Nahreen Tynngård

Preparation, storage and quality control of platelet concentrates

Patients with thrombocytopaenia need transfusions of platelet concentrates to prevent or stop bleeding. A platelet transfusion should provide platelets with good functionality. The quality of platelet concentrates (PCs) is affected by the preparation method and the storage conditions including duration of storage, type of storage container, and storage solution (plasma or an additive solution). Different in vivo and in vitro techniques can be used to analyse PCs. Platelets can be collected by apheresis technique, and from whole blood using either the buffy-coat or the platelet-rich plasma method. PCs can be gamma irradiated to prevent occurrence of graft-versus-host disease in the recipient. Pathogen inactivation procedures have been developed to prevent transmission of bacteraemia.

Effects of different blood components on clot retraction analysed by measuring elasticity with a free oscillating rheometer

Free oscillation rheometry (FOR) using the ReoRox® 4 instrument makes it possible, at bedside, to study the coagulation process in blood over time and gives information on clotting time and coagulum elastic properties. In order to find out how various factors influence the FOR analysis we studied the coagulation process and change of elasticity over time in non-anticoagulated and citrated blood samples, plasma samples with various platelet concentrations (0–200 × 109/l) and blood samples with various haematocrit (0–40%). Blood samples supplemented with fibrinogen were analysed to elucidate the importance of fibrinogen on elasticity. The importance of the GPIIb/IIIa receptor on platelets was investigated by comparing the elasticity development in blood samples in presence and absence of a GPIIb/IIIa receptor inhibitor, abciximab. Anticoagulation with citrate did not have major influence on the viscoelastic properties of the coagulum. Increasing number of platelets and increasing fibrinogen concentration resulted in higher elasticity while increasing haematocrit gave lower elasticity. Blood samples with GPIIb/IIIa receptor inhibitor had very low elasticity indicating the importance of functional GPIIb/IIIa receptors. In conclusion we consider FOR to be a useful method to study the elastic properties of the coagulum. Various factors such as the number of red blood cells and platelets as well as the fibrinogen concentration should be taken into consideration when evaluating the results. The ReoRox® 4 instrument had excellent measuring range and unusually small artefactual effects on clot elasticity induced by the instrument in comparison with published results on other instruments.

The quality of platelet concentrates produced by COBE Spectra and Trima Accel cell separators during storage for 7 days as assessed by in vitro methods

BACKGROUND: The quality of platelet (PLT) concentrates (PCs) can be evaluated with various in vitro methods. A new technique, free oscillation rheometry (FOR), can be used to monitor coagulation properties of PCs and gives information on clotting time and coagulum elasticity. This study compared the quality of apheresis PCs produced by the COBE Spectra and Trima Accel cell separators (both Gambro BCT) during storage for 7 days with in vitro tests including FOR.

Assays of different aspects of haemostasis - what do they measure?

Haemostasis is a complex process affected by many factors including both cellular and plasma components. It is a multistep process starting with platelet adhesion to damaged endothelium and ending in clot fibrinolysis. There are several methods available to study different aspects of haemostasis including adhesion, aggregation, coagulation and fibrinolysis. This review describes the different methods, what aspects of haemostasis they measure and their limitations. Methods discussed include methods to study adhesion (e.g. PFA-100, cone and platelet(let) analyzer and perfusion chambers) and aggregation (e.g. Multiplate, VerifyNow and Plateletworks). Furthermore the principles behind viscoelastic haemostatic assays are presented as well as methods that can analyse aspects of haemostasis in plasma or platelet-rich-plasma samples (thrombin generation, overall haemostasis potential and Thrombodynamics Analyzer).

Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of, and Correlations and Differences between aPTT, Anti-Factor Xa and Thrombin Generation Assays

Background Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin. Aim To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance. Method Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents. Results Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80–0.92). Conclusions aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is a promising method for monitoring LMWH’s.

Platelet Function Determined by Flow Cytometry: New Perspectives?

Flow cytometry enables studies of several different aspects of platelet function in response to a variety of platelet agonists. This can be done using only a small volume of whole blood, and also in blood with low platelet counts. These properties, together with the increasing number of flow cytometers available in hospitals worldwide, make flow cytometry an interesting option for laboratories interested in studies of platelet function in different clinical settings. This review focuses on practical issues regarding the use of flow cytometry for platelet function testing. It provides an overview of available activation markers, platelet agonists, and experimental setup issues. The review summarizes previous experience and factors important to consider to perform high-quality platelet function testing by flow cytometry. It also discusses its current use and possibilities and challenges for future use of flow cytometry in clinical settings.

The effect of gamma irradiation on the quality of apheresis platelets during storage for 7 days

BACKGROUND: This study compares the quality of gamma‐irradiated versus nonirradiated platelet (PLT) concentrates (PCs) during storage for 7 days as assessed by various in vitro methods. A new technique, free oscillation rheometry (FOR), which measures clotting time and coagulum elasticity, was also used to evaluate the PLT function.

