Thromboelastometry
Encyclopedia
Thromboelastometryis an established viscoelastic method for haemostatis testing in whole blood. TEM investigates the interaction of coagulation factors, their inhibitors
Enzyme inhibitor
An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides...

, anticoagulant drugs, blood cells, specifically platelets, during clotting and subsequent fibrinolysis
Fibrinolysis
Fibrinolysis is a process that prevents blood clots from growing and becoming problematic. This process has two types: primary fibrinolysis and secondary fibrinolysis...

. The rheological conditions mimic the sluggish flow of blood in veins. TEM is performed with the ROTEM whole blood analyzer (Tem Innovations GmbH, Munich) and is an enhancement of thrombelastography, originally described by H. Hartert in 1948.

While traditional thrombelastography is a global assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...

 for blood clotting disorders and drug effects, TEM is primarily used in combination with appropriate differential assays. They allow testing in the presence of therapeutic heparin
Heparin
Heparin , also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule...

 concentrations and provide differential diagnostic information to support decisions in therapy. In numerous publications the validity of the method is shown. Application of TEM at the point of care (POC) or in emergency laboratories is getting more and more popular. TEM detects both hypo- and hyperfunctional stages of the clotting process and is probably the only reliable rapid test for the diagnosis of hyperfibrinolysis
Hyperfibrinolysis
The fibrinolysis system is responsible for removing blood clots. Hyperfibrinolysis describes a situation with markedly enhanced fibrinolytic activity, resulting in increased, sometimes catastrophic bleeding. Hyperfibrinolysis can be caused by acquired or congenital reasons...

. In contrast to standard clotting tests, the fibrin
Fibrin
Fibrin is a fibrous, non-globular protein involved in the clotting of blood. It is a fibrillar protein that is polymerised to form a "mesh" that forms a hemostatic plug or clot over a wound site....

 stabilizing effect of factor XIII
Factor XIII
Factor XIII or fibrin stabilizing factor is an enzyme of the blood coagulation system that crosslinks fibrin.- Function :Factor XIII is a transglutaminase that circulates in the plasma as a heterotetramer of two catalytic A subunits and two carrier B subunits...

 contributes to the result. The rapid availability of results helps to discriminate surgical bleeding from a true haemostasis disorder and improves the therapy with blood products, factor concentrates, anticoagulants and protamine
Protamine
Protamines are small, arginine-rich, nuclear proteins that replace histones late in the haploid phase of spermatogenesis and are believed essential for sperm head condensation and DNA stabilization. They may allow for denser packaging of DNA in spermatozoon than histones, but they must be...

, hemostyptic and antifibrinolytic
Antifibrinolytic
Antifibrinolytics, such as aminocaproic acid and tranexamic acid are used as inhibitors of fibrinolysis. These lysine-like drugs interfere with the formation of the fibrinolytic enzyme plasmin from its precursor plasminogen by plasminogen activators which takes place mainly in lysine rich areas...

 drugs. Several reports confirm that application of TEM is cost effective by reducing the consumption of blood products.

Method

Blood (300 µl, anticoagulated with citrate
Citrate
A citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid. An example of the former, a salt is trisodium citrate; an ester is triethyl citrate.-Other citric acid ions:...

) is placed into the disposable cuvette using an electronic pipette. A disposable pin is attached to a shaft which is connected with a thin spring (the equivalent to Hartert’s torsion wire in thrombelastography) and slowly oscillates back and forth. The signal of the pin suspended in the blood sample is transmitted via an optical detector system. The test is started by adding appropriate reagents. The instrument measures and graphically displays the changes in elasticity at all stages of the developing and resolving clot. The typical test temperature is 37°C, but different temperatures can be selected, e.g. for patients with hypothermia.
In contrast to thrombelastography with its pendulum-like principle, the design of the TEM viscoelastic detection system (figure 1) makes it quite robust and insensitive against mechanical shocks or vibrations.

