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1.
Transfus Med Hemother ; 51(2): 66-75, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38584698

RESUMEN

Introduction: Before being implemented in daily clinical routine, new production strategies for platelet concentrates (PCs) must be validated for their efficacy. Besides in vitro testing, the establishment of new methods requires the labeling of platelets for in vivo studies of platelets' survival and recovery. Indocyanine green (ICG) is a Food and Drug Administration-approved near-infrared (NIR) fluorescent dye for diagnostic use in vivo, suitable for non-radioactive direct cell labeling of platelets. Methods: Platelets from PCs in storage solutions with different plasma concentrations were labeled with ICG up to concentrations of 200 µm. Whole blood (WB) was used as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. The impact of labeling processes was assessed by the quantification of CD62P expression and PAC-1 binding as platelet function markers. Platelet aggregation was analyzed by light transmission aggregometry. ICG-labeling efficiency and stability of platelets were determined by flow cytometry. Results: Platelets from PCs could be successfully labeled with 10 µm ICG after 1 and 4 days of storage. The best labeling efficiency of 99.8% ± 0.1% (immediately after labeling) and 81% ± 6.2% (after 24 h incubation with WB) was achieved by plasma replacement by 100% platelet additive solution for the labeling process. Since the washing process slightly impaired platelet function, ICG labeling itself did not affect platelets. Immediately after the ICG-labeling process, plasma was re-added, resulting in a recovered platelet function. Conclusion: We developed a Good Manufacturing Practice compatible protocol for ICG fluorescent platelet labeling suitable for survival and recovery studies in vivo as a non-radioactive labeling alternative.


We developed a simple, reproducible method according to Good Manufacturing Practice guidelines for labeling of platelets from platelet concentrates (PCs) with indocyanine green (ICG) as a non-radioactive alternative. PCs are medicinal drugs that are transfused to prevent or treat bleeding. They consist of the blood cells' platelets which are responsible for clotting processes in the body. Manufacturing procedures of PCs are continuously refined, and for in vivo testing, these platelets have to be labeled to track and to distinguish them from proband's or patient's own cells. Radioactive labeling, for a long time the gold standard for cell labeling, is no longer accepted. ICG is a fluorescent dye approved by the drug authorities and already used for diagnostic purposes in humans. We used ICG to label platelets from PCs. With our method, we achieved a labeling efficiency of 99.8%. We used whole blood (WB) as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. After the addition of ICG-labeled platelets to WB, the labeling efficiency decreased to 81% after 24 h. However, we were still able to distinguish the ICG-labeled platelets from the WB platelets. We could also show that platelet function was not impaired by the labeling processes. The good tolerance of ICG indicates a short path to clinical application in healthy volunteers and investigations of novel PC-manufacturing procedures. As a read-out system, flow cytometry systems equipped with NIR lasers and filters could offer the possibility of rapid visualization, cell tracking, re-isolation, and ex vivo studies.

2.
Pathogens ; 12(4)2023 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-37111436

RESUMEN

SARS-CoV-2 serosurveillance is important to adapt infection control measures and estimate the degree of underreporting. Blood donor samples can be used as a proxy for the healthy adult population. In a repeated cross-sectional study from April 2020 to April 2021, September 2021, and April/May 2022, 13 blood establishments collected 134,510 anonymised specimens from blood donors in 28 study regions across Germany. These were tested for antibodies against the SARS-CoV-2 spike protein and nucleocapsid, including neutralising capacity. Seroprevalence was adjusted for test performance and sampling and weighted for demographic differences between the sample and the general population. Seroprevalence estimates were compared to notified COVID-19 cases. The overall adjusted SARS-CoV-2 seroprevalence remained below 2% until December 2020 and increased to 18.1% in April 2021, 89.4% in September 2021, and to 100% in April/May 2022. Neutralising capacity was found in 74% of all positive specimens until April 2021 and in 98% in April/May 2022. Our serosurveillance allowed for repeated estimations of underreporting from the early stage of the pandemic onwards. Underreporting ranged between factors 5.1 and 1.1 in the first two waves of the pandemic and remained well below 2 afterwards, indicating an adequate test strategy and notification system in Germany.

