Centrifugation Protocol Research Articles

Evaluation of an alternative centrifugation protocol for reducing total turnaround time

Abstract Objectives Centrifugation is a key process that should be controlled to ensure an adequate sample quality. To achieve rapid, standardized, and consistent sample centrifugation, we aimed to evaluate an alternative centrifugation protocol and its impact on the results of 20 biochemical determinations in serum. Methods The study included 45 ambulatory patients. Two serum-separating tubes were collected from each patient (Becton Dickinson (BD) Vacutainer® SST™ II Advance, 8.5 mL Ref. 366468). One of the tubes was centrifuged at 2530 × g for 10 min (control method), while the other tube was centrifuged under alternative centrifugation conditions, namely 2530 × g for 7 min. Results The analysis of results revealed that calcium, total bilirubin, and magnesium exhibited a constant and proportional systematic bias. However, considering the proportional mean difference, all analytes met the desirable bias performance established by our laboratory, except for magnesium, which met the minimum bias criteria. Conclusions Our study demonstrates that both centrifugation protocols are interchangeable for measuring the studied analytes, thereby ensuring adequate sample quality.

Use of Advanced Platelet-Rich Fibrin for the Treatment of Multiple Adjacent Mucogingival Recessions: A Technical Report of the Fibrin-Assisted Soft-Tissue Promotion Protocol.

Platelet-rich fibrin (PRF) has had a marked impact on regenerative medicine due to its widespread ability to promote angiogenesis to defective tissues. Particularly in the dental field, evidence from randomized clinical trials has further shown that PRF facilitates greater soft-tissue regeneration when compared with hard tissues. Recently, the fibrin-assisted soft-tissue promotion (FASTP) technique has been developed as a means to promote soft-tissue regeneration of mucogingival recessions utilizing PRF. Within the present case report, a 28-year-old male presented with multiple adjacent mucogingival recessions in the maxilla ranging in probing depths (1-3 mm) and gingival recessions (1-5 mm). For optimal regenerative outcomes, the use of advanced PRF (A-PRF; 1300 RPM for 8 min) has been utilized to enhance regenerative outcomes by fully taking advantage of the low-speed centrifugation concept (LSCC). This case report highlights the latest surgical concepts, centrifugation protocols, and use of the LSCC to regenerate multiple adjacent mucogingival recessions in the esthetic zone.

Autologous platelet concentrates in root coverage procedures.

Gingival recessions are vastly prevalent among the general population. With regards to their treatment, recent advancements in periodontal and microsurgical procedures, focusing on minimal invasiveness and patient-centered therapies, have propelled a shift in their contemporary treatment, highlighting the field of biologics and bioactive mediators. Among different classes and types of biologics, autologous platelet concentrates (APCs), also referred to as autologous blood-derived products, are commonly used and preferred among many clinicians. These are essentially obtained via venipuncture (intravenous access) followed by centrifugation, for which numerous protocols and preparation methods have been used, leading to varieties of blood-derived products. In this review, via a systematic search, we explored the efficacy of the different utilized preparation methods and centrifugation protocols of APCs (e.g., platelet-rich plasma (PRP), platelet-rich fibrin (PRF), leucocyte-PRF, advanced-PRF, concentrated growth factor (CGF), etc.) for the treatment of type 1 gingival recessions (RT1, without interproximal attachment loss or noticeable tooth displacement), as well as their effectiveness relative to a common control (treatment with flap advancement alone without any additional material). Based on the available literature from randomized trials found in our systematic search, we observed that utilization of PRF can significantly enhance treatment outcomes when performing a coronally advanced flap, in terms of the amount of root coverage. The improvement in root coverage was further enhanced in the presence of baseline keratinized tissue width, and with an increasing relative magnitude (the more the baseline keratinized tissue width, the better the root coverage outcomes when using PRF). The efficacy of these products needs to be further explored with different graft substitutes and matrices, as well as relative to other commonly applied biologics, through well-conducted and adequately-powered randomized clinical trials.

Understanding and controlling the variables for stromal vascular fraction therapy.

