsed xenon (Xe) flash without any photoreactive compounds has been shown to inactivate a type of bacteria spiked into platelet (PLT) suspension in plasma, but enhanced the PLT storage lesion (PSL). Predicting reduction of PSL with increasing bactericidal ability, pulsed Xe flash was filtered through a band-stop filter, which excluded ultraviolet (UV)A, UVB, and visible light. Apheresis PLT concentrates (PCs) inoculated with bacteria were irradiated with filtered Xe flash (fXe treatment). For in vitro functional quality assessment, PLT aggregation and thrombin generation together with other assays that monitor the PSL were investigated. Staphylococcus aureus and Streptococcus dysgalactiae could be inactivated without regrowth during 6 days of storage. PC variables, such as PLT count, concentrations of soluble CD40 ligand, and ratio of aggregated PLTs, were not significantly different between fXe-treated and untreated PCs after 6 days of storage, while PAC-1 binding increased in the fXe-treated PLTs. Responsiveness of fXe-treated PLTs to ADP was maintained over a 6-day storage period as shown by the up regulation of P-selectin expression and induction of both integrin αIIbβ3 conformational change and PLT aggregation. The fXe-treated PLTs showed a sustained aggregation curve in response to ADP, whereas untreated PLTs transiently aggregated and then subsequently dissociated. Thrombin-generating kinetics of fXe-treated PLTs via PLT membrane surface were equivalent to those of untreated PLTs. The fXe treatment inactivated bacteria in apheresis PCs in plasma without additional chemical compounds. The fXe-treated PCs retained acceptable in vitro properties of PC quality and PLT functionality.

Role of platelet-rich plasma: The current trend and evidence

Platelets contain many important growth factors, which helps in healing. These lead researchers to inject high concentration of Platelets in plasma (platelet-rich plasma [PRP]) at injury site. There is a variety of PRP preparations produced by the different commercial equipment. The concentration of platelets should be more than 5 times the normal in PRP. PRP can be used for long list of conditions, for example, orthopedic, neurology, musculoskeletal, cardiology, dermatology, and plastic surgery. The research on PRP is still in infancy, and there is no consensus on platelet concentration, amount of PRP, which is the best technology in preparing PRP. There are many animal studies, which are showing encouraging results, but human studies are lacking. PRP is very promising treatment option which is nonsurgical. We need to wait for more concrete evidence to emerge to define its exact clinical role.

Microparticle and mitochondrial release during extended storage of different types of platelet concentrates

On activation, platelets release vesicles called microparticles (MPs). MPs are heterogeneous with regard to the presence or absence of mitochondria. We quantified MPs in platelet concentrates (PCs) taking their mitochondrial content into account. Platelet-rich plasma (PRP), buffy coat (BC) and apheresis (AP) PCs were tested through 7 days of storage. A combination of flow cytometry and spanning-tree progression analysis of density-normalized events (SPADE) was used to determine MP and mitochondrial release during storage. All the PC biochemical parameters complied with transfusion standards at all times. Platelet activation markers increased during storage and were higher for PRP than other types of PCs. Concentrations of MPs and extracellular mitochondria interpreted by SPADE algorithm were significantly higher in PRP than other in PCs and were stable throughout storage. The mode of preparation, rather than storage duration, impacts the release of MPs and mitochondria in PCs.

Reduction of bacteria and human immunodeficiency virus Type 1 infectivity of platelet suspension in plasma using xenon flash-pulse light in a bench-scale trial.

Current pathogen reduction systems for platelet concentrates (PCs) require addition of chemical compounds and/or reduction of plasma content in PCs. We have investigated a new method using xenon (Xe) flash-pulse light without additional compounds or plasma replacement. An aliquot of apheresis platelets (PLTs) in plasma inoculated with bacteria or human immunodeficiency virus Type 1 (HIV-1) was irradiated with Xe flash-pulse light (Xe flash phototreatment). Bacterial growth was monitored up to 6 days of storage, whereas HIV-1 infectivity was assayed just after treatment. Pairs of Xe flash-phototreated and untreated PCs were examined for PLT lesion during the storage period. Under the current conditions, a low titer (1.8 colony-forming units [CFUs]/mL) of Staphylococcus aureus did not proliferate during the 6-day storage period, but grew in some cases at high-titer (24.0 CFUs/mL) inoculation. HIV-1 infectivity was reduced by 1.8 log. PLT recovery of the treated PCs was lower than untreated ones. An increase of mean PLT volume and glucose consumption, together with a decrease of hypotonic shock response and pH, were enhanced by the treatment. CD62P- and PAC-1-positive PLTs increased after the treatment, indicating the induction of PLT activation. Among biologic response modifiers, soluble CD40 ligand was significantly increased in the treated PCs on Day 6. Xe flash phototreatment could prevent bacterial proliferation and reduce HIV-1 infectivity in 100% plasma PCs without any additional compounds, but enhanced PLT storage lesions. Further improvement is required to increase the potency of pathogen inactivation with reducing PLT damage.