3.
Transfusion ; 63(1): 217-228, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36453841

RESUMEN

BACKGROUND: Structural and biochemical changes in stored platelets are influenced by collection and processing methods. This international study investigates the effects of platelet (PLT) processing and storage conditions on HMGB1, sCD40L, and sCD62P protein levels in platelet concentrate supernatants (PCs). STUDY DESIGN/METHODS: PC supernatants (n = 3748) were collected by each international centre using identical centrifugation methods (n = 9) and tested centrally using the ELISA/Luminex platform. Apheresis versus the buffy coat (BC-PC) method, plasma storage versus PAS and RT storage versus cold (4°C) were investigated. We focused on PC preparation collecting samples during early (RT: day 1-3; cold: day 1-5) and late (RT: day 4-7; cold: day 7-10) storage time points. RESULTS: HMGB1, sCD40L, and sCD62P concentrations were similar during early storage periods, regardless of storage solution (BC-PC plasma and BC-PC PAS-E) or temperature. During storage and without PAS, sCD40L and CD62P in BC-PC supernatants increased significantly (+33% and +41%, respectively) depending on storage temperature (22 vs. 4°C). However, without PAS-E, levels decreased significantly (-31% and -20%, respectively), depending on storage temperature (22 vs. 4°C). Contrastingly, the processing method appeared to have greater impact on HMGB1 release versus storage duration. These data highlight increases in these parameters during storage and differences between preparation methods and storage temperatures. CONCLUSIONS: The HMGB1 release mechanism/intracellular pathways appear to differ from sCD62P and sCD40L. The extent to which these differences affect patient outcomes, particularly post-transfusion platelet increment and adverse events, warrants further investigation in clinical trials with various therapeutic indications.


Asunto(s)
Eliminación de Componentes Sanguíneos , Proteína HMGB1 , Humanos , Eliminación de Componentes Sanguíneos/métodos , Plaquetas/metabolismo , Conservación de la Sangre/métodos , Ligando de CD40/metabolismo , Proteína HMGB1/metabolismo , Transfusión de Plaquetas
4.
Blood Adv ; 6(14): 4162-4173, 2022 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-35561312

RESUMEN

Platelets within one individual display heterogeneity in reactivity, size, age, and expression of surface receptors. To investigate the combined intraindividual contribution of platelet size, platelet age, and receptor expression levels on the reactivity of platelets, we studied fractions of large and small platelets from healthy donors separated by using differential centrifugation. Size-separated platelet fractions were perfused over a collagen-coated surface to assess thrombus formation. Multicolor flow cytometry was used to characterize resting and stimulated platelet subpopulations, and platelet age was determined based on RNA and HLA-I labeling. Signal transduction was analyzed by measuring consecutive phosphorylation of serine/threonine-protein kinase Akt. Compared with small platelets, large platelets adhered faster to collagen under flow and formed larger thrombi. Among the large platelets, a highly reactive juvenile platelet subpopulation was identified with high glycoprotein VI (GPVI) expression. Elevated GPVI expression correlated with high HLA-I expression, RNA content, and increased platelet reactivity. There was a stronger difference in Akt phosphorylation and activation upon collagen stimulation between juvenile and older platelets than between large and small platelets. GPVI expression and platelet reactivity decreased throughout platelet storage at 22°C and was better maintained throughout cold storage at 4°C. We further detected higher GPVI expression in platelets of patients with immune thrombocytopenia. Our findings show that high GPVI expression is a feature of highly reactive juvenile platelets, which are predominantly found among the large platelet population, explaining the better performance of large platelets during thrombus formation. These data are important for studies of thrombus formation, platelet storage, and immune thrombocytopenia.