In regenerative medicine, the isolation of mesenchymal stromal cells (MSCs) from the adipose tissue's stromal vascular fraction (SVF) is a critical area of study. Our review meticulously examines the isolation process of MSCs, starting with the extraction of adipose tissue. The choice of liposuction technique, anatomical site, and immediate processing are essential to maintain cell functionality. We delve into the intricacies of enzymatic digestion, emphasizing the fine-tuning of enzyme concentrations to maximize cell yield while preventing harm. The review then outlines the filtration and centrifugation techniques necessary for isolating a purified SVF, alongside cell viability assessments like flow cytometry, which are vital for confirming the efficacy of the isolated MSCs. We discuss the advantages and drawbacks of using autologous vs allogeneic SVF sources, touching upon immunocompatibility and logistical considerations, as well as the variability inherent in donor-derived cells. Anesthesia choices, the selection between hypodermic needles vs liposuction cannulas, and the role of adipose tissue lysers in achieving cellular dissociation are evaluated for their impact on SVF isolation. Centrifugation protocols are also analyzed for their part in ensuring the integrity of the SVF. The necessity for standardized MSC isolation protocols is highlighted, promoting reproducibility and successful clinical application. We encourage ongoing research to deepen the understanding of MSC biology and therapeutic action, aiming to further the field of regenerative medicine. The review concludes with a call for rigorous research, interdisciplinary collaboration, and strict adherence to ethical and regulatory standards to safeguard patient safety and optimize treatment outcomes with MSCs.

Systematic Review of Platelet-Rich Plasma in Medical and Surgical Specialties: Quality, Evaluation, Evidence, and Enforcement.

Background: Platelet-rich plasma (PRP) is widely used in various medical and surgical specialties for its regenerative properties, including aesthetics (facial rejuvenation, hair restoration, and skin tightening) and orthopedics (treatment of tendinitis and osteoarthritis). However, the inconsistent literature on PRP's efficacy and safety leads to critical knowledge gaps. This systematic review evaluates quality control measures in PRP preparation and application and explores the regulatory environment governing its clinical use. Methods: Following PRISMA guidelines, a comprehensive search was conducted across multiple databases, including PubMed, EMBASE, and Web of Science, for studies published from January 2020 to April 2024. The review included randomized controlled trials (RCTs) involving human participants undergoing PRP treatment for aesthetic or regenerative purposes. Key parameters such as the PRP preparation methods, platelet concentration, and quality control measures were analyzed. The study protocol was registered with PROSPERO (ID: CRD42024557669). Results: Out of 75 RCTs involving 5726 patients, the review identified significant variability in PRP preparation methods and application techniques, including differences in centrifugation protocols and platelet concentration levels. A new evidence-based scoring system, the William-Eqram Scoring System for PRP Quality Reporting (WESS-PQR), was proposed to address these inconsistencies. Correlation analysis revealed a strong positive correlation (r = 0.79) between proper temperature control during preparation and PRP efficacy. Initial platelet count assessment showed a moderate positive correlation (r = 0.57) with efficacy. Conclusions: Standardized PRP preparation protocols and robust regulatory frameworks are urgently needed to ensure the safety and efficacy of PRP treatments. The proposed WESS-PQR scoring system can serve as a valuable tool for clinicians and researchers, promoting consistency and reliability in PRP applications.

Influence of haematological parameters on size of the advanced platelet-rich fibrin+ (A-PRF+) in the horse