Result

The primary result of TEM is a reaction curve which shows the elasticity over time when the clot forms or dissolves. This curve is also called a TEMogram. Four key parameters describe the clotting curve for clinical routine. More than 10 additional calculated parameters, including derivative
Derivative
In calculus, a branch of mathematics, the derivative is a measure of how a function changes as its input changes. Loosely speaking, a derivative can be thought of as how much one quantity is changing in response to changes in some other quantity; for example, the derivative of the position of a...

 curves which are useful in specific research applications, e.g. in hemophilia or thrombophilia or for the effects of recombinant FVIIa are available for research purposes.

Measurement parameters

CT (Clotting time):
The CT is the latency time from adding the start reagent to blood until the clot starts to form. Prolongation of CT may be a result of coagulation deficiencies, primarily coagulation factors, or heparin (dependent on the test used). A potential contribution of heparin can be detected by comparing INTEM- with HEPTEM CT data (see “reagents” below). A shortening of CT indicates hypercoagulability.

CFT (Clot formation time) and alpha-angle:
The alpha angle is the angle of tangent between 2 and the curve while CFT is the time from CT until a clot firmness of 20 mm point has been reached. These parameters denote the speed at which a solid clot forms and are primarily influenced by platelet
Platelet
Platelets, or thrombocytes , are small,irregularly shaped clear cell fragments , 2–3 µm in diameter, which are derived from fragmentation of precursor megakaryocytes.  The average lifespan of a platelet is normally just 5 to 9 days...

 function, but to a certain extent especially fibrinogen
Fibrinogen
Fibrinogen is a soluble plasma glycoprotein, synthesised by the liver, that is converted by thrombin into fibrin during blood coagulation. This is achieved through processes in the coagulation cascade that activate the zymogen prothrombin to the serine protease thrombin, which is responsible for...

 and coagulation factors contribute. A prolonged CFT (or a lower alpha-angle) is usually caused by poor platelet function, low platelet count, fibrin polymerization disorders or fibrinogen deficiency. Apparently also FXIII seems to be involved already in this phase. Higher concentrations of heparin
Heparin
Heparin , also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule...

 can also prolong CFT in the INTEM assay, but not in HEPTEM, EXTEM, FIBTEM or APTEM (see under “reagents”). A shortening of CFT (or a high alpha-angle) indicate hypercoagulability.

MCF (Maximum clot firmness):
MCF is the greatest vertical amplitude
Amplitude
Amplitude is the magnitude of change in the oscillating variable with each oscillation within an oscillating system. For example, sound waves in air are oscillations in atmospheric pressure and their amplitudes are proportional to the change in pressure during one oscillation...

 of the trace. It reflects the absolute strength of the fibrin and platelet clot. A low MCF is indicative of decreased platelet number or function, decreased fibrinogen level or fibrin polymerization disorders, or low activity of factor XIII. A mechanically weak clot represents a severe bleeding risk and should initiate immediate therapeutic steps. High doses of heparin can lower MCF in the INTEM assay, but not in HEPTEM, EXTEM, FIBTEM or APTEM (see under “reagents”).

A5, A 10, A15 or A20 value
These values describe the clot firmness (or amplitude) obtained after 10, 15 or 20 minutes and provide a forecast on the expected MCF value at an earlier stage already. A recent investigation has validated this approach for the A15 value in more than 800 cases during liver transplantation
Liver transplantation
Liver transplantation or hepatic transplantation is the replacement of a diseased liver with a healthy liver allograft. The most commonly used technique is orthotopic transplantation, in which the native liver is removed and replaced by the donor organ in the same anatomic location as the original...

. The advantage of the A15-values is obvious: It allows for a more rapid decision about therapeutic interventions.

LI 30 (Lysis Index after 30 minutes) and ML (Maximum Lysis)
The LI30 value is the percentage of remaining clot stability in relation to the MCF value at 30 min after CT. A similar value can also be calculated at other time points (45 or 60 min). The ML parameter describes the percentage of lost clot stability (relative to MCF, in %) viewed at any selected time point or when the test has been stopped. A low LI (X) value or a high ML value indicates hyperfibrinolysis. While in normal blood fibrinolysis activity is quite low, in clinical samples a more rapid loss of clot stability by hyperfibrinolysis may lead to bleeding complications which can be treated by the administration of antifibrinolytic
Antifibrinolytic
Antifibrinolytics, such as aminocaproic acid and tranexamic acid are used as inhibitors of fibrinolysis. These lysine-like drugs interfere with the formation of the fibrinolytic enzyme plasmin from its precursor plasminogen by plasminogen activators which takes place mainly in lysine rich areas...

 drugs.

