High fragmentation in platelet concentrates impacts the activation, procoagulant, and aggregatory capacity of platelets.

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.

What is the context?● Platelets are necessary to prevent and stop bleeding.● Conditions associated with a low platelet count in the circulation, such as during chemotherapy treatment for hematologic cancer, can result in life-threatening bleeding. To prevent this, platelets from blood donors are transfused to these patients.● The collection and preparation of platelet concentrates and subsequent storage before transfusion can affect the ability of the platelets to prevent bleeding.● In this study, we investigated platelet concentrates prepared from whole blood and how their activation capacity was affected by the preparation and storage period.What is new?● We found that the platelet concentrates contained mainly low activated platelets of normal size, but also smaller platelets and platelet fragments.● Unlike normal-sized platelets, small platelets and fragments exhibited hallmarks that are characteristic of pre-activation.● Some platelet concentrates contained a relatively high proportion of small platelets and fragments already directly following preparation.● Investigating several platelet activation markers, we found that platelet concentrates containing a high proportion of small platelets and platelet fragments showed lower activation capacity throughout the storage period.What is the impact?● We show that some platelet concentrates show lower activation capacity and might contain a substantial fraction of platelets with characteristics that might potentially trigger spontaneous blood coagulation. The variation between different concentrations is high, even though the preparation procedure is the same.● If these differences will affect the efficacy of platelet transfusion is an important area for future studies.

Variation in activation marker expression within the platelet population - a new parameter for evaluation of platelet flow cytometry data.

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.

Cryopreservation of buffy coat derived platelets: Paired in vitro characterization using uncontrolled versus controlled freezing rate protocols.

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, Δψ, 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.

Haemostatic responsiveness and release of biological response modifiers following cryopreservation of platelets treated with amotosalen and ultraviolet A light.

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.

Storage-induced change in platelet transfusion response evaluated by serial transfusions from one donor to one patient.

BACKGROUND: Storage of platelet concentrates (PCs) results in storage lesions with possible detrimental effects on platelet recovery after transfusion, which might affect their ability to prevent or arrest bleeding. The aim of this study was to compare the quality of PCs stored for 1 to 3 or 5 to 7 days by assessing the corrected count increment (CCI) after transfusion. To isolate the effects of storage time, we studied serial transfusions of PCs obtained from one donor and one donation, and transfused to one single recipient after storage for 1 to 3 days and 5 to 7 days. STUDY DESIGN AND METHODS: Platelets were obtained from one donor by apheresis, divided into two units (>240 × 109 platelets/unit) and stored for 1 to 3 and 5 to 7 days, respectively, before transfusion. The PCs were transfused on normal indications to patients undergoing treatment at the hematology ward. Platelet count was measured before and after transfusion. RESULTS: Thirty patients concluded the study according to the protocol. The mean storage time was 2.4 ± 0.7 and 5.7 ± 0.8 days for platelets transfused on Days 1 to 3 and 5 to 7, respectively. Storage for 5 to 7 days decreased the 1-hour transfusion response as compared to platelets stored 1 to 3 days, from a CCI of 17 ± 7 to 13 ± 5. Despite this decrease, 86% of the 5 to 7 days stored PCs resulted in a CCI above the cutoff value for a successful transfusion of 7.5, which was not significantly different to PCs stored for 1 to 3 days. CONCLUSION: Storage of PCs for 5 to 7 days only slightly altered the transfusion response.

Treatment of platelet concentrates with ultraviolet C light for pathogen reduction increases cytokine accumulation.

BACKGROUND: Pathogen reduction technologies use photoactive substances in combination with ultraviolet (UV) light to inactivate pathogens. A new method uses only UVC light for pathogen reduction. This study assesses the effects of UVC light treatment on cytokine release in platelet (PLT) concentrates (PCs). STUDY DESIGN AND METHODS: A PC with 35% plasma and 65% PLT additive solution (SSP+) was prepared from five buffy coats. Three such PCs were pooled and divided into 3 units. One unit was used as a nonirradiated control, the second was a gamma-irradiated control, and the third unit was treated with UVC light technology. Ten units of each type were investigated. Cytokine release was analyzed on Days 1, 5, and 7 of storage. Correlation between cytokines, PLT surface markers, and hemostatic properties was investigated. RESULTS: Swirling was well preserved and pH was above the reference limit of 6.4 during storage of PLTs in all groups. Cytokine levels increased during storage in all groups but to a larger degree in PCs treated with UVC light. Only weak correlation was found between cytokines and PLT surface markers (r < 0.5). However, several cytokines showed strong correlation (r > 0.6) with the PLTs' ability to promote clot retraction. CONCLUSION: UVC treatment resulted in increased release from PLT alpha granules as evident by a higher cytokine release compared to nonirradiated and gamma-irradiated PCs. The clinical relevance of these findings needs to be further evaluated.