PO-56 - Subsequent clot properties depend on the storage time of platelet concentrates

Despite platelets properties varies during storage, transfused platelets are included in blood clot in the same way as well as native. We assume that after transfusion the prevalence of platelets with changed activity lead to worse quality of blood clot in vivo. The aim was the in vitro study of platelet-dependent clot properties that are formed from some stored platelet concentrates (PCs). Twenty-six PCs in autologous plasma and 24 PCs in platelet additive solution SSP+ (MacoPharma, France) (70vol.%) were analized by thromboelastography with and without platelets activation, and by standard aggregometry. The testing were carried out at the day of proceeding, after 24 hours, and at 3rd and 5th days of storage. We used Trima Accel (Terumo BCT, USA) for the proceeding of platelets apheresis. We found that clot demonstrated gradual reduction of elasticity and deformability starting from second day to fifth day in stored PCs suspended in autologous plasma. From the third storage day platelets lost their meaning for clot properties. The platelets apheresis with next re-suspending in SSP+ solution leads to the depression for platelets aggregability. Actived platelets had no impact to clot properties during full storage time. Total decline of clot quality including low elasticity and impaired deformability were found starting from 3rd storage day compared to the day of proceeding. We assume that such properties of clot as both elasticity and deformability are forming in PCs at the day of proceeding. Further clot changes observed in PCs does not depend directly from platelets aggregability because clot forming are under other influences. The last are determined mainly by the coagulation what was no included in this study. Also obtained results confirms the 5th-days storage as a benefit independent from PCs proceeding method.

In vitro analysis of volume-reduced washed platelet concentrates stored in bicarbonated Ringer's solution containing less than 5% residual plasma.

Volume-reduced washed platelet (PLT) concentrates (PCs) can prevent circulatory overload and allergic reactions in patients undergoing PLT transfusions. For these reasons, they are in demand for paediatric settings and for patients at risk of circulatory overload. Here, we evaluated the quality of volume-reduced washed PCs stored for 5 days in a novel acetate-free PLT additive solution (PAS) containing glucose and bicarbonate (BRS-A) with <5% residual plasma protein. PCs from two apheresis donations were mixed and divided equally into control and test units. For the test unit (volume-reduced washed PCs), PLTs were washed and stored in 90 ml BRS-A with <5% plasma protein. PLTs in the control unit were stored in 200 ml 100% plasma without any washing manipulations. The in vitro properties of PLTs in both units were compared over a 5-day storage period. The procedure for volume-reduced washed PCs effectively removed >98% plasma protein in 100% plasma PCs and yielded an approximately twofold lower mean volume (91 ml) compared to that observed with the control units. Immediately after washing, the mean PLT concentration of the test units was 20·5 × 10(11) /l, twofold higher than that of the control units. The pH (37°C) levels in the test unit remained above 7·0 for 5 days. Glucose consumption and lactate production rates of the test units on days 1-3 were higher than those of the control units, leading to glucose exhaustion in the test unit by Day 3. Hypotonic shock responses and CD62P and CD42b expression levels in both units were comparable during 5-day storage. Considering the pH buffering capacity of BRS-A, a 90-ml volume may be acceptable for maintaining the in vitro quality of washed PLTs for at least 2 days.

Platelet-rich plasma and its derivatives as promising bioactive materials for regenerative medicine: basic principles and concepts underlying recent advances.