Asunto(s)
Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria , Púrpura Trombocitopénica Idiopática , Trombosis , Colágeno/metabolismo , Humanos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Proto-Oncogénicas c-akt , Trombosis/metabolismo
5.
Semin Hematol ; 59(2): 97-107, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35512907

RESUMEN

Vaccine-induced immune thrombotic thrombocytopenia (VITT; synonym, thrombosis with thrombocytopenia syndrome, is associated with high-titer immunoglobulin G antibodies directed against platelet factor 4 (PF4). These antibodies activate platelets via platelet FcγIIa receptors, with platelet activation greatly enhanced by PF4. Here we summarize the current concepts in the pathogenesis of VITT. We first address parallels between heparin-induced thrombocytopenia and VITT, and provide recent findings on binding of PF4 to adenovirus particles and non-assembled adenovirus proteins in the 2 adenovirus vector-based COVID-19 vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S. Further, we discuss the potential role of vaccine constituents such as glycosaminoglycans, EDTA, polysorbate 80, human cell-line proteins and nucleotides as potential binding partners of PF4. The immune response towards PF4 in VITT is likely triggered by a proinflammatory milieu. Human cell-line proteins, non-assembled virus proteins, and potentially EDTA may contribute to the proinflammatory state. The transient nature of the immune response towards PF4 in VITT makes it likely that-as in heparin-induced thrombocytopenia -marginal zone B cells are key for antibody production. Once high-titer anti-PF4 antibodies have been formed 5 to 20 days after vaccination, they activate platelets and granulocytes. Activated granulocytes undergo NETosis and the released DNA also forms complexes with PF4, which fuels the Fcγ receptor-dependent cell activation process, ultimately leading to massive thrombin generation. Finally, we summarize our initial observations indicating that VITT-like antibodies might also be present in rare patients with recurrent venous and arterial thrombotic complications, independent of vaccination.


Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Púrpura Trombocitopénica Idiopática , Trombosis , Ad26COVS1 , COVID-19/prevención & control , Vacunas contra la COVID-19/efectos adversos , ChAdOx1 nCoV-19 , Ácido Edético/efectos adversos , Humanos , Factor Plaquetario 4 , Púrpura Trombocitopénica Idiopática/inducido químicamente , Trombosis/inducido químicamente
6.
Sci Rep ; 12(1): 6229, 2022 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-35422472

RESUMEN

Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2-10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.


Asunto(s)
Conservación de la Sangre , Magnesio , Plaquetas , Conservación de la Sangre/métodos , Citoesqueleto , Magnesio/farmacología , Agregación Plaquetaria
7.
Transfus Med Hemother ; 49(5): 280-287, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-37969862

RESUMEN

Background: Plasma transfusion is one of the basic treatments in patients with major blood loss. The anti-A and anti-B antibodies contained in the plasma demand ABO blood group compatibility. This is limiting the use of plasma in emergency situations and can cause a shortage in the supply of plasma of certain blood groups. We developed a method for anti-A and anti-B depletion by adsorbing plasma isoagglutinins using red blood cells. Materials and Methods: Three units of fresh frozen plasma were thawed after quarantine storage, pooled, and an aliquot of red cell concentrate was added. After 2 h of incubation at room temperature antibody-red-cell complexes were removed by centrifugation, the isoagglutinin-depleted plasma was split into three units and deep frozen. Isoagglutinin titers, free hemoglobin, residual red cells, clotting factor activity, and sterility of plasma units were determined after isoagglutinin depletion and a double freeze-thawing procedure. Results: Anti-B titers in group A plasma were reduced from values of 1:64 to 1:1 or lower, anti-A titers in group B plasma decreased from values of 1:128 to at least 1:16. Postprocedure clotting factor activities were preserved with 88.0 ± 7.3% (factor V), 106.9 ± 11.4% (factor VIII), and 84.0 ± 7.5% (factor XI) fulfilling the quality control requirements. No residual red cells were found, but free hemoglobin slightly increased to 53.7 ± 5.2 µmol/L. All units were sterile. Discussion: We described a method for the production of anti-A- and anti-B-depleted plasma in a closed system that uses standard equipment. The resulting isoagglutinin-depleted plasma may allow for blood group independent plasma transfusion.

8.
Blood ; 138(22): 2256-2268, 2021 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-34587242

RESUMEN

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.


Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Autoanticuerpos/inmunología , COVID-19/prevención & control , Proteínas de la Cápside/efectos adversos , ChAdOx1 nCoV-19/efectos adversos , Contaminación de Medicamentos , Vectores Genéticos/efectos adversos , Células HEK293/inmunología , Inmunoglobulina G/inmunología , Factor Plaquetario 4/inmunología , Púrpura Trombocitopénica Idiopática/etiología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/efectos adversos , Adenoviridae/inmunología , Animales , Complejo Antígeno-Anticuerpo/ultraestructura , Autoanticuerpos/biosíntesis , Síndrome de Fuga Capilar/etiología , Proteínas de la Cápside/inmunología , Línea Celular Transformada , ChAdOx1 nCoV-19/química , ChAdOx1 nCoV-19/inmunología , ChAdOx1 nCoV-19/toxicidad , Dispersión Dinámica de Luz , Epítopos/química , Epítopos/inmunología , Trampas Extracelulares/inmunología , Extravasación de Materiales Terapéuticos y Diagnósticos/etiología , Vectores Genéticos/inmunología , Células HEK293/química , Humanos , Imagenología Tridimensional , Inmunoglobulina G/biosíntesis , Inflamación , Ratones , Microscopía/métodos , Activación Plaquetaria , Proteómica , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/inmunología , Trombosis de los Senos Intracraneales/diagnóstico por imagen , Trombosis de los Senos Intracraneales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Cultivo de Virus
9.
Transfus Med Hemother ; 48(3): 148-153, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-34177418

RESUMEN

INTRODUCTION: In the light of the ongoing SARS-CoV-2 pandemic, convalescent plasma is a treatment option for CO-VID-19. In contrast to usual therapeutic plasma, the therapeutic agents of convalescent plasma do not represent clotting factor activities, but immunoglobulins. Quarantine storage of convalescent plasma as a measure to reduce the risk of pathogen transmission is not feasible. Therefore, pathogen inactivation (e.g., Theraflex®-MB, Macopharma, Mouvaux, France) is an attractive option. Data on the impact of pathogen inactivation by methylene blue (MB) treatment on antibody integrity are sparse. METHODS: Antigen-specific binding capacity was tested before and after MB treatment of plasma (n = 10). IgG and IgM isoagglutinin titers were tested by agglutination in increasing dilutions. Furthermore, the binding of anti-EBV and anti-tetanus toxin IgG to their specific antigens was assessed by ELISA, and IgG binding to Fc receptors was assessed by flow cytometry using THP-1 cells expressing FcRI and FcRII. RESULTS: There was no significant difference in the isoagglutinin titers, the antigen binding capacity of anti-EBV and anti-tetanus toxin IgG, as well as the Fc receptor binding capacity before and after MB treatment of plasma. CONCLUSION: MB treatment of plasma does not inhibit the binding capacity of IgM and IgG to their epitopes, or the Fc receptor interaction of IgG. Based on these results, MB treatment of convalescent plasma is appropriate to reduce the risk of pathogen transmission if quarantine storage is omitted.

10.
Blood ; 137(26): 3656-3659, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-33945605

RESUMEN

Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.


Asunto(s)
Autoanticuerpos/sangre , Vacunas contra la COVID-19/efectos adversos , COVID-19/prevención & control , Citometría de Flujo/métodos , Inmunoglobulina G/sangre , Activación Plaquetaria/inmunología , Factor Plaquetario 4/inmunología , Púrpura Trombocitopénica Idiopática/diagnóstico , Receptores de IgG/inmunología , SARS-CoV-2 , Vacunación/efectos adversos , Especificidad de Anticuerpos , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Vacunas contra la COVID-19/inmunología , ChAdOx1 nCoV-19 , Heparina/efectos adversos , Heparina/inmunología , Humanos , Técnicas para Inmunoenzimas , Inmunogenicidad Vacunal , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Selectina-P/análisis , Púrpura Trombocitopénica Idiopática/etiología , Púrpura Trombocitopénica Idiopática/inmunología
11.
Blood ; 138(4): 299-303, 2021 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-33988688