The advanced-PRF+ (A-PRF+) is a platelet concentrate, showing a higher concentration of growth factors, an increased number of cells and looser structure of the fibrin clot than leukocyte-PRF. A high variability in the size of PRF associated with patients, haematological features and centrifugation protocols was reported. The aims of this study were to evaluate the feasibility of A-PRF+ production in the field and the correlation between haematological parameters, macroscopic and microscopic features in equine A-PRF+. Samples from twenty Standardbred horses (3–7 years) were harvested with glass tubes without anticoagulants, previously heated at 37 °C. Blood samples were centrifugated at 1300 rpm for 8 min with a fixed-angle centrifuge and a horizontal centrifuge in the field, at a temperature of 15–17 °C. Clots were measured and placed on the Wound Box® for a 2-min compression. Membranes were measured and fixed in 10% formalin for histological examination. Clot and membrane surface did not differ between sex and centrifuge. Haematological parameters did not show a significant correlation to clot and membrane size. Membranes obtained from both centrifugation protocols showed a loose fibrin structure and cells evenly distributed throughout the clot. Tubes' warming was effective to obtain A-PRF+ clots from all samples, regardless the environmental temperature. Further studies are needed to evaluate the influence of other blood molecules on the A-PRF+ structure and size.

3D printable and biocompatible PEDOT:PSS-ionic liquid colloids with high conductivity for rapid on-demand fabrication of 3D bioelectronics

3D printing has been widely used for on-demand prototyping of complex three-dimensional structures. In biomedical applications, PEDOT:PSS has emerged as a promising material in versatile bioelectronics due to its tissue-like mechanical properties and suitable electrical properties. However, previously developed PEDOT:PSS inks have not been able to fully utilize the advantages of commercial 3D printing due to its long post treatment times, difficulty in high aspect ratio printing, and low conductivity. We propose a one-shot strategy for the fabrication of PEDOT:PSS ink that is able to simultaneously achieve on-demand biocompatibility (no post treatment), structural integrity during 3D printing for tall three-dimensional structures, and high conductivity for rapid-prototyping. By using ionic liquid-facilitated PEDOT:PSS colloidal stacking induced by a centrifugal protocol, a viscoplastic PEDOT:PSS-ionic liquid colloidal (PILC) ink was developed. PILC inks exhibit high-aspect ratio vertical stacking, omnidirectional printability for generating suspended architectures, high conductivity (~286 S/cm), and high-resolution printing (~50 µm). We demonstrate the on-demand and versatile applicability of PILC inks through the fabrication of 3D circuit boards, on-skin physiological signal monitoring e-tattoos, and implantable bioelectronics (opto-electrocorticography recording, low voltage sciatic nerve stimulation and recording from deeper brain layers via 3D vertical spike arrays).

A lateral flow immunoassay for the rapid identification of Candida auris from isolates or directly from surveillance enrichment broths.

Candida auris is a recently discovered yeast with a multi-drug resistant profile associated with high mortality rates. The rapid identification of Candida auris in hospital settings is crucial to allow appropriate therapeutic and rapid implementation of infection management measures. The aim of this study was to develop a lateral flow immunoassay (LFIA) for the rapid identification of Candida auris. Highly specific monoclonal antibodies were obtained by immunizing mice with membrane proteins from Candida auris which were then used to develop a LFIA whose performance was assessed by testing 12 strains of Candida auris and 37 strains of other Candida species. Isolates were grown on either Sabouraud dextrose, CHROMagarTM Candida Plus or HardyCHROMTM Candida + auris agar plates. The strains were also cultured on salt sabouraud-dextrose with chloramphenicol or a commercially available Salt-Sabouraud Dulcitol Broth with chloramphenicol and gentamicin, and processed using a simple centrifugation protocol to recover a pellet. Finally, the colonies or yeast extract were transferred to the LFIA to determine the specificity and sensitivity of the assay. The LFIA reached 100% specificity and sensitivity from solid agar plates. For both enrichment broths, some Candida non-auris species were able to grow, but the LFIA remained 100% specific. The use of a dextrose-based sabouraud broth resulted in earlier identification with the LFIA, with most of the Candida auris strains detected at 24 h. The developed LFIA prototype represents a powerful tool to fight the emerging threat of Candida auris. Clinical validation represents the next step.

Essential principles for blood centrifugation.

