A cross-sectional analysis of the effects of various centrifugation speeds and inclusion of the buffy coat in platelet-rich plasma preparation

Introduction: Platelet-rich plasma is an autologous blood preparation which is used in various medical specialties because of its regenerative properties. There is a wide variation in platelet-rich plasma preparation protocols and attaining the ideal platelet yield (>1 million platelets/μL) in a clinic setting can be challenging. We aimed at analyzing the centrifuge spin rates at which to attain an ideal platelet-rich plasma yield and also to study the effect of inclusion of the buffy coat after the first spin on the final platelet concentration in platelet-rich plasma. Methods: Seventy-five whole blood samples were obtained and divided into two groups – (1) leukocyte-rich platelet-rich plasma group and (2) leukocyte-poor platelet-rich plasma group. Samples in both groups were centrifuged using the dual spin method, at one of three centrifugation speed combinations (initial “soft” spin and second “hard” spin speeds, respectively): (1) 100 g/400 g, (2) 350 g/1350 g and (3) 900 g/1800 g. Platelet, red blood cell (RBC) and white blood cell (WBC) counts in both groups were compared. Results: The 100 g/400 g spin gave a high platelet yield (increase of 395.4 ± 111.1%) in the leukocyte-poor-platelet-rich plasma group, while in the leukocyte-rich platelet-rich plasma group both 100 g/400 g and 350 g/1350 g spins resulted in significantly higher yields with an increase of 691.5 ± 316.3% and 738.6 ± 193.3%, respectively. Limitations: The study was limited by a smaller sample size in the pure platelet-rich plasma (leukocyte-poor platelet-rich plasma) group. Conclusion: Ideal platelet yields can be achieved with both the 100 g/400 g as well as the 350 g/1350 g spins using the buffy coat inclusion method while the 100 g/400 g spin for “pure” platelet-rich plasma accomplishes a near-ideal platelet count with significantly reduced contamination with other cells.

Download Full-text

Evaluation of allogeneic freeze-dried platelet lysate in cartilage exposed to interleukin 1-β in vitro

BMC Veterinary Research ◽

10.1186/s12917-019-2118-z ◽

2019 ◽

Vol 15 (1) ◽

Cited By ~ 3

Author(s):

Livia Camargo Garbin ◽

C. Wayne McIlwraith ◽

David D. Frisbie

Keyword(s):

Musculoskeletal Diseases ◽

Platelet Rich Plasma ◽

Autologous Blood ◽

Interleukin 1 ◽

Platelet Lysate ◽

Freeze Dried ◽

Platelet Concentration ◽

High Concentrations ◽

Disease Modifying Treatment

Abstract Background Platelet-rich plasma (PRP) as well as other platelet-derived products have been used as a potential disease-modifying treatment for musculoskeletal diseases, such as osteoarthritis (OA). The restorative properties of such products rely mainly on the high concentrations of growth factors, demonstrating encouraging results experimentally and clinically. Yet, the autologous blood-derived nature of the PRP product lead to limitations that precludes it’s widespread use. The main limitations for PRP use are; product variability, the need for minimum laboratory settings in most cases, and the need for storage at low temperatures to preserve its properties. Based on these limitations, the objective of this study was to investigate an allogeneic off-the-shelf platelet lysate (PL) in cartilage exposed to interleukin 1β (IL-1β). For this purpose, blood and cartilage were harvested from eight skeletally mature and healthy horses. Blood was processed into PL aliquots and divided into three groups (Frozen, Freeze-dried and Filtered freeze-dried), used in autologous and allogeneic conditions and in three different concentrations (1.5, 3 and 6-fold). Different PL preparations were then applied in cartilage culture with interleukin-1 beta and cultured for 10 days. Cartilage and media samples were collected and analyzed for total GAG and 35SO4-labeled GAG content. Results No significant differences between the controls and PL groups in cartilage and media were demonstrated. The effects of PL on cartilage matrix were concentration dependent and intermediate concentrations (3-fold) in PL showed increased 35SO4-labelled GAG in cartilage. Conclusion In conclusion, the allogeneic freeze-dried PL presented equivalent effects compared to frozen autologous PL. Intermediate platelet concentration on average demonstrated improved results, demonstrating less GAG loss compared to other concentrations.