Assessment of Haemostasis in Disseminated Intravascular Coagulation by Use of Point-of-Care Assays and Routine Coagulation Tests, in Critically Ill Patients; A Prospective Observational Study.

BACKGROUND: Disseminated intravascular coagulopathy (DIC) relates to the consumption of coagulation factors and platelets with bleeding and micro thrombosis events. AIM: The aim of this study was to compare haemostasis parameters in critically ill patients with DIC versus patients without DIC, and in survivors versus non-survivors over time. Correlations between the DIC-score, the degree of organ failure and the haemostasis were assessed. METHOD: Patients admitted to the intensive care unit with a condition known to be associated with DIC and with an expected length of stay of >3 days were included. Routine laboratory tests, prothrombin time, activated partial thromboplastin time, platelet count, fibrinogen concentration and D-dimer were measured. Coagulation and platelet function were assessed with two point-of-care devices; Multiplate and ROTEM. DIC scores were calculated according to the International Society on Thrombosis and Haemostasis and Japanese Association for Acute Medicine. RESULTS: Blood was sampled on days 0-1, 2-3 and 4-10 from 136 patients with mixed diagnoses during 290 sampling events. The point-of-care assays indicated a hypocoagulative response (decreased platelet aggregation and reduced clot strength) in patients with DIC and, over time, in non-survivors compared to survivors. Patients with DIC as well as non-survivors had decreased fibrinolysis as shown by ROTEM. DIC scores were higher in non-survivors than in survivors. CONCLUSIONS: Patients with DIC displayed signs of a hypocoagulative response and impaired fibrinolysis, which was also evident over time in non-survivors. Patients with DIC had a higher mortality rate than non-DIC patients, and DIC scores were higher in non-survivors than in survivors.

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.

Thromboelastometry versus free-oscillation rheometry and enoxaparin versus tinzaparin: an in-vitro study comparing two viscoelastic haemostatic tests' dose-responses to two low molecular weight heparins at the time of withdrawing epidural catheters from ten patients after major surgery.

BACKGROUND: Monitoring low molecular weight heparins (LMWH's) in the perioperative period is prudent in patients at high risk of coagulative complications, especially when the patient has an epidural catheter requiring withdrawal, which is associated with the risk of spinal haematoma. The aim of this study was to evaluate the in vitro dose-responses of two different LMWH's on two different viscoelastic haemostatic tests, using blood sampled from patients with normal routine coagulation parameters, on the day after major surgery when their epidural catheters were due to be withdrawn. METHODS: Enoxaparin or tinzaparin were added in vitro to blood from ten patients who had undergone oesophageal resection, to obtain plasma concentrations of approximately 0, 0.5, 1.0 and 1.5 IU/mL. Coagulation was monitored using thromboelastometry (ROTEM®) using the InTEM® activating reagent; and free oscillation rheometry (FOR: ReoRox®), activated using thromboplastin. Clot initiation was measured using ROTEM-CT, ReoRox-COT1 and ReoRox-COT2. Clot propagation was measured using ROTEM-CFT, ROTEM-Alpha Angle and ReoRox-Slope. Clot stability was measured using ROTEM-MCF and ReoRox-G'max, and clot lysis was measured using ROTEM-ML and ReoRox-ClotSR. RESULTS: Clot initiation time assessed by thromboelastometry and FOR was prolonged by increasing concentrations of both LMWH's (P < 0.01). Equivalent doses of tinzaparin in international units (anti-FXa units) per millilitre prolonged clot initiation more than enoxaparin (P < 0.05). There was significant inter-individual variation - the ranges of CT and COT1 at LMWH-concentrations of 0 and 1.5 IU/mL overlapped. None of the tests reflecting clot formation rate or stability showed a dose-response to either LMWH but clot lysis showed a tentative negative dose-response to the LMWH's. CONCLUSIONS: Clot initiation time's dose-dependent prolongation by LMWH's in this study agrees with previous research, as does tinzaparin's stronger anti-coagulative effect than enoxaparin at equivalent levels of anti-FXa activity. This casts doubt on the validity of using anti-FXa assays alone to guide dosage of LMWH's. The significant inter-individual variation in dose-response suggests that the relationship between dose and effect in the postoperative period is complicated. While both ROTEM and FOR may have some role in postoperative monitoring, more research is needed before any conclusion can be made about their clinical usefulness.

