This is a semiquantitative assay. The diagnostic potential of euglobulin lysis times is limited by the extreme variation in lysis times among healthy individuals.6 Both hypofibrinogenemia and factor XIII deficiency may result in a shortened lysis time. In the case of hypofibrinogenemia, the shortened time is due to the decreased amount of fibrin to be lysed. In factor XIII deficiency, the clot is not stabilized by covalent cross-linking of fibers and can be readily lysed by plasmin. Traumatic venipuncture, prolonged stasis, or incorrect sample preparation may invalidate test results.
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The euglobulin fraction of the plasma refers to plasma proteins that precipitate at low pH and decreased ionic strength.6 In the ELT test, plasma inhibitors of fibrinolysis are physically removed and the reaction of fibrinogen, plasminogen, and plasminogen activators are assayed. The precipitate also includes tissue plasminogen activators, plasminogen, plasmin, and fibrinogen. The inhibitors of lysis, α2-antiplasmin, and α2-macroglobulin, do not precipitate. In the test system, the euglobulin precipitate is redissolved in buffer, and clotting is initiated with calcium. The assay is performed in a microtiter plate. The time required for the intrinsic plasmin to lyse the fibrin clot equates to the euglobulin lysis time.
The euglobulin lysis time (ELT) test provides an overview of the fibrinolytic system function by measuring the time it takes for an in vitro clot to dissolve in the absence of plasmin inhibitors.6 The fibrinolytic system is initiated following activation of the contact factors in the coagulation cascade. Fibrinolytic system activation leads to the production of plasmin, a proteolytic enzyme capable of degrading fibrin and fibrinogen as well as other plasma proteins. Increased fibrinolytic activity is suggested by clot lysis that occurs in less than two hours. A shortened ELT result implies excessive fibrinolytic activity that may be primary or could be secondary to inflammation, malignancy, trauma, fracture, liver disease, or thrombolytic therapy. Clinical bleeding is a possible consequence of excessive fibrinolysis. Excessive fibrinolysis may result from increased levels of tissue plasminogen activator (TPA), increased plasmin activity, decreased levels of plasminogen activator inhibitor-1 (PAI-1), or decreased α2-antiplasmin activity. The effect of fibrinolysis is the production of plasma fibrin(ogen) degradation products (FDP or FSP) and D-dimer fragments. A prolonged ELT result implies a defect in the fibrinolytic system such as elevated plasminogen activator inhibitor (PAI-1) levels, elevated levels of α2-antiplasmin, a plasminogen deficiency, or decreased tissue plasminogen activator (TPA) activity. The ELT may also be prolonged if the fibrinogen level exceeds 600 mg/dL. Inadequate fibrinolysis may be associated with superficial or deep vein thrombosis, pulmonary embolism, coronary thrombosis, transient ischemic attack, or stroke.
In addition, a preliminary study in 40 healthy volunteers and 43 adult patients referred for investigation of a bleeding disorder was conducted to compare GFC and ECLT assays in their ability to classify samples with shortened or prolonged clot lysis times. Disagreements between ECLT and GFC were observed for 23 samples (out of 83), most of them minor.
Furthermore, as fibrinolysis is a very slow physiological process because of low or even no circulating levels of free plasminogen activators (fully complexed to PAI-1 and thus inactive) [7], two laboratory tips have to be used to speed up the process, leading to two different kinds of coagulolytic assays: on the one hand removal of most inhibitors (i.e. euglobulin clot lysis time, which relies on a very binding and time-consuming processing of plasma), and on the other hand addition of a low quantity of t-PA (i.e. global fibrinolytic capacity of plasma). Both approaches have pros and cons: the first modifies the baseline equilibrium and does not reflect physiological conditions, the second is sensitive to the amount of t-PA added, which should be sufficient to shorten lysis time but low enough not to alter the balance between all the actors of the fibrinolytic system [7, 8].
Euglobulin clot lysis time (ECLT), also known as von Kaulla assay, is a coagulolytic assay historically thought to detect what is still usually referred to as hyperfibrinolysis. This assay is commonly performed in our laboratory as part of the diagnostic workup for bleeding disorders, but suffers from four major limitations: (i) it is time-consuming, (ii) samples have to be drawn and processed on melting ice [9, 10], (iii) there is no harmonized and standardized procedure between laboratories, (iv) and as it is performed with a plasma sample artificially depleted in fibrinolysis inhibitors (mostly alpha-2-antiplasmin with less than 10% left, and PAI-1 and TAFI with respectively 42% and 38% left [11]); it does not reflect the whole in vivo phenomenon. To minimize inter-operator variability about the determination of the result, our center uses a photometer device, the Mevatronic T800 (MevaTronics, Belgium) [12], to record turbidity changes and to determine the clot lysis time.