Download Full-text

Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation

Blood ◽

10.1182/blood-2012-05-427856 ◽

2012 ◽

Vol 120 (18) ◽

pp. 3837-3845 ◽

Cited By ~ 83

Author(s):

Matthew F. Whelihan ◽

Vicentios Zachary ◽

Thomas Orfeo ◽

Kenneth G. Mann

Keyword(s):

Blood Cells ◽

Thrombin Generation ◽

Marked Decrease ◽

Platelet Rich Plasma ◽

Buffy Coat ◽

Glycophorin A ◽

Initiation Phase ◽

Platelet Concentration ◽

Corn Trypsin Inhibitor ◽

Prothrombin Activation

Abstract Prothrombin activation can proceed through the intermediates meizothrombin or prethrombin-2. To assess the contributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)–activated blood, immunoassays were developed that measure the meizothrombin antithrombin (mTAT) and α-thrombin antithrombin (αTAT) complexes. We determined that Tf-activated blood produced both αTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn trypsin inhibitor–treated blood was fractionated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initiation phase and a marked decrease in the rate and extent of αTAT formation. Only the addition of RBCs to PRP was capable of normalizing αTAT generation. FACS on glycophorin A–positive cells showed that approximately 0.6% of the RBC population expresses phosphatidylserine and binds prothrombinase (FITC Xa·factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that supports prothrombin activation through the meizothrombin pathway.

Download Full-text

Higher Physiologic Platelet Counts in Whole Blood Are Not Associated With Improved ACL Cross-sectional Area or Signal Intensity 6 Months After Bridge-Enhanced ACL Repair

Orthopaedic Journal of Sports Medicine ◽

10.1177/2325967120927655 ◽

2020 ◽

Vol 8 (7) ◽

pp. 232596712092765 ◽

Cited By ~ 1

Author(s):

Christina Freiberger ◽

Ata M. Kiapour ◽

Shanshan Liu ◽

Rachael N. Henderson ◽

Samuel Barnett ◽

...

Keyword(s):

Extracellular Matrix ◽

Magnetic Resonance ◽

Blood Cell ◽

Signal Intensity ◽

Cross Sectional Area ◽

Sectional Area ◽

Acl Repair ◽

Cross Sectional ◽

Platelet Concentration ◽

Male Sex

Background: A bridge-enhanced anterior cruciate ligament (ACL) repair (BEAR) procedure places an extracellular matrix implant, combined with autologous whole blood, in the gap between the torn ends of the ligament at the time of suture repair to stimulate healing. Prior studies have suggested that white blood cell (WBC) and platelet concentrations significantly affect the healing of other musculoskeletal tissues. Purpose/Hypothesis: The purpose of this study was to determine whether concentrations of various blood cell types placed into a bridging extracellular matrix implant at the time of ACL repair would have a significant effect on the healing ligament cross-sectional area or tissue organization (as measured by signal intensity). We hypothesized that patients with higher physiologic platelet and lower WBC counts would have improved healing of the ACL on magnetic resonance imaging (MRI) (higher cross-sectional area and/or lower signal intensity) 6 months after surgery. Study Design: Cohort study; Level of evidence, 2. Methods: A total of 61 patients underwent MRI at 6 months after bridge-enhanced ACL repair as part of the BEAR II trial. The normalized signal intensity and average cross-sectional area of the healing ligament were measured from a magnetic resonance stack obtained using a gradient echo sequence. The results were stratified by sex, and univariate and multivariate regression analyses determined significant correlations between blood cell concentrations on these 2 magnetic resonance parameters. Results: In unadjusted analyses, older age and male sex were associated with greater healing ligament cross-sectional area ( P < .04) but not signal intensity ( P > .15). Adjusted multivariable analyses indicated that in female patients, a higher monocyte concentration correlated with a higher ACL cross-sectional area (β = 1.01; P = .049). All other factors measured, including the physiologic concentration of platelets, neutrophils, lymphocytes, basophils, and immunoglobulin against bovine gelatin, were not significantly associated with either magnetic resonance parameter in either sex ( P > .05 for all). Conclusion: Although older age, male sex, and monocyte concentration in female patients were associated with greater healing ligament cross-sectional area, signal intensity of the healing ligament was independent of these factors. Physiologic platelet concentration did not have any significant effect on cross-sectional area or signal intensity of the healing ACL at 6 months after bridge-enhanced ACL repair in this cohort. Given these findings, factors other than the physiologic platelet concentration and total WBC concentration may be more important in the rate and amount of ACL healing after bridge-enhanced ACL repair.