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-response 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.

In vitro function of platelets treated with ultraviolet C light for pathogen inactivation: a comparative study with nonirradiated and gamma-irradiated platelets.

BACKGROUND: During storage of platelet concentrates (PCs) replication of contaminating pathogens might occur, which can be prevented by various pathogen inactivation (PI) methods using photoactive substances in combination with ultraviolet (UV) light. A new method uses only UVC light for PI without photoactive substances. This study evaluates the in vitro function, including hemostatic properties (clot formation and elasticity), of platelets (PLTs) treated with UVC light. STUDY DESIGN AND METHODS: A PC with 35% plasma and 65% PLT additive solution (SSP+) was prepared from five buffy coats. Three PCs were pooled and divided into 3 units. One unit was used as a nonirradiated control, the second was a gamma-irradiated control, and the third unit was treated with UVC light. In vitro variables including analysis of coagulation by free oscillation rheometry were analyzed on Days 1, 5, and 7 of storage. Ten units in each group were investigated. RESULTS: Swirling was well preserved, and the pH level was higher than the reference limit (6.4) during storage of PLTs in all groups. Glycolysis and PLT activation were higher for UVC-treated PLTs but the clot-forming capacity was unaffected. However, immediately after UVC treatment, the clot elastic properties were slightly affected. Hypotonic shock response decreased immediately after UVC treatment but recovered partly during the storage period. CONCLUSION: UVC treatment affected the in vitro properties, but PLT quality and storage stability were well preserved for up to 7 days, and the in vitro hemostatic capacity of UVC-treated PLTs was only minimally altered. The clinical relevance of these changes needs to be evaluated in controlled trials.

Platelet adhesion changes during storage studied with a novel method using flow cytometry and protein-coated beads.

The aim of the present study was to set up and evaluate a novel method for studies of platelet adhesion and activation in blood and platelet suspensions such as platelet concentrate (PC) samples using protein-coated polystyrene beads and flow cytometry. To demonstrate its usefulness, we studied PCs during storage. PCs were prepared by aphaeresis technique (n = 7). Metabolic variables and platelet function was measured on day 1, 5, 7 and 12 of storage. Spontaneous and TRAP-6-induced adhesion to fibrinogen- and collagen-coated beads was analyzed by flow cytometry. P-selectin and phosphatidyl serine (PS) expression was assessed on platelets bound to beads as well as on non-adherent platelets. Platelet adhesion to fibrinogen beads had increased by day 12 and adhesion to collagen beads at day 7 of storage (p < 0.05). TRAP-6 stimulation significantly increased the platelet adhesion to fibrinogen beads (p < 0.05) as well as the P-selectin and PS exposure on platelets bound to beads (p < 0.01) during the first 7 days of storage, but by day 12, significant changes were no longer induced by TRAP-6 stimulation. We demonstrate that our adhesion assay using protein-coated polystyrene beads can be used to assess the adhesion properties of platelets during storage without the addition of red blood cells. Therefore it may offer a useful tool for future studies of platelet adhesive capacity in transfusion medicine and other settings.

Low dose of hydroxyethyl starch impairs clot formation as assessed by viscoelastic devices.

OBJECTIVE: High doses of the synthetic colloid hydroxyethyl starch (HES) used for plasma expansion have been associated with impaired haemostasis and hypocoagulation. Less is known about effects on clot formation in the low haemodilutional range (< 40%). This study evaluated the effects of low haemodilution with HES and albumin on coagulation using two different viscoelastic methods. METHODS: Clot formation was studied in vitro in healthy donor blood after 10% and 30% haemodilution with 60 g/L HES 130/0.4 or 50 g/L albumin with free oscillation rheometry (FOR) and rotational thromboelastography. RESULTS: Clotting time was not significantly affected at 10% haemodilution but was prolonged with both substances at 30% dilution (p < 0.01-0.001). The effect was significantly more pronounced with HES than with albumin. The elasticity of the clot was slightly reduced at 10% dilution with albumin, more pronounced at 10% dilution with HES (p < 0.05), further reduced at 30% dilution with albumin and to a still greater extent at 30% dilution with HES (p < 0.05). With albumin the functional activity of fibrinogen was not reduced in excess of the dilutional effect. HES in contrast produced a further reduction in clot elasticity than caused by mere dilution at both 10% and 30% dilutions (p < 0.001). CONCLUSIONS: There is an adverse effect on clot formation even at low grade haemodilution with both albumin and HES. The effect on coagulation is significantly more pronounced with HES than with albumin.