TEM assays

Initially, thrombelastography was simply performed with whole blood without adding reagents (except calcium when citrate anticoagulated blood was used). This provides a global overview about all phases of clot formation, stabilization and degradation. In the case of monocausal haemostasis disorders, the resulting reaction curves may be quite typical; however, under most clinical conditions this approach has severe limitations. In reality various effects overlap, including haemodilution or application of high doses of parenteral
Parenteral
Parenteral is a route of administration that involves piercing the skin or mucous membrane. Parenteral nutrition refers to providing nutrition via the veins.-Etymology:...

 anticoagulants. High doses of heparin often prevent clot formation at all. Absence of a controlled activation step leads to inferior reproducibility and very long test times which are not acceptable for POC applications.
The assays for ROTEM analysis help to get a rapid differentiation between various potential haemostasis defects or anticoagulant drug effects and allow for a rapid differential diagnosis. They form the base for selecting a therapeutic strategy. Several diagnostic algorithms have been proposed and were clinically validated. Application of this strategy helps to minimize the exposure of patients to allogeneic blood products which have certain risks; and it saves costs. Numerous research applications have used the technique as well.

Tests

INTEM
This test mildly activates the contact phase of haemostasis. The result is influenced by coagulation factors, platelets, fibrinogen and heparin. Low molecular weight heparin is detected at higher concentrations. In the absence of heparin, INTEM is a screening test for the haemostasis system. It is used for therapeutic decisions regarding the administration of fresh frozen plasma, coagulation factors, fibrinogen or platelets.

HEPTEM
This assay represents an INTEM assay performed in the presence of heparinase, a heparin (or LMWH) degrading enzyme. It allows the identification of haemostasis deficiencies even in the presence of heparin and represents an INTEM test without interference by heparin or heparin like anticoagulants. The difference between HEPTEM and INTEM CT-value comparison confirms the presence of heparin, e.g. accidental exposure.

EXTEM
test mildly activates haemostasis via the physiological activator tissue factor. The result is influenced by extrinsic coagulation factors, platelets and fibrinogen. EXTEM is a screening test for the (extrinsic) haemostasis system. This assay is not influenced by heparin (heparin inhibitor included in the EXTEM reagent). It is used for therapeutic decisions regarding the administration of fresh frozen plasma, coagulation factors, fibrinogen or platelets. EXTEM is also the base activator for FIBTEM and APTEM.

FIBTEM
test is an EXTEM based assay for the fibrin part of the clot. FIBTEM eliminates the platelet contribution of clot formation by inhibiting the platelets irreversibly with cytochalasin D
Cytochalasin D
Cytochalasin D is a member of the class of mycotoxins known as cytochalasins. Cytochalasin D is an alkaloid produced by Helminthosporium and other molds....

, a potent inhibitor
Enzyme inhibitor
An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides...

 of actin
Actin
Actin is a globular, roughly 42-kDa moonlighting protein found in all eukaryotic cells where it may be present at concentrations of over 100 μM. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans...

 polymerization which disrupts actin microfilaments, an essential part of a cytoskeleton-mediated contractibility apparatus of the platelet. The use of cytochalasin is more favorable than using glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...

 IIb/ IIIa inhibitors which block platelet incompletely, especially at higher platelet counts. FIBTEM allows for the detection of fibrinogen deficiency or fibrin polymerization disorders, e.g. induced by certain plasma expanders, and may identify rapidly the need to substitute fibrinogen. FIBTEM results correlate well in many cases with the Clauss fibrinogen assay, but is additionally influenced by fibrin polymerization disorders which cannot reliably be detected with clotting tests.