RESUMEN

Vaccination using the adenoviral vector COVID-19 vaccine ChAdOx1 nCoV-19 (AstraZeneca) has been associated with rare vaccine-induced immune thrombotic thrombocytopenia (VITT). Affected patients test strongly positive in platelet factor 4 (PF4)/polyanion enzyme immunoassays (EIAs), and serum-induced platelet activation is maximal in the presence of PF4. We determined the frequency of anti-PF4/polyanion antibodies in healthy vaccinees and assessed whether PF4/polyanion EIA+ sera exhibit platelet-activating properties after vaccination with ChAdOx1 nCoV-19 (n = 138) or BNT162b2 (BioNTech/Pfizer; n = 143). In total, 19 of 281 participants tested positive for anti-PF4/polyanion antibodies postvaccination (All: 6.8% [95% confidence interval (CI), 4.4-10.3]; BNT162b2: 5.6% [95% CI, 2.9-10.7]; ChAdOx1 nCoV-19: 8.0% [95% CI, 4.5% to 13.7%]). Optical densities were mostly low (between 0.5 and 1.0 units; reference range, <0.50), and none of the PF4/polyanion EIA+ samples induced platelet activation in the presence of PF4. We conclude that positive PF4/polyanion EIAs can occur after severe acute respiratory syndrome coronavirus 2 vaccination with both messenger RNA- and adenoviral vector-based vaccines, but many of these antibodies likely have minor (if any) clinical relevance. Accordingly, low-titer positive PF4/polyanion EIA results should be interpreted with caution when screening asymptomatic individuals after vaccination against COVID-19. Pathogenic platelet-activating antibodies that cause VITT do not occur commonly following vaccination.


Asunto(s)
Autoanticuerpos/inmunología , Vacunas contra la COVID-19/efectos adversos , COVID-19/prevención & control , Factor Plaquetario 4/inmunología , Polielectrolitos , Púrpura Trombocitopénica Trombótica/etiología , Vacunación/efectos adversos , Adulto , Enfermedades Asintomáticas , Autoanticuerpos/sangre , Vacuna BNT162 , ChAdOx1 nCoV-19 , Femenino , Personal de Salud , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Activación Plaquetaria , Púrpura Trombocitopénica Trombótica/inmunología , Seroconversión , Trombofilia/etiología
14.
Lab Chip ; 20(13): 2306-2316, 2020 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-32458864

RESUMEN

Without cellular blood products such as platelet concentrates (PC), red blood cell concentrates (RCC), and hematopoietic stem cells (HPSC) modern treatments in medicine would not be possible. An unresolved challenge is the assessment of their quality with minimal cell manipulation. Minor changes in production, storage conditions, or blood bag composition may impact cell function, which can have important consequences on product integrity. This is especially relevant for personalized medicine, such as autologous T-cell therapy. Today a robust methodology that globally determines cell status directly before transfusion or transplantation is lacking. We demonstrate that measuring viscoelastic characteristics of peripheral blood cells using real-time deformability cytometry (RT-DC) provides comprehensive information on product quality, which is not accessible using conventional quality control tests. In addition, RT-DC requires few cells, a minimal sample volume and has a rapid turnaround time. We compared RT-DC to standard in vitro quality assays assessing: i) PC after storage at 4 °C and room temperature; ii) magnetic nanoparticle labeled platelets; iii) RCC stored in blood bags with different plasticizers; iv) RCC after gamma irradiation; and v) HPSC after cryopreservation with 5% or 10% dimethyl sulfoxide, respectively. Additionally, we evaluated the engraftment time of patients' platelets and leukocytes after transplantation of HPSC products. Our results demonstrate that label-free mechano-phenotyping can be used as a potential biomarker for quality assessment of cell-based pharmaceutical products.