Currently, autologous platelet concentrates (APCs) are frequently used for soft- and hard-tissue regeneration, not only within the oral cavity, but also extra-orally including chronic wounds, burns, joints, dermatological conditions, among others. The benefits of APCs are largely influenced by the treatment strategy but also their preparation. This paper therefore discusses in detail: the physical properties of blood cells, the basic principles of blood centrifugation, the impact of the centrifugation protocol (rotations/revolutions per minute, g-force, variation between centrifuges), the importance of timing during the preparation of APCs, the impact of the inner surface of the blood tubes, the use/nonuse of anticoagulants within APC tubes, the impact of the patient's hematocrit, age, and gender, as well as the important requirements for an optimal centrifugation protocol. All these variables indeed have a significant impact on the clinical outcome of APCs.

Enhancing pneumococcal bacteraemia diagnosis: A comparative assessment of culture-independent assays (MALDI–TOF–MS Sepsityper® module and a lateral flow inmunochromatography test)

IntroductionPneumococcal bacteraemia is a major contributor to global morbidity and mortality. Traditional culture-based methods lack sensitivity and are time-consuming. This study aimed to assess the effectiveness of two culture-independent assays, the MALDI–TOF–MS Sepsityper® module and the lateral flow inmunochromatography test (LFICT) with the Standard F® Streptococcus pneumoniae, directly from positive blood culture (BC) bottles. MethodsA prospective study was conducted from December 2021 to July 2022. For all BC positives for S. pneumoniae a double centrifugation protocol was implemented. The resulting pellet was subsequently processed using both techniques. ResultsThe LFICT showed exceptional performance with 100% sensitivity and specificity, outperforming the MALDI–TOF–MS Sepsityper® module, which achieved 85.2% sensitivity and 100% specificity. Nevertheless, the combination of these assays offers a robust and comprehensive approach to diagnosis. ConclusionsThe simultaneous use of both techniques offers a promising alternative that can be integrated into routine practices directly from BC samples.

Leukocytes within Autologous Blood Concentrates Have No Impact on the Growth and Proliferation of Human Primary Osteoblasts: An In Vitro Study.

Platelet-rich fibrin (PRF) is a widely used autologous blood concentrate in regenerative medicine. This study aimed to characterize the cellular composition and distribution of different PRF matrices generated by high (710 g) and low (44 g) relative centrifugal forces (RCFs) and to analyze their bioactivity on human primary osteoblasts (pOBs). PRF was separated into upper layer (UL) and buffy coat (BC) fractions, and their cellular contents were assessed using histological and immunohistochemical staining. The release of platelet-derived growth factor (PDGF) and transforming growth factor (TGF-β) was quantified using an ELISA. Indirect PRF treatment on pOBs was performed to evaluate cell viability and morphology. A histological analysis revealed higher quantities of leukocytes and platelets in the low-RCF PRF. TGF-β release was significantly higher in the low-RCF PRF compared to the high-RCF PRF. All PRF fractions promoted pOB proliferation regardless of the centrifugation protocol used. The low-RCF PRF showed higher TGF-β levels than the high-RCF PRF. These findings contribute to understanding the cellular mechanisms of PRF and provide insights into optimizing PRF protocols for bone regeneration, advancing regenerative medicine, and improving patient outcomes.

Small-scale perfusion mimic cultures in the ambr250 HT bioreactor system

Perfusion cell cultures generate higher viable cell densities (VCD) and volumetric productivity compared to fed-batch cultures. However, due to the limited availability of small-scale perfusion models, perfusion systems are seldom used to produce licensed biotherapeutics. This study evaluated two small-scale perfusion mimic protocols to bridge the research-to-production gap. Shake flasks and the ambr250 HT were used to compare centrifugation and in situ gravity settling protocols. The centrifugation protocol achieved a peak VCD >50 million cells/mL and is well-suited to media formulation and feeding strategy comparisons. The in situ gravity settling protocol achieved ∼20 million cells/mL and is well-suited to cell productivity and stability studies. The cell retention steps resulted in temporary DO and pH changes but did not affect the overall culture's health. Further, both protocols were able to recover from an imposed long-duration DO stress, although the centrifugation protocol cultures had higher cell specific oxygen consumption rates indicative of higher culture stress. Both protocols sustained the cultures for 39 days with stable cell specific productivities (∼27 pg/cell·day). Overall, this study demonstrated the feasibility of two economic small-scale perfusion mimic protocols in a standard small-scale bioreactor system, which could be translated to other multi-unit small-scale bioreactor systems.

