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- Tynngård, Nahreen, 1976-, et al.
(författare)
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Variation in activation marker expression within the platelet population - a new parameter for evaluation of platelet flow cytometry data
- 2022
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Ingår i: Platelets. - : Taylor & Francis Group. - 0953-7104 .- 1369-1635. ; 33:8, s. 1113-1118
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Tidskriftsartikel (refereegranskat)abstract
- In flow cytometry, individual cells are investigated. Platelet activation is normally reported in form of percentage of platelets expressing the marker (positive platelets) and/or mean/median fluorescence intensity (MFI) for the entire analyzed population. None of these take into account the variance of the marker expression between individual platelets. This can be obtained as data on coefficient of variation (CV). This study explores if CV provides additional information regarding platelet function. Samples from platelet concentrates (PCs) prepared by apheresis- (n = 13) and interim platelet unit (IPU) technique (n = 26) and stored for 6-7 days were included and compared. Spontaneous- and agonist-induced expression of activation markers (fibrinogen binding and exposure of P-selectin, LAMP-1, and CD63) was investigated as percentage positive platelets, MFI and CV. Spontaneous expression of P-selectin as percentage positive platelets and MFI was higher for IPU PCs than apheresis PCs, which in contrast had higher agonist-induced activation. CV for spontaneous fibrinogen binding and P-selectin exposure was larger for apheresis PCs, while IPU PCs generally had larger CV for P-selectin, LAMP-1, and CD63 after agonist stimulation. Our findings show that CV adds additional information when assessing platelet activation by providing data on the variation in activation responses within the platelet population.
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| 2. |
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| 3. |
- Tynngård, Nahreen, 1976-, et al.
(författare)
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Cryopreservation of buffy coat derived platelets: Paired in vitro characterization using uncontrolled versus controlled freezing rate protocols
- 2021
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Ingår i: Transfusion. - : WILEY. - 0041-1132 .- 1537-2995. ; 61:2, s. 546-556
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Tidskriftsartikel (refereegranskat)abstract
- Background Cryopreserved platelets show a reduced recovery and viability after freezing and thawing including several ultrastructural and phenotypic deteriorations compared with liquid-stored platelets. It is suggested that using Controlled-Rate Freezing (CRF) can reduce variability and optimize the functionality profile for cells. The objective of the study is to compare cellular, metabolic, phenotypic and functional effects on platelets after cryopreservation using different freezing rate protocols. Study Design and Methods To evaluate the possible effects of different freezing rate protocols a two-experimental study comparing diverse combinations was tested with a pool and split design. Uncontrolled freezing of platelets in materials with different thermal conductivity (metal vs cardboard) was evaluated in experiment 1. Experiment 2 evaluated uncontrolled vs a controlled-rate freezing protocol in metal boxes. All variables were assessed pre and post cryopreservation. Results Directly after thawing, no major differences in platelet recovery, LDH, ATP, Delta psi, CD62P, CD42b, platelet endothelial cell adhesion molecule and sCD40L were seen between units frozen with different thermal conductivity for temperature. In contrast, we observed signs of increased activation after freezing using the CRF protocol, reflected by increased cell surface expression of CD62P, PAC-1 binding and increased concentration of LDH. Agonist induced expression of a conformational epitope on the GPIIb/IIIa complex and contribution to blood coagulation in an experimental rotational thromboelastometry setup were not statistically different between the groups. Conclusion The use of a uncontrolled freezing rate protocol is feasible, creating a platelet product comparable to using a controlled rate freezing equipment during cryopreservation of platelets.
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| 4. |
- Tynngård, Nahreen, 1976-
(författare)
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Free oscillation rheometry in the assessment of platelet quality
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Platelets play an important role in the haemostatic process in order to seal damaged blood vessels. The platelets form a platelet plug at the damaged area and prevent blood loss. Once the damage to the vessel wall has been covered, the platelets retract the coagulum, to allow the blood to flow freely in the vessel. Free oscillation rheometry (FOR) can be used for analysis of coagulation as measured by clotting time and changes in clot elasticity (G'). Clot G' provides information about the fibrin network in the coagulum and the platelets’ ability to retract the coagulum. FOR analysis is performed using the ReoRox® 4 instrument. The blood sample is added to a cylindrical sample cup, which is set into free oscillation. The frequency and damping of the oscillation is recorded over time as the blood coagulates. The change in G' is calculated from the frequency and damping measured. Patients with malignant haematological diseases are often thrombocytopaenic and require platelet transfusions to prevent or stop bleeding. To ensure good haemostatic function in the recipient it is important that the quality of the platelets used for transfusion is well preserved. The aim of this thesis was to determine the quality of platelet concentrates (PCs), during storage, using various in vitro methods, including FOR, and to investigate how various preparation processes affect the quality. We also investigated whether FOR can be used to evaluate the haemostatic status in subjects at risk for thrombosis or bleeding as well as how the haemostatic status was affected by a platelet transfusion.We show that FOR can provide information about the coagulation properties in subjects at risk of thrombosis (pregnant women) or bleeding (thrombocytopaenic patients). We also show that the coagulation as measured by FOR is influenced by red blood cells and the fibrinogen concentration. However, the presence of functional platelets accounted for 90% of the G'. Furthermore we present data that FOR can provide information on the haemostatic effect of platelet transfusions and on the function of the transfused platelets.PCs produced by two different cell separators showed similar quality during storage for 7 days as assessed by FOR analysis. Leukocytes in the PCs can cause transfusion-associated graft-versus-host disease which can be prevented by gamma irradiation of the PCs. Gamma irradiation did not affect the quality of PCs during 7 days of storage as analysed by FOR. The clotting time was unchanged during the storage period. The capacity of platelets to retract the coagulum was reduced from days 1 to 5 of storage as seen by a prolonged time to reach maximum G' and the reduced mean change in G' per minute. However, if sufficient time is allowed for the platelets to regain their function, the clot will be fully retracted (as seen by a well maintained maximum G'). The FOR parameters were similar for 5- and 7-day old PCs, which, combined with other in vitro tests (e.g. hypotonic shock response, changes in pH, swirling, lactate and glucose), support the prolongation of the platelet storage period to 7 days. Intercept™ treatment of PCs can be performed to inhibit replication of contaminating bacteria in PCs. Intercept™ treatment of PCs did not diminish the clot-promoting capacity of the platelets as assessed by FOR clotting time.In conclusion, FOR is a promising method for assessing hyper- and hypocoagulability. It can provide information on the haemostatic effect of platelet transfusions and the function of the transfused platelets. FOR was also shown to be useful for analysing PC quality during different preparation and storage conditions.
