Euglobin Lysis Time

TEST 

Euglobulin Lysis Time 

METHOD 

TEST EVALUATION 

Don Jacobs, M.D. 

Venous blood is collected in a plasticized tube containing sodium citrate 

(approximately 1 part citrate/9 parts plasma). Platelet poor plasma is obtained by 

centrifugation at 4C. The plasma is diluted with distilled H2O (1:10) and acidified 

by the addition of acetic acid or CO2 to a pH of 5.9. This step causes precipitation 

of the euglobulins: plasminogen activators, plasminogen, plasmin, fibrinogen, 

factor V, and factor VIE. The inhibitors of fibrinolysis (anti-activators and antiplasmins) 

remain in the supernatant and are discarded. The euglobulin pellet is 

resuspended in buffer solution. Thrombin or calcium is added to the euglobulin 

suspension to form a clot. The specimen is incubated in a 37° water bath and 

observed every fifteen minutes until clot lysis is complete. Normal values are 

between 3 and 20 hrs. Lysis times less than 1 hour are indicative of severe 

fibrinolysis. The significance of an elevated lysis time has not been fully evaluated. 

The spontaneous fibrinolytic activity of plasma may be mathematically expressed as 

1000/ECL time (min). 

The significance of an elevated lysis time has not been fully evaluated. 

DIRECT ACTIVATION 

f a c t o r X I I 

urokinase 

hepar i no i ds 

s y n t h e t i c o r g a n i c c . 

v a c c i n e s p y r o g e n s 

N A - n i c o t i n i c d e r . 

s t r e p t o k i n a s e 

hepar i no i ds 

INDIRECT ACTIVATION 

PLASMINOGEN 

PL. 

» r 

ACTIVATOR 

* 

PLASMIN 

4 < 

Ipl. proactivatorI ' 

DIRECT INHIBITION 

ant i pI asm i n 

p a n c r e a t i c i n h i b i t o r 

FIBRIN 

.FIBRINOGEN, 

-I 

F D P 

ant i act i vator 

parotid inhibitor 

£ a m i n o c a p r o i c a c . 

t r a m e x a m i c a c i d 

INDIRECT INHIBITION 

FIG. 1. Activation and inhibition of the fibrinolytic system.

INTRODUCTION 

Page 2 

The fibrinolytic system is a critical regulator of fibrin deposition and thrombosis. 

The components of the system are diagrammed above. Plasminogen, probably 

synthesized in the liver, is a circulating inactive proenzyme which is found in 

concentrations of 10-15 mg/dl. Plasminogen is converted into the active serine 

protease plasmin by a variety of plasminogen activators. These activators are 

widely distributed in a variety of tissues (tissue activator), plasma (activated 

Hageman factor/Kallikrein system) and urine (Urokinase). In addition to endogenous 

activators, a variety of bacterial products and pyrogens interact with the 

fibrinolytic system. The plasminogen activator of greatest physiologic importance 

is the tissue activator synthesized in venous endothelium and the vasa vasorum of 

muscular arteries. The release mechanism of the vascular-derived activator is 

poorly understood. To counterbalance the powerful proteolytic effects of plasmin, 

there are numerous fibrinolytic inhibitors which are normally present in 

approximately 30-fold excess concentration. These circulating anti-plasmins and 

anti-activators include alpha-1-antitrypsin, alpha-2-macroglobulin, antithrombin m, 

Cl-inhibitor, and alpha-2-anti-plasmin. 

The spontaneous fibrinolytic activity of blood may be measured by simply observing 

the time for clot lysis of whole blood collected in a glass tube (Whole Blood Clot 

Lysis Time). Due to the presence of natural inhibitors of fibrinolysis, clot lysis will 

occur only in cases of profoundly elevated fibrinolysis. In the Dilute Plasma Clot 

Lysis Test, dilution of the plasma results in differential depression of the inhibitors 

/#^ and allows lysis to occur in normal subjects or cases of slightly elevated fibrinolysis. 

^ The lysis time will generally be 18-24 hours. The ECL test is based on the 

concentration and precipitation of the fibrinolytic factors in the "euglobulin'1 

fraction with removal of the fibrinolytic inhibitors in the supernatant. Thus with 

most inhibitors removed, lysis time will be shortened. The ECL test is felt to 

reflect the spontaneous fibrinolytic activity of plasma and most closely corresponds 

to the level of plasminogen activators. 