Comparative analysis of the effects of different purification methods on the yield and purity of cow milk extracellular vesicles

AbstractIsolation of extracellular vesicles (EV) has been developing rapidly in parallel with the interest in EVs. However, commonly utilized protocols may not suit more challenging sample matrixes and could potentially yield suboptimal results. Knowing and assessing the pitfalls of isolation procedure to be used, should be involved to some extent for EV analytics. EVs in cow milk are of great interest due to their abundancy and large‐scale availability as well as their cross‐species bioavailability and possible use as drug carriers. However, the characteristics of milk EVs overlap with those of other milk components. This makes it difficult to isolate and study EVs individually. There exists also a lack of consensus for isolation methods. In this study, we demonstrated the differences between various differential centrifugation‐based approaches for isolation of large quantities of EVs from cow milk. Samples were further purified with gradient centrifugation and size exclusion chromatography (SEC) and differences were analyzed. Quality measurements were conducted on multiple independent platforms. Particle analysis, electron microscopy and RNA analysis were used, to comprehensively characterize the isolated samples and to identify the limitations and possible sources of contamination in the EV isolation protocols. Vesicle concentration to protein ratio and RNA to protein ratios were observed to increase as samples were purified, suggesting co‐isolation with major milk proteins in direct differential centrifugation protocols. We demonstrated a novel size assessment of vesicles using a particle mobility analyzer that matched the sizing using electron microscopy in contrast to commonly utilized nanoparticle tracking analysis. Based on the standards of the International Society for Extracellular Vesicles and the quick checklist of EV‐Track.org for EV isolation, we emphasize the need for complete characterization and validation of the isolation protocol with all EV‐related work to ensure the accuracy of results and allow further analytics and experiments.

Three Milliliters of Peripheral Blood Is Sufficient for Preparing Liquid Platelet-Rich Fibrin (PRF): An In Vitro Study.

Platelet-rich fibrin (PRF) has assumed an important role in supporting tissue regeneration in different fields. To date, the standard protocol for liquid PRF requires at least 10 mL of peripheral blood. The present study aimed to analyze the composition, growth factor release, and effects on the cell proliferation of PRF samples produced using 3 mL vs. 10 mL of peripheral blood in vitro. Peripheral venous blood from six healthy donors was used to prepare liquid PRF using either 3 mL or 10 mL tubes. Three different centrifugation protocols were used according to the low-speed centrifugation concept. The cellular distribution was evaluated using immunohistology and automated cell count. ELISA was used to determine the release of different growth factors (EGF, TGF-β1, and PDGF) and interleukin 8 at different time points. Primary human osteoblasts (pOBs) were cultivated for 7 days using PRF-conditioned media acquired from either 3 mL or 10 mL of peripheral blood. The results showed that 3 mL of peripheral blood is sufficient to produce a liquid PRF concentrate similar to that acquired when using 10 mL blood. The concentrations of platelets and leukocytes were comparable regardless of the initial blood volume (3 mL vs. 10 mL). Similarly, the release of growth factors (EGF, TGF-β1, and PDGF) and interleukin 8 was often comparable in both groups over 7 days. The cultivation of pOBs using PRF-conditioned media showed a similar proliferation rate regardless of the initial blood volume. This proliferation rate was also similar to that of pOBs treated with 20% FBS-conditioned media. These findings validated the use of 3 mL of peripheral blood to generate liquid PRF matrices according to the low-speed centrifugation concept, which may open new application fields for research purposes such as in vivo experiments and clinical applications such as pediatric surgery.