APTEM
test is an EXTEM based assay in which fibrinolysis
Fibrinolysis
Fibrinolysis is a process that prevents blood clots from growing and becoming problematic. This process has two types: primary fibrinolysis and secondary fibrinolysis...

 is inhibited by aprotinin
Aprotinin
The drug aprotinin , is the bovine version of the small protein basic pancreatic trypsin inhibitor, or BPTI, which inhibits trypsin and related proteolytic enzymes. Under the trade name Trasylol, aprotinin was used as a medication administered by injection to reduce bleeding during complex surgery,...

 in the reagent. A significant improvement of the clot in APTEM compared to EXTEM allows to detect fulminant hyperfibrinolysis. Therefore the test helps in identifying the necessity of administrating antifibrinolytic
Antifibrinolytic
Antifibrinolytics, such as aminocaproic acid and tranexamic acid are used as inhibitors of fibrinolysis. These lysine-like drugs interfere with the formation of the fibrinolytic enzyme plasmin from its precursor plasminogen by plasminogen activators which takes place mainly in lysine rich areas...

 drugs. Furthermore, APTEM enables the estimation if an antifibrinolytic therapy alone normalizes coagulation or if additional measures have to be taken (e.g. administration of fibrinogen or platelets).

Quality Control

Quality control is an important requirement in laboratory and POC testing. An automatic internal electronic control procedure is implemented in the ROTEM whole blood analyzer. Furthermore, biological control materials at 2 different levels are available and should be used in order to document quality and validity of results.

Clinical application

TEM is successfully used in the near patient assessment of haemostasis. The method allows detection of complex haemostasis disorders (available in most coagulopathies) within a few minutes and thus enables rapid therapeutic intervention. Whole blood TEM is sensitive to haemostasis affecting agents such as plasma expanders or acidosis
Acidosis
Acidosis is an increased acidity in the blood and other body tissue . If not further qualified, it usually refers to acidity of the blood plasma....

 while the effects of these agents are hardly identified by plasma based laboratory tests.
TEM-guided transfusion
Blood transfusion
Blood transfusion is the process of receiving blood products into one's circulation intravenously. Transfusions are used in a variety of medical conditions to replace lost components of the blood...

 of blood products or factor concentrates in cardiac, hepatic and major orthopedic surgery is the main application of the method. Moreover, it is successfully used in the complex situation of (poly)trauma
Physical trauma
Trauma refers to "a body wound or shock produced by sudden physical injury, as from violence or accident." It can also be described as "a physical wound or injury, such as a fracture or blow." Major trauma can result in secondary complications such as circulatory shock, respiratory failure and death...

, or in decision making for of alternative therapy such as antifibrinolytic drug administration .
The clinical benefits of TEM have resulted in major savings and publications recommend that viscoelastic methods such as TEM should be used in surgical procedures where major blood loss followed by the need for transfusions can be expected.

Limitations of TEM

Like any other haemostasis evaluating method, TEM (and thrombelastography) have limitations which need to be considered when interpreting the results. The typical assays are not responsive for the effect of von Willebrand factor or platelet antagonists such as aspirin
Aspirin
Aspirin , also known as acetylsalicylic acid , is a salicylate drug, often used as an analgesic to relieve minor aches and pains, as an antipyretic to reduce fever, and as an anti-inflammatory medication. It was discovered by Arthur Eichengrun, a chemist with the German company Bayer...

 or thienopyridines (e.g. clopidogrel
Clopidogrel
Clopidogrel is an oral, thienopyridine class antiplatelet agent used to inhibit blood clots in coronary artery disease, peripheral vascular disease, and cerebrovascular disease. It is marketed by Bristol-Myers Squibb and Sanofi-Aventis under the trade name Plavix. The drug works by irreversibly...

), and only supratherapeutic doses of GPIIb/IIIa antagonists may influence results. The sensitivity for coagulation factor deficiencies, including those induced by oral anticoagulation, is less pronounced as compared to clotting assays. Therefore TEM is not ment to replace laboratory assays such as INR or factor assays. However, due to the rapid availability of differential diagnostic information, TEM has become an established method in surgical procedures where blood losses can be expected.
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