TECHNICAL PROBLEMS 

Fibrinolytic activity is increased when plasma comes in contact with glass, probably 

due to the activation of the Hageman factor /Kallikrein system which is capable of 

generating plasmin. Thus, glass contact must be minimized. Venous occlusion is 

known to cause release of plasminogen activators from venous endothelium and 

particular attention to blood drawing technique is necessary. The method of 

precipitation of the euglobulin fraction is critical and the ECL times will change 

significantly with slight variations in pH and plasma dilution. Most (62%) of the 

fibrinogen is precipitated with the euglobulins, but this will vary if pH is not 

controlled. Some anti-plasmin activity, probably Cl-inactivator, will co-precipitate 

with the euglobulins even if pH and ionic strength of the plasma dilution are 

carefully standardized. A recent modification of the ECL test utilizes the addition 

of sodium flufenamate to the euglobulin fraction to neutralize any remaining Clinactivator 

(not done in the UCSF lab). Precipitation of euglobulins in the presence 

of dextran sulfate polymer will markedly increase the recovery of plasminogen 

activator and pro-plasminogen activator. Heparin and EACA (epsilon-amino-caproic 

acid) remain in the supernatant. Most authors strongly advise against storage of the

Page 3 

plasma prior to test performance. Storage at 20°C or 36°C will markedly decrease 

fibrinolytic activity. However, storage at -20°C may not affect outcome. For 

reproducible results, the test should probably be run within two hours of blood 

collection. The type of anticoagulant used will affect results. Citrate is 

recommended. EDTA will increase fibrinolysis whereas heparin will reduce it. 

Variations in the ECL time will occur with alterations in the fibrinogen level since 

the patient's fibrin clot is the substrate in the assay. Care should be taken to avoid 

undue handling of the tubes during performance of the ECL test. The endpoint 

(complete clot dissolution) is usually easy to read, although difficulties may occur in 

lipemic sera. 

CLINICAL APPLICATIONS 

The ECL test and related assays have been used to investigate a variety of factors 

which influence spontaneous fibrinolysis. Epinephrine, stress, and exercise will 

markedly increase fibrinolytic activity either by neuronally mediated release of 

plasminogen activator or through their effects on vascular permeability. ACTH and 

corticosteroids potentiate the effects of epinephrine in stimulating activator 

release. Numerous drugs, including procainamide, nicotinic acid, sulfonylureas, 

phenformin, reserpine, pitressin, androsterone and DDAVP will increase fibrinolytic 

activity. Some of these may find therapeutic application as antithrombotic agents. 

Various surgical procedures produce a biphasic response in fibrinolysis. Many 

neurologic procedures, including ventriculography and pneumoencephalography, have 

been shown to markedly increase spontaneous fibrinolysis and on occasion provoke 

severe bleeding. Post-operatively, fibrinolysis usually decreases, reaching a nadir on 

postoperative days 1 and 2. Studies have indicated that large rapid falls in 

fibrinolytic activity as measured by the ECL test are associated with increased 

incidence of post-operative deep venous thrombosis. In approximately 95% of 

normal subjects, venous occlusion stimulates fibrinolysis in blood collected from the 

occluded vein. This potentiation of fibrinolysis with occlusion is more marked in 

arm than in leg veins. Subjects with idiopathic deep venous thrombosis frequently 

show no such enhancement of fibrinolysis upon venous occlusion. Release of 

plasminogen activators from venous endothelium of occluded vessels may be 

physiologically important in controlling thrombosis in situations of stasis. 

Fibrinolysis has also been reported to be reduced in individuals with multiple risk 

factors for myocardial infarction and in patients with peripheral atherosclerotic 

disease, vasculitis, and Raynaud's phenomenon. 

Thrombolytic agents such as Urokinase and Streptokinase markedly accelerate 

fibrinolysis through direct and indirect mechanisms (see chart). The ECL test was 

formerly used to monitor patients receiving thrombolytic therapy for acute 

myocardial infarction. Currently, however, only PT, PTT, TT, platelets and 

fibrinogen are commonly monitored during intracoronary streptokinase infusion. 

The ECL test has been used to monitor patients receiving urokinase therapy for the 

treatment of DVT. 

The ECL test has been used in the investigation of primary fibrinogenolysis 

(hyperplasminemia). Various tissues, including prostate, uterus, and colon appear to 

be exceptionally rich in plasminogen activator substances. Surgical manipulation of 

these organs may give rise to a consumptive coagulopathy based on widespread

Page 4 

intravascular activation of fibrinolysis. Plasmin has a wide range of substrates, 

including factors V and VIII, and will interfere with coagulation by multiple 

mechanisms. Production of abnormal quantities of plasminogen activator has also 

been described in certain tumors and acute leukemias. In primary fibrinogenolysis, 

there will be a marked increase in the spontaneous fibrinolytic activity of plasma. 

Increased fibrinolytic activity may also be seen, although usually to a lesser extent, 

in DIC. 