Asunto(s)
Eliminación de Componentes Sanguíneos , Preparaciones Farmacéuticas , Plaquetas , Conservación de la Sangre , Criopreservación , Humanos , Leucocitos
15.
Nat Commun ; 10(1): 415, 2019 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-30679420

RESUMEN

In life sciences, the material properties of suspended cells have attained significance close to that of fluorescent markers but with the advantage of label-free and unbiased sample characterization. Until recently, cell rheological measurements were either limited by acquisition throughput, excessive post processing, or low-throughput real-time analysis. Real-time deformability cytometry expanded the application of mechanical cell assays to fast on-the-fly phenotyping of large sample sizes, but has been restricted to single material parameters as the Young's modulus. Here, we introduce dynamic real-time deformability cytometry for comprehensive cell rheological measurements at up to 100 cells per second. Utilizing Fourier decomposition, our microfluidic method is able to disentangle cell response to complex hydrodynamic stress distributions and to determine viscoelastic parameters independent of cell shape. We demonstrate the application of our technology for peripheral blood cells in whole blood samples including the discrimination of B- and CD4+ T-lymphocytes by cell rheological properties.


Asunto(s)
Forma de la Célula , Citometría de Flujo/métodos , Microfluídica/métodos , Reología/métodos , Análisis de la Célula Individual/métodos , Células Sanguíneas/citología , Forma de la Célula/efectos de los fármacos , Citocalasina D/farmacología , Elasticidad , Células HL-60/efectos de los fármacos , Humanos , Hidrodinámica , Modelos Biológicos , Fenotipo , Viscosidad
16.
Haematologica ; 104(1): 207-214, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30115655

RESUMEN

Platelet transfusion has become essential therapy in modern medicine. Although the clinical advantage of platelet transfusion has been well established, adverse reactions upon transfusion, especially transmission of bacterial infection, still represent a major challenge. While bacterial contamination is favored by the storage of platelets at room temperature, cold storage may represent a solution for this important clinical issue. In this study, we aimed to clarify whether plasma has protective or detrimental effects on cold-stored platelets. We investigated the impact of different residual plasma contents in apheresis-derived platelet concentrates, stored at 4°C or room temperature, on platelet function and survival. We found that platelets stored at 4°C have higher expression of apoptosis marker compared to platelets stored at room temperature, leading to accelerated clearance from the circulation in a humanized animal model. While cold-induced apoptosis was independent of the residual plasma concentration, cold storage was associated with better adhesive properties and higher response to activators. Interestingly, delta (δ) granule-related functions, such as ADP-mediated aggregation and CD63 release, were better preserved at 4°C, especially in 100% plasma. An extended study to assess cold-stored platelet concentrates produced under standard care Good Manufacturing Practice conditions showed that platelet function, metabolism and integrity were better compared to those stored at room temperature. Taken together, our results show that residual plasma concentration does not have a cardinal impact on the cold storage lesions of apheresis-derived platelet concentrates and indicate that the current generation of additive solutions represent suitable substitutes for plasma to store platelets at 4°C.


Asunto(s)
Apoptosis , Plaquetas/metabolismo , Conservación de la Sangre , Frío , Plasma/metabolismo , Agregación Plaquetaria , Tetraspanina 30/metabolismo , Adenosina Difosfato/farmacología , Animales , Plaquetas/citología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Soluciones Farmacéuticas/farmacología , Transfusión de Plaquetas
17.
ACS Appl Mater Interfaces ; 10(34): 28314-28321, 2018 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-30067021

RESUMEN

Magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. Platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (HSA). However, the optimal HSA density coated on particles and the uptake mechanism of single particles in platelets remain unclear. Here, we utilized single-molecule force spectroscopy (SMFS) and other complementary methods to characterize the interaction of particles when interacting with platelets and to determine the optimal HSA amount required to coat particles. An HSA concentration of 0.5-1.0 mg/mL for coating particles is most efficient for platelet labeling. Binding pathways could be elucidated by linking a single HSA particle to SMFS tips via polyethylene glycol (PEG) linkers of different lengths and allowing them to interact with immobilized platelets on the substrate. Depending on the PEG length (i.e., short ∼2 nm, medium ∼30 nm, and long ∼100 nm), particles interact differently with platelets as shown by one, two, or three force distributions, which correspond up to three different binding pathways, respectively. We propose a model that the short PEG linker allows the particle to interact only with the platelet membrane, whereas the medium and long PEG linkers promote the particle to transfer from open canalicular system to another target inside platelets. Our study optimizes magnetic platelet labeling and provides details of particle pathways in platelets.