Download Full-text

Fatty Acids Composition of Blood Cell Membranes and Peripheral Inflammation in the PREDIMED Study: A Cross-Sectional Analysis

Nutrients ◽

10.3390/nu11030576 ◽

2019 ◽

Vol 11 (3) ◽

pp. 576 ◽

Cited By ~ 5

Author(s):

Jananee Muralidharan ◽

Christopher Papandreou ◽

Aleix Sala-Vila ◽

Nuria Rosique-Esteban ◽

Montserrat Fitó ◽

...

Keyword(s):

Fatty Acids ◽

Blood Cell ◽

Inflammatory Markers ◽

Cell Membranes ◽

Cross Sectional ◽

Negatively Associated ◽

Cross Sectional Analysis ◽

Ifn Γ ◽

Sectional Analysis ◽

Predimed Study

There is limited evidence from epidemiological studies for the inflammatory or anti-inflammatory properties of fatty acids in blood cell membranes. Therefore, this study examined associations between baseline (n = 282) and 1-year (n = 143) changes in the levels of fatty acids in blood cell membranes with circulating inflammatory markers in older adults at high cardiovascular risk. The data for this cross-sectional analysis was obtained from a case-control study within the PREDIMED study. Linear regression with elastic net penalty was applied to test associations between measured fatty acids and inflammatory markers. Several fatty acids were associated with interferon-γ (IFNγ) and interleukins (ILs) IL-6, IL-8, and IL-10 at baseline and additionally also with IL-1b at 1 year. Omega-6 fatty acids were consistently positively associated with pro-inflammatory IL-6 and IL-8 at baseline. Omega-3 fatty acids including C20:5n3 and C18:3n3 were negatively associated with IFN-γ at 1 year. It is interesting to note that the cis and trans forms of C16:1n7 at 1 year were oppositely associated with the inflammatory markers. C16:1n7trans was negatively associated with IFN-γ, IL-6, IL-8, IL-10, and IL-1b, whereas C16:1n7cis was positively associated with IL-1b. This study adds to the growing body of evidence suggesting potential differences in inflammatory or anti-inflammatory properties of fatty acids in blood cell membranes.

Download Full-text

P–600 Ovarian function following intraovarian injection of autologous platelet rich plasma (APRP) in women with low functional ovarian reserve

Human Reproduction ◽

10.1093/humrep/deab130.599 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

D Barad ◽

S K Darmon ◽

A Benor ◽

N Gleicher

Keyword(s):