In vitro properties of platelets stored in a small container for pediatric transfusion.

BACKGROUND: The quality of a platelet (PLT) concentrate (PC) is affected by the number of PLTs in relation to the size and gas permeability of the container. This study evaluates the in vitro function, including hemostatic properties (clot formation and elasticity), of PLTs stored in a container of standard or small size. STUDY DESIGN AND METHODS: PCs with 30% plasma and 70% PLT additive solution were prepared from buffy coats. Two PCs were pooled and divided into the following containers: 1 unit and ½ a unit into a 1.8-L container (reference container) and ½ a unit into a 0.45-L container (test container). In a second set of experiments » of a unit was stored in the reference and test containers. Swirling, PLT count, blood gases, metabolic variables, PLT activation markers, hypotonic shock response (HSR), and coagulation by free oscillation rheometry were analyzed during 7 days of storage. RESULTS: Swirling was well preserved and pH was acceptable (6.4-7.4) during storage of PLTs in both containers. Glycolysis and PLT activation were higher when storing ½ and » of a unit in the reference container and storage of » of a unit in the reference container resulted in the largest decrease in HSR. The clotting time was similar whereas the clot elasticity was slightly lower for PLTs when stored as ½ and » of a unit in the reference container. CONCLUSION: Storage of a low number of PLTs benefits by storage in a small container in terms of better maintained in vitro properties.

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

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.

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

BACKGROUND: Haemodilution 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. METHODS: Blood 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. RESULTS: Hydroxyethyl 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. CONCLUSIONS: Both 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.

In vitro properties of platelets stored in three different additive solutions.

BACKGROUND: New platelet (PLT) additive solutions (PASs) contain compounds that might improve the storage conditions for PLTs. This study compares the in vitro function, including hemostatic properties (clot formation and elasticity), of PLTs in T-Sol, Composol, or SSP+ during storage for 5 days. STUDY DESIGN AND METHODS: Fifteen buffy coats were pooled and divided into three parts. PLT concentrates (PCs) with 30% plasma and 70% PAS (T-Sol, Composol, or SSP+) were prepared (n = 10). Swirling, PLT count, blood gases, metabolic variables, PLT activation markers, and coagulation by free oscillation rheometry (FOR) were analyzed on Days 1 and 5. RESULTS: Swirling was well preserved and pH acceptable (6.4-7.4) during storage for all PASs. Storage of PLTs in T-Sol led to a decrease in PLT count whereas the number of PLTs was unchanged in Composol or SSP+ PCs. PLTs in T-Sol showed higher glucose metabolism than PLTs in Composol or in SSP+. At the end of storage PLTs in T-Sol had higher spontaneous activation and lower ability to respond to an agonist than PLTs in Composol or SSP+. PLTs in all the PASs had a similar ability to promote clot formation and clot elasticity. CONCLUSION: Storage of PLTs in Composol or in SSP+ improved the quality of PCs in terms of better maintained PLT count, lower glucose metabolism, lower spontaneous activation, and improved response to a PLT agonist compared to PLTs in T-Sol. PLTs stored in the various PASs had similar hemostatic properties. These findings make Composol and SSP+ interesting alternatives as PASs.

Platelet quality after washing: the effect of storage time before washing.

BACKGROUND: Patients who have experienced anaphylactic transfusion reactions receive washed platelet (PLT) concentrates (PCs) where the plasma has been substituted with a PLT additive solution. This study compares the in vitro quality of PCs washed at the beginning of the storage period (Day 1) to PCs washed at the end of storage (Day 7). STUDY DESIGN AND METHODS: PLTs were prepared by the buffy coat procedure. Two concentrates were pooled and then split to obtain an identical pair of PCs. One of the PCs was washed with T-Sol on Day 1 and the other on Day 7 of storage. Swirling, blood gases, and metabolic variables were analyzed before washing. Analyses of surface expression of CD62P and coagulation by free oscillation rheometry (FOR) were performed before and after washing. RESULTS: pH was acceptable in all PCs. Washing on Days 1 and 7 increased the CD62P surface expression. The FOR variables clotting time and clot retraction were not influenced by washing on either day. Washing resulted in a decrease in the number of PLTs and the decrease was larger on Day 7 compared to Day 1. CONCLUSIONS: PLTs washed on Days 1 and 7 of storage are effected by washing in a similar manner. However, a larger loss of PLTs occurred during washing on Day 7.