Differentiation of large extracellular vesicles in oral fluid: Combined protocol of small force centrifugation and sedimentation pattern analysis

AbstractExtracellular vesicles (EVs) in biofluids are highly heterogeneous entities in terms of their origins and physicochemical properties. Considering the application of EVs in diagnostic and therapeutic fields, it is of extreme importance to establish differentiating methods by which focused EV subclasses are operationally defined. Several differentiation protocols have been proposed; however, they have mainly focused on smaller types of EVs, and the heterogeneous nature of large EVs has not yet been fully explored. In this report, to classify large EVs into subgroups based on their physicochemical properties, we have developed a protocol, named EV differentiation by sedimentation patterns (ESP), in which entities in the crude large EV fraction are first moved through a density gradient of iodixanol with small centrifugation forces, and then the migration patterns of molecules through the gradients are analysed using a non‐hierarchical data clustering algorithm. Based on this method, proteins in the large EV fractions of oral fluids clustered into three groups: proteins shared with small EV cargos and enriched in immuno‐related proteins (Group 1), proteins involved in energy metabolism and protein synthesis (Group 2), and proteins required for vesicle trafficking (Group 3). These observations indicate that the physiochemical properties of EVs, which are defined through low‐speed gradient centrifugation, are well associated with their functions within cells. This protocol enables the detailed subclassification of EV populations that are difficult to differentiate using conventional separation methods.

Fibrin Network and Platelets Densities in Platelet-Rich Fibrin (PRF) Membranes Produced from Plastic Tubes Without Additives: A New Approach to PRF Clinical Use.

The present study aimed to investigate plastic tubes without additives as alternatives to glass and silica-coated plastic tubes, in the production of PRF membranes. Nine blood samples were collected from eight volunteers (n = 8) separated into three groups, according to tube material: glass, silica-coated plastic, and plastic without additives. In each group, the samples were centrifuged using different relative centrifugation forces: L-PRF (700g/12min), A-PRF (200g/14min), and A-PRF + (200g/8min). The generated membranes were evaluated by histomorphometry, considering the fibrin network, platelet aggregates, and cellular morphology, by light microscopy. The ultrastructural cellular morphology integrity was evaluated by transmission electron microscopy. The L-PRF (p < 0.019) and A-PRF (p < 0.001) membranes showed a significantly lower fibrin network density in plastic tubes without additives compared to glass and silica-coated plastic tubes. Plastic tubes without additives revealed a significantly higher platelet percentage, regardless of the protocol (p < 0.005). In all groups, TEM analysis showed preserved normal morphological ultrastructure, maintaining the integrity of cellular components. Plastic tubes without additives offer a viable alternative for producing PRF membranes. They exhibited a higher platelet density and demonstrated fibrin network and cellular morphology similar to those of glass and silica-coated plastic tubes, irrespective of the centrifugation protocol.

CHARACTERIZATION OF PHYSICAL AND CELLULAR PROPERTIES OF MEMBRANES OF L-PRF PRODUCED BY DIFFERENT CENTRIFUGES/ CENTRIFUGATION PROTOCOLS.

Leukocyte and platelet-rich fibrin (L-PRF), an essential source of cytokines and growth factors, has been widely used to regulate the healing process. The quality of the L-PRF obtained is dependent on technical factors and the centrifugation protocol used. Thus, the present study aimed to macro and microscopically evaluate the formation of the L-PRF membrane/clot in two centrifuge models and protocols recommended by manufacturers. Four blood samples from 8 healthy volunteers undergoing rehabilitation surgery with dental implants were used. In group 1 of the Montserrat centrifuge (model 80-2B 15mL) 400g of centrifugal force were used for 10 minutes, 27ºC, and 2700 rpm, while in group 2 of the Intra Spin-Intra-Lock centrifuge (IntraSpin™, Intra-Lock Iberia) 400g of centrifugal force were used for 12 minutes, 27ºC and 2700 rpm. After obtaining 32 L-PRF clots/membranes, the pieces were recorded, measured, and weighed, separating the L-PRF from the supernatant/clot of red blood cells, remaining in the tubes. The specimens were then histologically processed for Hematoxylin/Eosin (H/E) staining and each slide was divided into three called areas: proximal, center, and distal. The presence of leukocytes was analyzed semi-quantitatively in each area. The results revealed no significant macroscopic differences in L-PRF between the two groups. However, the central areas of the L-PRF membranes produced in group 2 showed higher intensity of leukocytes (p=0.012), as well as the presence of concomitant leukocytes in at least 2 of the evaluated areas (p=0.003). In the proximal area, no significant differences were observed between the two groups. The results allow us to postulate that technical protocol factors influence the presence and distribution of leukocytes between the L-PRF membranes, obtained in different centrifuges.