Growth Factors ◽

Ovarian Reserve ◽

Ovarian Function ◽

Endometrial Thickness ◽

Platelet Rich Plasma ◽

Controlled Trial ◽

Autologous Blood ◽

Poor Response ◽

Medical Specialties ◽

Menstruating Women

Abstract Study question Does exposure of ovaries to autologous growth factors in platelet rich plasma (APRP) affect the pituitary ovarian axis? Summary answer Within 60 days after injection, growing follicle numbers and estrogen levels increased, though FSH did not change, with effects most pronounced in still menstruating women. What is known already APRP is extracted from a patient’s autologous blood and delivers growth factors. It is widely used in several medical specialties and has in infertility practice been reported to increase folicle/egg numbers if injected into ovaries and improve endometrial thickness/implantation if used for perfusion of the endometrium. Study design, size, duration Prospective observational cohort study of women with low functional ovarian reserve, followed for 60 days after subcortical injection of ovaries. Participants/materials, setting, methods 44 women with prior poor response to ovulation induction, FSH > 12 mIU/mL and AMH < 1.0 ng/mL. APRP was prepared using Regen Lab PRP Kit which is approved by the US-FDA. 1.0–1.5 ml of PRP was injected into the cortex of each ovary divided among 7 to 10 injection sites. Participants were followed every three days with monitoring for estradiol, FSH and follicle growth for the first two weeks after PRP and then weekly. Main results and the role of chance 21/43 patients still regularly menstruated (subgroup A, age 43.9 ± 5.1 years); 23/43 (subgroup B, age 42.6 ± 6.2 years) were amenorrheic for a median of 6 months. In A, AMH, FSH and estradiol were 0.18 ± 0.20 ng/mL, 37.5 ± 47.6 mIU/mL, and 100.2 ± 73.4 pg/mL, while in B they were 0.06 ± 0.11 ng/mL, 73.0 ± 44.8 mIU/mL and 66.7 ± 57.6 pg/mL. Following APRP, A-patients demonstrated increased estradiol to 211 ± 193.7 pg/mL (P = 0.029) while B-patients only demonstrated a trend to 98.1 ± 86.5 (P = 0.09). Among A patients, 14/21 (66.7%) entered IVF cycles and 5/21 (23.8%) reached retrieval. So-far 1 patients established an ongoing clinical pregnancy. Among B patients 8/23 (34.8%) entered IVF cycles and only 2/23 (8.7%) reached retrieval and none achieved pregnancy. Limitations, reasons for caution This observational study was only carried out to estimate possible effects of APRP treatments. Based on these observations, we are now conducting a randomized controlled trial, limited to cycling women under age 45 years [registration # NCT04278313]. Wider implications of the findings: PRP appears to have limited ability to affect ovarian reserve of older, and especially amenorrheic women. It may, however, exert more favorable effects on still menstruating women. Promotion of APRP treatment as “ovarian rejuvenation,” however, appears to be an inappropriate choice of words. Trial registration number N/A

Download Full-text

Leukocyte-derived ratios are associated with late-life any type dementia: a cross-sectional analysis of the Mugello study

GeroScience ◽

10.1007/s11357-021-00474-3 ◽

2021 ◽

Author(s):

Gemma Lombardi ◽

Roberto Paganelli ◽

Michele Abate ◽

Alex Ireland ◽

Raffaele Molino-Lova ◽

...

Keyword(s):

Body Mass Index ◽

Blood Cell ◽

Lymphocyte Count ◽

Cross Sectional ◽

Lymphocyte To Monocyte Ratio ◽

Cell Counts ◽

Blood Cell Counts ◽

Diagnosis Of Dementia ◽

White Blood Cell Counts ◽

Sectional Analysis

Abstract Immunosenescence, vascular aging, and brain aging, all characterized by elevated levels of inflammatory markers, are thought to share a common pathogenetic pathway: inflamm-aging. Retrospective cross-sectional analysis was conducted using data from the Mugello study (Tuscany, Italy), a representative Italian cohort of free-living nonagenarians. to assess the association between specific peripheral inflammation markers derived from white blood cell counts, and the diagnosis of dementia. All the variables of interest were reported for 411 subjects (110 males and 301 females) out of 475 enrolled in the study. Anamnestic dementia diagnosis was obtained from clinical certificate and confirmed by a General Practitioner, whereas leukocyte ratios were directly calculated from white blood cell counts. Body mass index and comorbidities were considered potential confounders. Diagnosis of any type dementia was certified in 73 cases (17.8%). Subjects affected by dementia were older, more frequently reported a previous stroke, had lower body mass index, and lower Mini-Mental-State-Examination score. Moreover, they had a higher lymphocyte count and lymphocyte-to-monocyte ratio compared to the non-demented nonagenarians. We found that higher levels of lymphocyte counts are cross-sectionally associated with a clinical diagnosis of dementia. Furthermore, lymphocyte-to-monocyte ratio is directly associated with any type of dementia, independently of age, sex, lymphocyte count, and comorbidities. Lymphocyte-to-monocyte ratio may be considered a marker of immunological changes in the brain of dementia patients; moreover, it is low-cost, and easily available, thus enabling comparisons among different studies and populations, although the timeline and the extent of lymphocyte-to-monocyte ratio role in dementia development must be further investigated.