In addition, 40 healthy volunteers and 43 adult patients referred for investigation of a bleeding disorder were enrolled to compare GFC and ECLT assays in their ability to classify samples with shortened or prolonged clot lysis times.
Euglobulin clot lysis time (ECLT) was assessed with fresh and frozen c-PDP samples using the Mevatronic T800 (MevaTronics, Belgium) [12]. Briefly, 500 µL of c-PDP was diluted 1:20 with acetic acid 0.014%, incubated 15 min at 4 C and centrifuged 15 min at 1,500 g at C. Supernatant was discarded and the pellet was dissolved in 500 µL of Owren-Koller buffer (Stago, Asnières-sur-Seine, France). 400 µL were then transferred to a cuvette and the cuvette was introduced into the Mevatronic T800. Clot formation was triggered by 100 µL of CaCl2 0.025 M (Stago, Asnières-sur-Seine, France) and light transmittance was continuously recorded at a wavelength of 890 nm. The digital signal was then converted into a light transmission curve and its primary and secondary derivatives by the dedicated software [12]. Locally established reference interval ranges from 131 to 523 min.
Distribution of samples according to the assay performed. The lower and upper limits used for ECLT and GFC correspond to the limits of locally established reference intervals. Samples located in zones A3, B2 and C1 show agreement between the lysis times provided by the two assays whereas samples located in zones A2, B3 and C2 show disagreements, most of them minor
A high proportion of patients (up to 75%) referred for a mild bleeding tendency shows no lab abnormalities and are diagnosed with a bleeding disorder of undefined cause (BDUC) [30]. A recent survey focusing on current practice for diagnosis and management of patients with unclassified bleeding disorders in United Kingdom [31] showed that only 37% and 2% of the centres assayed PAI-1 and/or alpha-2-antiplasmin or performed euglobulin clot lysis time respectively to exclude other disorders before making a diagnosis of an unclassified bleeding disorder in a patient with a significant bleeding history. The laboratory work-up of a bleeding tendency would benefit a lot from a fibrinolysis assay sensitive enough to qualitative and/or quantitative defects in the fibrinolysis system, sufficiently robust to not be (too much) affected by preanalytical constraints (such as a freeze-thawing cycle or the sampling temperature), and standardised enough to provide comparable results among laboratories.
Finally, compared to ECLT there are fewer pre-analytical constraints since samples no longer need to be processed at 4 C nor to be depleted in fibrinolysis inhibitors, leading to a shorter TAT compatible with emergency settings. However, we have observed disagreements between the two methods with samples with shortened or prolonged clot lysis times according to ECLT but within reference interval according to GFC, and conversely samples with prolonged clot lysis times according to GFC but within reference interval according to ECLT.
To perform the euglobulin clot lysis time, samples have to be collected and processed on melting ice [9, 10] to preserve plasma t-PA and PAI-1 from degradation and to prevent spontaneous t-PA/PAI-1 complex formation. In contrast, the processing temperature has no influence on GFC, because plasma t-PA and active PAI-1 are stabilized and protected from early degradation or neutralization by interactions with all the other actors of the fibrinolytic system [2, 34].
In a broader context, several tests are used to monitor clot lysis time: some of them display different results according to the gender, whereas for others there is no difference [20, 42]. This could be explained, at least in part, by a combination of two factors. First, the fibrinolytic system is tightly regulated and equilibrium results from a balance between inhibitors (such as PAI-1, α2-antiplasmin and α2-macroglobulin, and thrombin activatable fibrinolysis inhibitor (TAFI)) and activators (such as t-PA), with lower levels of plasma PAI-1, t-PA and t-PA/PAI-1 complexes in women before menopause [42,43,44,45,46].
First, the procedure we use to perform ECLT assay does not involve addition of exogenous thrombin in contrast to GFC assay. Thrombin is a key enzyme in coagulation, with both procoagulant (activation of factors V, VIII and XI, conversion of soluble fibrinogen into fibrin clot, activation of factor XIII) and anticoagulant effect (activation of protein C). For three decades now, thrombin is also known as playing a pivotal role between coagulation and fibrinolysis by converting TAFI into activated TAFI (TAFIa), a carboxypeptidase acting as a potent attenuator of fibrinolysis by counteracting the conversion of plasminogen into plasmin [47]. An addition of exogenous human thrombin, as is the case in the GFC assay, could therefore hide a defective activation of TAFI by the patient's endogenous thrombin, leading to a falsely normal or prolonged clot lysis time. 2ff7e9595c
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