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Changes in platelet morphology and function during 24 hours of storage.
Abstract: For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according to a standardized protocol and examined immediately after blood collection, four hours and twenty four hours later. The capability of platelets to adhere and form stable aggregates (PFA100, closure time) was examined in sodium citrate anticoagulated whole blood (WB) using the agonists equine type I collagen and epinephrine bitartrate (collagen/epinephrine) as well as equine type I collagen and adenosine-5'-diphosphate (collagen/ADP). Circulating platelets were quantified at each time point. Morphology of platelets and platelet aggregates were visualized microscopically and measured using an electric field multi-channel counting system (CASY). The percentage of activated platelets was assessed by means of P-selectin (CD62P) expression of circulating platelets. Furthermore, platelet factor 4 (PF4) release was measured in platelet poor plasma (PPP) at each time point. Whole blood PFA100 closure times increased after stimulation with collagen/ADP and collagen/epinephrine. Twenty four hours after blood collection, both parameters were prolonged pathologically above the upper limit of the reference range. Numbers of circulating platelets, measured in PRP, decreased after four hours, but no longer after twenty four hours. Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL - 40 fL) decreased over time. Immediately after blood collection, no debris or platelet aggregates could be visualized microscopically. After four hours, first debris and very small aggregates occurred. After 24 hours, platelet aggregates and also debris progressively increased. In accordance to this, the CASY system revealed an increase of platelet aggregates (up to 90 μm diameter) with increasing storage time. The percentage of CD62P positive platelets and PF4 increased significantly with storage time in resting PRP. When soluble ADP was added to stored PRP samples, the number of activatable platelets decreased significantly over storage time. The present study reveals the importance of a consequent standardization in the preparation of WB and PRP. Platelet morphology and function, particularly platelet reactivity to adherent or soluble agonists in their surrounding milieu, changed rapidly outside the vascular system. This knowledge is of crucial interest, particularly in the field of biomaterial development for cardiovascular applications, and may help to define common standards in the in vitro hemocompatibility testing of biomaterials.
Publication Date: 2014-09-18 PubMed ID: 25227196DOI: 10.3233/CH-141876Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research analyses how the morphology and function of platelets change during a 24-hour storage process. The findings emphasize the need for the standardization of protocols employed in the preparation of “platelet rich plasma” used in in vitro studies; this is to mitigate discrepancies in results arising from diverse protocols since platelet aging during prolonged storage times could lead to underestimation of material thrombogenicity.
Research Methodology
- In this study, blood was collected from seemingly healthy subjects following a standardized protocol and examined immediately, and then reassessed four hours and twenty-four hours later.
- The ability of platelets to stick and form stable aggregates was evaluated using different compounds known as ‘agonists’ in sodium citrate anticoagulated whole blood (WB).
- The number of circulating platelets was measured at each time point, with changes in platelet morphology and aggregation visualized under a microscope and measured using an electric field multi-channel counting system (CASY).
- The study also evaluated the percentage of activated platelets by assessing the P-selectin (CD62P) expression.
- The release of platelet factor 4 (PF4) was also monitored over time in platelet poor plasma (PPP).
Research Findings
- The study revealed an increase in whole blood PFA100 closure times after stimulation with two different types of agonist combinations.
- Prolonged parameters were observed 24 hours post blood collection, exceeding the upper limit of the reference range.
- While the number of circulating platelets decreased after four hours, no further decline was noted after twenty-four hours.
- Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL – 40 fL) both decreased over time.
- Microscopic evaluation showed no platelet debris or aggregation immediately after blood collection; however, slight aggregates and debris appeared after four hours and increased significantly after 24 hours.
- The rate of activated platelets, identified by CD62P expression, and PF4 increased over time during storage in resting PRP.
- When soluble ADP was added to stored PRP samples, the activatable platelet numbers declined substantially over the storage period.
Conclusion and Implications
The study emphasizes the necessity of standardized methods in the preparation of Whole Blood (WB) and Platelet Rich Plasma (PRP). It was found that the physical properties and functioning of platelets — especially their reactivity — changed quickly outside the vascular system. This has important implications, particularly in the creation of biomaterials for cardiovascular applications, as it will help to establish common standards for in vitro hemocompatibility testing of these materials.
Cite This Article
APA
Braune S, Walter M, Schulze F, Lendlein A, Jung F.
(2014).
Changes in platelet morphology and function during 24 hours of storage.
Clin Hemorheol Microcirc, 58(1), 159-170.
https://doi.org/10.3233/CH-141876 Publication
Researcher Affiliations
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany Institute of Chemistry, University of Potsdam, Potsdam, Germany.
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany Institute of Chemistry, University of Potsdam, Potsdam, Germany.
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany.
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany Institute of Chemistry, University of Potsdam, Potsdam, Germany.
- Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany.
MeSH Terms
- Adult
- Biocompatible Materials / chemistry
- Blood Platelets / cytology
- Blood Platelets / metabolism
- Blood Preservation / methods
- Citrates / chemistry
- Collagen / chemistry
- Epinephrine / chemistry
- Female
- Humans
- Male
- Middle Aged
- P-Selectin / chemistry
- Platelet Aggregation
- Platelet-Rich Plasma / metabolism
- Sodium Citrate
- Specimen Handling
- Time Factors
- Young Adult
Citations
This article has been cited 7 times.- Kerstholt M, van de Schoor FR, Oosting M, Moorlag SJCFM, Li Y, Jaeger M, van der Heijden WA, Tunjungputri RN, Dos Santos JC, Kischkel B, Vrijmoeth HD, Baarsma ME, Kullberg BJ, Lupse M, Hovius JW, van den Wijngaard CC, Netea MG, de Mast Q, Joosten LAB. Identifying platelet-derived factors as amplifiers of B. burgdorferi-induced cytokine production. Clin Exp Immunol 2022 Oct 21;210(1):53-67.
- Lu Q, Nehrer JM, Li J, Jamiolkowski MA, Rinaldi JE, Malinauskas RA. Platelet and leukocyte count assay for thrombogenicity screening of biomaterials and medical devices: Evaluation and improvement of ASTM F2888 test standard. J Biomed Mater Res B Appl Biomater 2021 Dec;109(12):2259-2267.
- Piao J, Yoo C, Kim S, Whang YW, Choi CU, Shin S. Performance comparison of aspirin assay between Anysis and VerifyNow: Assessment of therapeutic platelet inhibition in patients with cardiac diseases. Clin Hemorheol Microcirc 2021;79(2):327-334.
- McMahon SR, Chava S, Taatjes-Sommer HS, Meagher S, Brummel-Ziedins KE, Schneider DJ. Variation in platelet expression of FcγRIIa after myocardial infarction. J Thromb Thrombolysis 2019 Jul;48(1):88-94.
- Jamiolkowski MA, Hartung MC, Malinauskas RA, Lu Q. An In Vitro Blood Flow Loop System for Evaluating the Thrombogenicity of Medical Devices and Biomaterials. ASAIO J 2020 Feb;66(2):183-189.
- Schneider DJ, Taatjes-Sommer HS, DiBattiste PM, Palla KS, Shovah T, Biswas S, Ohrnberger J. Assessing prognosis by quantifying FcγRIIa on fixed platelets. Bioanalysis 2024;16(19-20):1025-1032.
- Inoue M, Ohwada M, Watanabe N. The shear rate promotes pinocytosis of extracellular dextran in platelets. Clin Hemorheol Microcirc 2024;87(2):237-247.
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