Download Full-text

The Effect of Autologous Platelet-Rich Plasma on Bronchial Stump Tissue Granulation after Pneumonectomy: Experimental Study

ISRN Surgery ◽

10.1155/2013/864350 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Eleftherios Spartalis ◽

Periklis Tomos ◽

Petros Konofaos ◽

Grigorios Karagkiouzis ◽

Georgia Levidou ◽

...

Keyword(s):

Platelet Rich Plasma ◽

Healing Process ◽

Bronchopleural Fistula ◽

White Blood Cells ◽

Buffy Coat ◽

Optimum Number ◽

Therapeutic Effects ◽

Bronchial Stump ◽

Platelet Concentration ◽

Autologous Platelet Rich Plasma

Objectives. Recent advances in perioperative management, antibiotics, and surgical materials, including mechanical staplers, have decreased the operative risk of pulmonary resection. However, bronchopleural fistula can still occur in some instances, the occurrence often being lethal. This study investigated whether platelet-rich plasma (PRP) promotes granulation of the bronchial stump after pneumonectomy. Methods. Ten pigs were randomized into two groups: (A) control or non-PRP group (pneumonectomy) and (B) PRP group (pneumonectomy and PRP application). PRP was obtained by spinning down the animal’s own blood and collecting the buffy coat containing platelets and white blood cells. Results. Increased platelet concentration triggered the healing process. The percentage of granulation tissue formed at the stumps was significantly higher in the PRP group of animals. This observation was confirmed when statistical analysis using Mann-Whitney U test was performed (P=0.0268). Conclusions. PRP is easily produced with minimal basic equipment and is useful in accelerating granulation of the bronchial stump, although the timing and optimum number of applications in humans require further study. Autologous PRP is a safe, feasible, and reliable new healing promoter with potential therapeutic effects.

Download Full-text

White blood cell fragments in platelet concentrates prepared by the platelet-rich plasma or buffy-coat methods

Vox Sanguinis ◽

10.1111/j.1423-0410.2005.00634.x ◽

2005 ◽

Vol 88 (4) ◽

pp. 275-277 ◽

Cited By ~ 10

Author(s):

M. J. Dijkstra-Tiekstra ◽

C. E. van der Schoot ◽

R. N. I. Pietersz ◽

H. W. Reesink

Keyword(s):

Blood Cell ◽

White Blood Cell ◽

Platelet Rich Plasma ◽

Buffy Coat ◽

Platelet Concentrates ◽

Cell Fragments

Download Full-text

Comparison of Platelet Concentrates Prepared from Platelet Rich Plasma-Platelet Concentrate and Buffy Coat Poor-Platelet Concentrate on Storage Days 1 and 5

Songklanagarind Medical Journal ◽

10.31584/smj.2017.35.1.617 ◽

2016 ◽

Vol 35 (2) ◽

pp. 5

Author(s):

Darinnat Buathong ◽

Jarin Buakaew ◽

Chutarat Nukfon

Keyword(s):