Comparative Evaluation of Platelet-derived Growth Factor Isoforms between Platelet-rich Fibrin Prepared with Three Different Centrifugation Protocols

Comparative Evaluation of Platelet-derived Growth Factor Isoforms between Platelet-rich Fibrin Prepared with Three Different Centrifugation Protocols

Gamma-Camera Direct Imaging of the Plasma and On/Intra Cellular Distribution of the 99mTc-DPD-Fe3O4 Dual-Modality Contrast Agent in Peripheral Human Blood.

The radiolabeled iron oxide nanoparticles constitute an attractive choice to be used as dual-modality contrast agents (DMCAs) in nuclear medical diagnosis, due to their ability to combine the benefits of two imaging modalities, for instance single photon emission computed tomography (SPECT) with magnetic resonance imaging (MRI). Before the use of any DMCA, the investigation of its plasma extra- and on/intra cellular distribution in peripheral human blood is of paramount importance. Here, we focus on the in vitro investigation of the distribution of 99mTc-DPD-Fe3O4 DMCA in donated peripheral human blood (the ligand 2-3-dicarboxypropane-1-1-diphosphonic-acid is denoted as DPD). Initially, we described the experimental methods we performed for the radiosynthesis of the 99mTc-DPD-Fe3O4, the preparation of whole blood and blood plasma samples, and their incubation conditions with 99mTc-DPD-Fe3O4. More importantly, we employed a gamma-camera apparatus for the direct imaging of the 99mTc-DPD-Fe3O4-loaded whole blood and blood plasma samples when subjected to specialized centrifugation protocols. The direct comparison of the gamma-camera data obtained at the exact same samples before and after their centrifugation enabled us to clearly identify the distribution of the 99mTc-DPD-Fe3O4 in the two components, plasma and cells, of peripheral human blood.

Separation of platelets by size in a microfluidic device based on controlled incremental filtration.

The significant biological and functional differences between small and large platelets suggested by recent studies could have profound implications for transfusion medicine. However, investigating the relationship between platelet size and function is challenging because separating platelets by size without affecting their properties is difficult. A standard approach is centrifugation, but it inevitably leads to premature activation and aggregation of separated platelets. This paper describes the development and validation of a microfluidic device based on controlled incremental filtration (CIF) for separating platelets by size without the cell damage and usability limitations associated with centrifugation. Platelet samples derived from whole blood were used to evaluate the dependence of the CIF device separation performance on design parameters and flow rate, and to compare the properties of PLT fractions generated by the CIF device with those produced using a centrifugation protocol in a split-sample study. This was accomplished by quantifying the platelet size distribution, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet activation before and after processing for all input and output samples. The 'large platelet' fractions produced by the CIF device and the centrifugation protocol were essentially equivalent (no significant difference in MPV and P-LCR). Platelets in the 'small platelet' fraction produced by the CIF device were significantly smaller than those produced by centrifugation (lower MPV and P-LCR). This was because the CIF 'small platelet' fraction was contaminated by much fewer large platelets (∼2-times lower recovery of >12 fL platelets) and retained the smallest platelets that were discarded by the centrifugation protocol. There was no significant difference in platelet activation between the two methods. However, centrifugation required a substantial amount of additional anticoagulant to prevent platelet aggregation during pelleting. Unlike centrifugation, the CIF device offered continuous, flow-through, single-step processing that did not cause platelet aggregation. Such a capability has the potential to accelerate the basic studies of the relationship between platelet size and function, and ultimately improve transfusion practice, particularly in the pediatric setting, where the need for low-volume, high-quality platelet transfusions is most urgent.