There are no studies providing detailed comparisons of coagulation and fibrinolytic 

parameters in primary fibrinogenolysis versus DIC. The utility of the ECL test in 

making this distinction is uncertain. In clinical practice, DIC should be ruled out on 

clinical and laboratory grounds before primary defects in fibrinogenolysis are 

suspected. There is little agreement on the specific clinical indications for 

employing the ECL test in general patient management. At present, the test 

appears useful mainly as an investigative and research tool. Additionally, the 

variety of factors sited above which affect fibrinolysis detract from the specificity 

and diagnostic utility of the ECL test. 

FURTHER DEVELOPMENTS 

In the ECL test, the patient's own fibrin clot serves as the substrate for fibrinolysis. 

Different substrates may also be used, including casein (caseinolysis) and synthesis 

esters (esterolysis). The most important modification of the ECL time is the Fibrin 

^ Plate assay in which a sample of plasma or euglobulin fraction is placed directly on 

the surface of a petrie dish containing human or bovine fibrin. At approximately 20 

hours, the zone size of fibrinolysis is measured. There is a relatively good 

correlation between assays of fibrinolysis as measured in the fibrin plate and the 

ECL test. The fibrin plate assay has a more precise and easily quantifiable endpoint 

and demands less technician intervention. However, it is not suitable for stat 

determinations. A recent modification of the ECL test used the addition of radiolabelled 

fibrinogen to the euglobulin fraction. The rate of radioactivity release is 

proportional to the fibrinolytic activity of the test plasma. This technique is very 

precise and has been used to investigate fibrinolytic activity in families with mild 

bleeding tendencies. For many research and clinical purposes, the ECL test has 

been supplanted by recently developed assays based on a chromogenic substrate (S- 

2251) which is a specific substrate for plasmin and plasminogen activated 

streptokinase. Using the chromogenic substrate technique, plasminogen activator, 

plasminogen, plasmin, and anti-plasmins may be easily and readily quantified. 

REFERENCES 

1. Allen RA. Blood sampling techniques for fibrinolytic studies. Thromb Res 

23:1-2. 

2. Von Kaulla KN, von Kaulla E. "Remarks on the Euglobulin Lysis Time." In: 

JF Davidson (ed.), Progress in Chemical Fibrinolysis and Thrombolysis, Vol 1. 

Raven Press: New York, 1975.

fifik^ 

Page 5 

3. Griffiths NJ, et al. Plasma fibrinolytic inhibitors post operation. Surg 

Gynecol Obst 144:673-676, 1977. 

4. Knight MTN, et al. Fibrinolytic response to surgery. Lancet 2:370-373, 1977. 

5. Gader AMA. The potentiating effect of corticosteroids on the fibrinolytic 

system in response to I.V. infusion of adrenalin in man. Haemostasis 17:353- 

356, 1982. 

6. Heimburger N. "Plasminogen Assay: a review and evaluation of various 

methods." In: JF Davidson (ed.), Progress in Chemical Fibrinolysis and 

Thrombolysis, Vol 1. Raven Press: New York, 1975. 

7. Prentice CRM. Basis of antifibrinolytic therapy. J Clin Pathol (Suppl.) 33 

(suppl 14) pp. 35-40. 

8. Horvath AE. The 125I-Fibrinogen Euglobulin Clot Lysis Test. Am J Clin 

Pathol 70(2):271-274, 1978. 

9. Latallo ZS, et al. Assessment of plasma fibrinolytic system with use of 

chromogenic substrate. Haemostasis 7:138-145, 1978. 

10. Blix S. Studies on the fibrinolytic system in the euglobulin fraction of human 

plasma. Scand J Clin Lab Invest 13(suppl 58), 1961. 

11. Arneson H, et al. "A comparison between the ECL time and the fibrin plate 

method for the estimation of fibrinolytic activity after venous stasis." In: JF 

Davidson (ed.), Progress in Fibrinolysis, Vol 5. Churchill Livingstone: 

Edinburgh, 1981. 

12. Nilsson IM, et al. "Physiology of Fibrinolysis." In: DL Kline, KNN Reddy 

(eds.), Fibrinolysis, CRC Press: Boca Raton, Florida, 1980. 

13. Theiss W, et al. Systemic fibrinolytic activity and inhibitor levels during 

treatment of deep venous thrombosis with urokinase and streptokinase. 

Thrombosis and Haemostasis 5(3):664-668, 1983. 

14. Kluft C. Studies on the fibrinolytic system in human plasma: quantitative 

determination of plasminogen activator and pro-activator. Thrombosis and 

Haemostasis 41:365-383, 1979. 

15. Von Kaulla KN, Schultz RL. Methods for evaluation of human fibrinolysis. 

Am J Clin Pathol 29:104-112, 1958.

Euglobin Lysis Time

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