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| 5. |
- Tynngård, Nahreen, 1976-, et al.
(författare)
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Haemostatic responsiveness and release of biological response modifiers following cryopreservation of platelets treated with amotosalen and ultraviolet A light
- 2020
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Ingår i: Blood Transfusion. - : SIMTIPRO SRL. - 1723-2007. ; 18:3, s. 191-199
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Tidskriftsartikel (refereegranskat)abstract
- Background - Due to the risk of replication of contaminating pathogens, platelets have a limited storage time of 5 days, which can be prolonged to 7 days by the use of pathogen inactivation technologies. Cryopreservation (CP) may be an alternative to permit longer storage periods and increased availability. However, the preparation of platelets can result in secretion of biological response modifiers (BRM), which can cause adverse transfusion reactions in the recipient. We investigated the impact of CP on platelet function and release of BRM in untreated (conventional) and pathogen-inactivated (PI) platelet concentrates. Materials and methods - Twelve buffy coat-derived platelet units were treated with amotosalen and ultraviolet A light to inactivate pathogens. Twelve untreated units were used as controls. The 24 units were cryopreserved and in vitro variables were analysed before and after CP. The in vitro variables investigated included platelet surface receptors and activation markers by flow cytometry, and coagulation time by viscoelastography. A panel of BRM, including cytokines, was investigated. Results - CP of both conventional and PI platelets resulted in a significant increase of BRM with similar increases of most of the BRM after CP of conventional and PI platelet concentrates. The increase in some of the BRM correlated significantly with shortened coagulation time, increased P-selectin expression, reduced mitochondrial transmembrane potential, and reduced capacity to respond to stimulation with ADP and collagen. Discussion - Cryopreservation of both conventional and PI platelets results in secretion of BRM. The increase in some of the BRM correlated with changes in platelet function variables and suggests that BRM release is affected, in part, in a similar way by CP as are changes in platelet function variables. PI with amotosalen and ultraviolet A light in combination with CP did not affect the release of immunomodulatory factors more than CP alone did.
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| 6. |
- Tynngård, Nahreen, et al.
(författare)
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High fragmentation in platelet concentrates impacts the activation, procoagulant, and aggregatory capacity of platelets
- 2023
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Ingår i: Platelets. - : Taylor & Francis. - 0953-7104 .- 1369-1635. ; 34:1
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Tidskriftsartikel (refereegranskat)abstract
- Platelets are transfused to patients to prevent bleeding. Since both preparation and storage can impact the hemostatic functions of platelets, we studied platelet concentrates (PCs) with different initial composition in regard to platelet fragmentation and its impact on storage-induced changes in activation potential. Ten whole blood derived PCs were assessed over 7 storage days. Using flow cytometry, platelet (CD41+) subpopulations were characterized for activation potential using activation markers (PAC-1, P-selectin, and LAMP-1), phosphatidylserine (Annexin V), and mitochondrial integrity (DiIC1(5)). Aggregation response, coagulation, and soluble activation markers (cytokines and sGPVI) were also measured. Of the CD41+ events, the PCs contained a median of 82% normal-sized platelets, 10% small platelets, and 8% fragments. The small platelets exhibited procoagulant hallmarks (increased P-selectin and Annexin V and reduced DiIC1(5)). Normal-sized platelets responded to activation, whereas activation potential was decreased for small and abolished for fragments. Five PCs contained a high proportion of small platelets and fragments (median of 28% of CD41+ events), which was significantly higher than the other five PCs (median of 9%). A high proportion of small platelets and fragments was associated with procoagulant hallmarks and decreased activation potential, but, although diminished, they still retained some activation potential throughout 7 days storage.
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