Free oscillation rheometry monitoring of haemodilution and hypothermia and correction with fibrinogen and factor XIII concentrates.

BackgroundHaemodilution and hypothermia induce coagulopathy separately, but their combined effect on coagulation has not been widely studied. Fibrinogen concentrate can correct dilutional coagulopathy and has an additional effect when combined with factor XIII concentrate. However, their effect on dilutional coagulopathy concomitant with hypothermia has not been studied previously. Free oscillation rheometry – FOR (Reorox®) – is a novel viscoelastic haemostatic assay that has not been studied in this context before.MethodsBlood from 10 healthy volunteers was diluted by 33% with hydroxyethyl starch or Ringer’s acetate solutions. Effects of fibrinogen added in vitro with and without factor XIII were studied at 33°C and 37°C. Coagulation velocity (coagulation time) and clot strength (elasticity) were assessed with FOR. Coagulation was initiated in vitro with thromboplastin alone, or thromboplastin plus a platelet inhibitor.ResultsHydroxyethyl starch increased the coagulation time and decreased clot strength significantly more than Ringer’s acetate solution, both in the presence and absence of a platelet inhibitor. There was a significant interaction between haemodilution with hydroxyethyl starch and hypothermia, resulting in increased coagulation time. After addition of fibrinogen, coagulation time shortened and elasticity increased, with the exception of fibrinogen-dependent clot strength (i.e., elasticity in the presence of a platelet inhibitor) after hydroxyethyl starch haemodilution. Factor XIII had an additional effect with fibrinogen on fibrinogen-dependent clot strength in blood diluted with Ringer’s acetate solution. Hypothermia did not influence any of the coagulation factor effects.ConclusionsBoth haemodilution and mild hypothermia impaired coagulation. Coagulopathy was more pronounced after haemodilution with hydroxyethyl starch than with Ringer’s acetate. Addition of fibrinogen with factor XIII was unable to reverse hydroxyethyl starch induced clot instability, but improved coagulation in blood diluted with Ringer’s acetate solution. Fibrinogen improved coagulation irrespective of hypothermia.

Free oscillation rheometry detects changes in clot properties in pregnancy and thrombocytopenia

Improved methods are needed to identify patients at risk for thrombotic or bleeding events. Free oscillation rheometry (FOR) is a technique that offers information on coagulation, based on contributions of all blood components, by measurement of clotting time and changes in clot elasticity. This is the first study that evaluates FOR parameters in subjects likely to represent hypercoagulability (pregnant women) and hypocoagulability (thrombocytopenic patients). Clotting time and blood clot elasticity were measured by FOR in blood samples obtained from women in different pregnancy trimesters (n = 58), in thrombocytopenic patients before and after a platelet transfusion (n = 20) and in healthy blood donors (n = 60). The clotting time was shorter and the clot elasticity higher in pregnant women compared to the non-pregnant female blood donors. The elasticity was higher in late pregnancy compared to early pregnancy. Compared to the blood donors, the thrombocytopenic patients had lower elasticity, which was increased by a platelet transfusion, but there was no difference in clotting time. The results suggest that FOR can provide new information on the haemostatic status of patients at risk of thrombotic or bleeding events as well as information on the haemostatic effect of a platelet transfusion.

Monitoring fibrinolysis in whole blood by viscoelastic instruments: A comparison of ROTEM and ReoRox.

Abstract Objective. Increased fibrinolysis with the risk of bleeding is a consequence of thrombolytic therapy and can also be seen in clinical situations such as acute trauma. Thrombelastography and thrombelastometry are viscoelastic coagulation instruments that can detect higher degrees of fibrinolysis; hyperfibrinolysis. A newer viscoelastic instrument is the ReoRox, which uses free oscillation rheometry to detect clot formation, strength and fibrinolysis. The ReoRox has a new test for detection of fibrinolysis, called ReoLyse. The aim of this study was to compare ReoRox with its new ReoLyse test with rotational thrombelastometry (ROTEM) in the monitoring of in vitro-induced fibrinolysis. Methods. Whole blood from 10 healthy volunteers was mixed with tissue plasminogen activator (t-PA) to obtain seven different plasma concentrations (0, 0.25, 0.5, 0.75, 1, 3 and 5 μg/mL). Whole blood samples with the different t-PA plasma concentrations were analyzed with ROTEM EXTEM and FIBTEM tests, ReoRox standard test Fib1 (clot formation/strength) and ReoLyse (fibrinolysis) tests. Results. The fibrinolysis variables with the best dose-response effect were the ReoRox ReoLyse lysis variables and ROTEM EXTEM Time to complete lysis. However, these variables only detected high t-PA levels (> 1 μg/mL). Conclusions. The new ReoRox ReoLyse test provides more information on fibrinolysis compared to the ReoRox Fib1 program. Neither ReoRox nor ROTEM could detect lower degrees of fibrinolysis. ReoRox is a valuable alternative to ROTEM to study high degrees of fibrinolysis and should be evaluated in clinical situations with increased fibrinolysis and during therapeutic thrombolysis.