Asunto(s)
Nanopartículas de Magnetita , Transporte Biológico , Plaquetas , Humanos , Nanopartículas , Tamaño de la Partícula , Polietilenglicoles , Albúmina Sérica
18.
ACS Appl Mater Interfaces ; 9(40): 34666-34673, 2017 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-28945336

RESUMEN

Platelets are the smallest blood cells and important for hemostasis. Platelet concentrates (PC) are medicinal products transfused to prevent or treat bleeding. Typically, platelets in PCs are assessed by in vitro tests for their function. However, in vivo testing of these platelets is highly desirable. To distinguish transfused platelets from patients or probands own cells after PC transfusions within the scope of clinical studies, platelets need to be efficiently labeled with minimal preactivation prior to transfusion. Here we report on a method for improved cell uptake of ferucarbotran magnetic nanoparticles contained in Resovist, an FDA-approved MRI contrast agent, by modifying the nanoparticle shell with human serum albumin (HSA). Both HSA-ferucarbotran nanoparticles and magnetically labeled platelets were produced according to EU-GMP guidelines. Platelet function after labeling was evaluated by light transmission aggregometry and by determination of expression of CD62P as platelet activation marker. Magnetic labeling does not impair platelet function and platelets showed reasonable activation response to agonists. Platelet survival studies in NOD/SCID-mice resulted in comparable survival behavior of magnetically labeled and nonlabeled platelets. Additionally, labeled platelets can be recovered from whole blood by magnetic separation.


Asunto(s)
Nanopartículas de Magnetita , Animales , Plaquetas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Transfusión de Plaquetas
19.
Nanomedicine ; 8(5): 537-44, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22024199

RESUMEN

Platelets play a dominant role in the pathogenesis of bleeding disorders and cardiovascular pathology (e.g., myocardial infarction). Nonradioactive labeling of platelets may offer several clinical applications, ranging from survival studies of transfused platelet concentrates to studies on the pathogenesis of stroke. We used ferucarbotran superparamagnetic nanoparticles (NPs) for cell labeling. Platelets incorporated these NPs by endocytosis (without linkers or binding agents). Flow cytometry using FITC-conjugated magnetic NPs showed ex vivo labeling of about 98% of platelets; NPs were predominantly located inside the platelet granules as confirmed by fluorescence microscopy and transmission electron microscopy. Iron concentrations of 2 pg per platelet were reached as determined by atomic absorption spectroscopy. This will enable sensitive ex vivo determination of transfused labeled platelets, allowing survival studies. In vitro, labeled platelets gave a clear signal by 7 Tesla magnetic resonance tomography. Magnetic labeling of platelets may offer a new tool for diagnosis and research in transfusion medicine and cardiovascular medicine. FROM THE CLINICAL EDITOR: In this study a platelet labeling method is discussed and described for in vivo and ex vivo applications, using a binder or linker free fluorescent superparamagnetic iron nanoparticle system. Magnetic labeling of platelets may offer a new tool for diagnosis and research in transfusion medicine and cardiovascular medicine.


Asunto(s)
Plaquetas/química , Rastreo Celular/métodos , Dextranos/química , Nanopartículas de Magnetita/química , Endocitosis , Compuestos Férricos/química , Humanos , Imagen por Resonancia Magnética , Coloración y Etiquetado
20.
Sensors (Basel) ; 9(6): 4022-33, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-22408511

RESUMEN

Measurements of magneto-optical relaxation signals of magnetic nanoparticles functionalized with biomolecules are a novel biosensing tool. Upon transmission of a laser beam through a nanoparticle suspension in a pulsed magnetic field, the properties of the laser beam change. This can be detected by optical methods. Biomolecular binding events leading to aggregation of nanoparticles are ascertainable by calculating the relaxation time and from this, the hydrodynamic diameters of the involved particles from the optical signal. Interaction between insulin-like growth factor 1 (IGF-1) and its antibody was utilized for demonstration of the measurement setup applicability as an immunoassay. Furthermore, a formerly developed kinetic model was utilized in order to determine kinetic parameters of the interaction. Beside utilization of the method as an immunoassay it can be applied for the characterization of diverse magnetic nanoparticles regarding their size and size distribution.

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