Blood Cell ◽

White Blood Cell ◽

Platelet Rich Plasma ◽

Buffy Coat ◽

Platelet Concentrate ◽

P Value ◽

Platelet Concentrates ◽

Hypotonic Shock ◽

Shock Response ◽

The Mean

Objective: The purpose of this study is to assess the quality of platelet concentrates on storage days 1 and 5 prepared by platelet rich plasma-platelet concentrate (PRP-PC) and buffy coat poor-platelet concentrate (BC-PC) methods comparing to the American Association of Blood Banks (AABB) recommendations.Material and Method: Totally of 120 platelet concentrates (PC) units on storage days 1 and 5 (60 of PRP-PC triple blood bag and 60 of BC-PC quadruple AS-5 blood bag) were separated from whole blood donations at Songklanagarind Hospital. The prepared PC were assessed with 5 parameters such as volume, platelet count, white blood cell count per unit, pH, swirling phenomenon score and hypotonic shock response. The independent t-tests, paired Student’s t-tests and SPSS program were utilized in statistical analysis step.Results: The mean±standard deviation (S.D.) of each parameter were as follow : (1) Volume of PRP-PC and BC-PC met the standard (40-70 ml). (2) All of the platelet concentrates met the standard (≥ 5.5x1010/unit). The mean±S.D.: PRP-PC and BC-PC (day 1) were 6.820±1.480 x1010 and 7.010±1.300 x1010/unit (p-value=0.260), while PRP-PC and BC-PC (day 5) were 6.620±1.160x1010 and 6.720± 1.150x1010/unit (p-value=0.040). (3) The white blood cell in platelet concentrates met the standard (<0.2x1010/unit). The mean±S.D.: PRP-PC and BC-PC (day 1) were 0.030±0.017 x1010and 0.026±0.019x1010/unit (p-value=0.040), while PRP-PC and BC-PC (day 5) were0.033±0.013x1010 and 0.027±0.019x1010/unit (p-value= 0.580). (4) The pH of all units (PRP-PC and BC-PC) met the standard (≥6.2). The mean±S.D.: PRP-PC and BC-PC (day 1) were 7.430±0.330 and 7.750±0.160 (p-value=0.006), while PRP-PC and BC-PC (day 5) were 7.590±0.350 and 7.620±0.280 (p-value=0.710). The swirling phenomenon score and hypotonic shock response were the same as standard AABB and were not statistically difference.Conclusion: The quality of PRP-PC and BC-PC after storing on days 1 and 5 as follow (1) Volume of PRP-PC and BC-PC met the standard. (2) The platelet count per unit of PRP-PC and BC-PC (day 1), PRP-PC and BC-PC (day 5) were not statistically difference. (3) The white blood cell count per unit of PRP-PC and BC-PC (day 1) were statistically difference, while PRP-PC and BC-PC (day 5) were not statistically difference. (4) The pH of PRP-PC and BC-PC (day 1) were statistically difference, while PRP-PC and BC-PC (day 5) were not statistically difference. The swirling phenomenon score and hypotonic shock response of PRP-PC and BC-PC were not statistically difference. Platelet concentrates of both method storing on days 1 and 5 fulfilled the quality guideline of AABB.

Download Full-text

Comparative Evaluation of the Angiogenic Potential of Hypoxia Preconditioned Blood-Derived Secretomes and Platelet-Rich Plasma: An In Vitro Analysis

Biomedicines ◽

10.3390/biomedicines8010016 ◽

2020 ◽

Vol 8 (1) ◽

pp. 16 ◽

Cited By ~ 2

Author(s):

Philipp Moog ◽

Katharina Kirchhoff ◽

Sanjar Bekeran ◽

Anna-Theresa Bauer ◽

Sarah von Isenburg ◽

...

Keyword(s):

Platelet Rich Plasma ◽

Autologous Blood ◽

Aortic Ring ◽

Peripheral Blood Cell ◽

Angiogenic Factor ◽

Angiogenic Potential ◽

Tissue Repair And Regeneration ◽

Platelet Concentration ◽

Vitro Analysis

Blood-derived factor preparations are being clinically employed as tools for promoting tissue repair and regeneration. Here we set out to characterize the in vitro angiogenic potential of two types of frequently used autologous blood-derived secretomes: platelet-rich plasma (PRP) and hypoxia preconditioned plasma (HPP)/serum (HPS). The concentration of key pro-angiogenic (VEGF) and anti-angiogenic (TSP-1, PF-4) protein factors in these secretomes was analyzed via ELISA, while their ability to induce microvessel formation and sprouting was examined in endothelial cell and aortic ring cultures, respectively. We found higher concentrations of VEGF in PRP and HPP/HPS compared to normal plasma and serum. This correlated with improved induction of microvessel formation by PRP and HPP/HPS. HPP had a significantly lower TSP-1 and PF-4 concentration than PRP and HPS. PRP and HPP/HPS appeared to induce similar levels of microvessel sprouting; however, the length of these sprouts was greater in HPP/HPS than in PRP cultures. A bell-shaped angiogenic response profile was observed with increasing HPP/HPS dilutions, with peak values significantly exceeding the PRP response. Our findings demonstrate that optimization of peripheral blood cell-derived angiogenic factor signalling through hypoxic preconditioning offers an improved alternative to simple platelet concentration and release of growth factors pre-stored in platelets.

Download Full-text