Over the past decade, platelet-rich plasma (PRP), a platelet-concentrated plasma fraction, has been widely investigated and applied to regenerative medicine. The clinical utility of PRP is supported by evidence that PRP contains high concentrations of platelet-related growth factors and normal concentrations of plasma-derived fibrinogen, both of which contribute synergistically to the regenerative process. Additionally, its superior cost-efficacy versus conventional therapies is attractive to many clinicians. However, current disadvantages of PRP include a relatively complicated preparation procedure and variable operator-dependent efficacy. An additional disadvantage is the use of bovine thrombin, an animal-derived biological, as a coagulant. Many of these disadvantages are overcome by recent advances in preparation procedures and devices; for example, Joseph Choukroun simplified the platelet-rich fibrin preparation procedure and improved handling efficiency without the aid of animal-derived factors. With advancements in cell processing technology, there has been a general shift in cell therapy from autologous to allogeneic treatment; however, autologous PRP therapy will not easily be replaced by allogeneic treatment in the near future. Therefore, to provide more predictable regenerative therapy outcomes using autologous PRP, further investigations should address developing a standardized procedure for PRP preparation to augment its efficacy and potency, independent of donor variability. We would then propose that operators and clinicians prepare PRP according to the standardized protocol and to carefully evaluate the clinical scenario (i.e., recipient factors comprising skeletal defects) to determine which factor(s) should be added to PRP preparations. This careful approach will lead to improved clinical outcomes for patients.

Influence of Different Methods Preparation on Platelet Activation in Stored Platelet Concentrates

Background: Platelet concentrates are routinely manufactured from whole blood by differential centrifugation. During this storage period under blood bank conditions, biochemical, structural and functional changes occur, a process that is also known as platelet storage lesion. Their quality was assessed using the following parameters: platelets, leukocytes and erythrocytes counts, pH, CD63, lactate dehydrogenase and Annexin V. Materials and Methods: In this experimental study, 25 platelet concentrates prepared with platelet rich plasma-platelet concentrates, 25 units via buffy coat and apheresis-derived platelet methods. The percentages of Annexin V, CD63 expression, lactatedehydrogenase, platelet, leukocytes counts and pH were evaluated. Results: During storage for up to 5 days, no significant pH, difference was observed among all three type of platelet concentrates (p>0.05). The mean leukocytes count buffy coat units, platelet rich plasmaplatelet concentrates and apheresis-derived units were comparable and statistically significant difference was observed (p<0.05). During storage for up to 5days platelet concentrates units displayed significant an increase in lactatedehydrogenase, CD63 and Annexin V expressions, as compared with buffy coat units and apheresisderived units preparation on day 5 (p<0.05). Discussion: The kinetics of CD63 and annexin V levels are influenced by the method used to prepare platelets for storage. The different levels of CD63, annexin V and lactatedehydrogenase in three types of units clearly demonstrating a progressive activation process of platelet concentrates units exceeds that of buffy coat and apheresis-derived units. Further clinical studies will be necessary to determine whether meet the quality criteria or is superior in predicting in vivo viability.

THE INFLUENCE OF THE CENTRIFUGATION SETTINGS ON AB QUANTIFICATION IN PLASMA

Platelets have been pointed as major contributors to the Aβ levels in blood. Centrifugal force and spin time are two pre-analytical factors which determine the final composition of the different blood fractions. These parameters should be strictly standardized to reduce variability in plasma Aβ quantification for diagnostic use.In this study, we have evaluated the influence of these two parameters on plasma platelet concentration and Aβ 1-40 and Aβ 1-42 levels. Peripheral blood from five healthy donors was obtained by venipuncture and collected in polypropylene vials with EDTA. Each sample was divided in different tubes and centrifuged at 4°C under diverse conditions. After centrifugation, plasma fractions were collected and platelet concentration was measured by using a hematologic analyzer. Levels of Aβ 1-40 and Aβ 1-42 were quantified by ELISA in undiluted plasma (UP) and diluted plasma (DP) fractions. Selected aliquots were filtered to obtain platelet-free plasma samples as a control. In the sample processed at 400g for 15', most of the platelets remain in the plasma fraction (156.4% ± 19.9 of the peripheral blood concentration). The number of platelets in plasma decreased proportionally as centrifugal force and spin time increase. Complete elimination of platelets from plasma required 4000g for 30 min. Regarding the DP fraction, no differences were found in levels of Aβ 1-40 and Aβ 1-42 among the samples processed with different centrifugation protocols. In UP samples, the levels of Aβ 1-40 (and to a lesser extent those of Aβ 1-42) were higher in samples centrifuged with progressively more energetic protocols. These differences were observed as well in platelet-free (filtered) plasma samples. Platelet concentration in plasma is dependent on centrifugation force and spin time. The presence of platelets in plasma does not appear to interfere with the determination of A β in plasma. However, centrifugation protocol affects Aβ quantification in UP, indicating that plasma matrix components may influence in the analysis.