Euglobulin Lysis Times: an Update*

Euglobulin Lysis Times: An Update*

A. GLASSMAN, M.D., M. ABRAM, M.D., G. BAXTER, B.S., and A. SWETT

Department of Laboratories, Vanderbilt University Medical Center, Nashville, TN 37232

ABSTRACT There are several clot lysis tests available to assess the presence of increased fibrinolysis in a clinical situation. These include whole blood, plasma, and “eugloblin” lysis tests. The euglobulin lysis test (ELT) is actually a modified plasma clot lysis time, which detects increased plas minogen activation and subsequent fibrinolysis. It does not detect the activity of antiplasmins. The purpose of this study was to determine ELT for a group of 25 ambulatory normals. There were 10 males and 15 females, ranging in age from 25 to 56 years. Citrate anticoagulated samples were obtained immediately after application of a tourniquet and 10 minutes after application of a sphygmomanometer inflated to midway between systolic and diastolic pressure. The citrated plasma was precipitated with cold ace tic acid. The precipitate contained fibrinogen, plasminogen, and plasmino gen activators, with fibrinolytic inhibitors theoretically removed. The pre cipitate was redissolved and the euglobulin solution was clotted with thrombin. The clotted sample was then incubated at 37°C and was observed at 10 minute intervals for clot lysis. Results: The reference range of onset of fibrinolysis was from 1.25 to 12 hours, with a mean of 3.78 hours ± 2.45 hours (1 S.D.). Venostasis (use of the sphygmomanometer) resulted in an ELT that was shorter than the non-venostasis specimens in 11 of 25 specimens. This is thought to be secondary to in vivo activation of plas minogen. Conclusion: The average euglobulin lysis time in 25 ambulatory normals is approximately 3.5 hours. The ELTs vary over a broad range (1.25 to 12.0 hours) in normal ambulatory volunteers. Interpretation of shortened ELTs must be made cautiously in the clinical situation.

Several clot lysis tests are available. clot lysis can be done on whole blood, These are used to assess the presence of plasma, and by a method know as the increased fibrinolysis in a variety of clini “euglobulin” lysis test (ELT), which is cal situations.3 The determinations for actually a modified plasma clot lysis time. It is thought that this test detects increased plasminogen activation with * Send reprint requests to: Armand B. Glassman, subsequent fibrinolysis.2,5 However, the M.D., Clinical Laboratories, The Vanderbilt Clinic 4605, 1161 21st Avenue South, Nashville, TN activity of anti-plasmins cannot be 37232-5310. detected with this method. 329 0091-7370/93/0900-0329 $00.60 © Institute for Clinical Science, Inc. 330 GLASSMAN, ABRAM, BAXTER, AND SWETT To perform the euglobulin lysis test, patient’s diastolic and systolic pressure inhibitors of plasminogen activation and readings, and maintaining that pressure plasmin are removed via precipitation for 10 minutes prior to drawing with solutions of low ionic strength and the sample. low pH. The precipitate is solubilized in The test is run as follows. (1) Label one an appropriate buffer and subsequently 13 x 100 mL glass tube for each plasma clotted through the addition of thrombin. specimen to be tested. One tube for the Clot lysis is reported as the time required normal control and one tube for the for the clot to begin to dissolve.1,5 abnormal control should also be pre The purpose of this study is to deter pared. (2) Add 0.5 mL of each patient mine the ELT for a group of ambulatory plasma specimen or the normal control to normal volunteers. The ELT was deter the correspondingly labelled tube. (3) mined for these subjects both without Reconstitute one vial of plasminogen venostasis and with venostasis (produced activator with 1 mL of H20. This should by sphygmomanometer) for 10 minutes be mixed and poured into a vial of unre prior to drawing the specimen. The 10 constituted plasma control, which will minute venostasis period was to assess serve as the abnormal fibrinolysis con whether or not plasminogen activation trol. (4) As soon as the reconstitution is might take place if a tourniquet were left complete, add 0.5 mL of abnormal fibri in place too long prior to obtaining nolysis control to the abnormal control the specimen.4 tube. (5) Add six mL of cold (2°-8°C) deionized water to each tube. (6) Add exactly 0.1 mL 1% acetic acid to each Method tube. (7) Mix by inversion. (8) Place tubes in the refrigerator 2°—8°C) for 10 minutes Twenty-five ambulatory normals were to allow complete euglobulin precipita used as the volunteer study group, tion. (9) Remove the tubes and centrifuge including 10 males and 15 females rang at 3000 rpm for three minutes to sedi ing in age from 25 to 56 years. Specimens ment the euglobulin precipitate. (10) were collected in 4.5 or 3.0 mL sodium After centrifugation, pour off and discard citrate tubes and were mixed well and the supernatant fluid. With tubes still transported on ice prior to determination. inverted, thoroughly blot the excess Anticoagulated blood was spun at 3,000 supernate from the inside of the tubes rpm for 10 to 15 minutes. The citrated with a cotton-tip applicator or twisted plasma was carefully removed so as not to kim-wipe. (11) Add 0.35 mL (350 |xl) disturb the buffy coat, and the plasma phosphate buffer saline to each tube and was determined to be free of red cells and shake gently until all precipitate is redis platelets. Samples were run within 30 solved. Make sure that no undissolved minutes of collection. If testing was to be “button” of precipitate remains. (12) delayed for a longer period of time, sam Reconstitute 1 vial of buffered thrombin ples were frozen in aliquots of 1.0 mL with 2.0 mL deionized H20 . (13) Using a each at — 70°C. separate pipette tip for each tube, add 25 Pre- and post-venostasis specimens jxl of reconstituted buffered thrombin were drawn. To obtain the pre-venostasis and mix immediately to clot the dis sample, a tourniquet was applied and solved precipitates. (14) Place tubes in blood was drawn as soon after application 37°C water bath and start timing lysis. as possible. The post-venostasis sample After 30 seconds, verify clotting by gently was obtained by inflating a sphygmoma tilting tubes. (15) Observe for clot lysis at nometer to a point halfway between the 10 minute intervals by gently tilting EUGLOBULIN LYSIS TIMES: AN UPDATE 33 1 tubes. The end point is the time required TABLEI for complete degradation of the clot to Euglobulin Lysis Time: Females small fibrin strands or particles. In routine patients, watch the clot for at Pre-stasis Lysis Post-stasis Lysis least two hours at 10 minute intervals or # Time in Hours Time in Hours until the clot dissolves, whichever comes first. Record the end point as the time the 1 4.00 4.00 clot dissolves or greater than two hours. 2 2.00 0.92 In pre- and post-stasis specimens, watch 3 2.58 2.58 the clot at 10 minute intervals until the 4 5.17 4.92 clots dissolve or for eight hours, which 5 3.42 3.67 ever comes first. Record the end point as 6 2.42 2.00 the time the clot dissolves or greater than 7 1.67 1.50 eight hours. 8 4.92 4.58 9 4.83 1.08 10 1.33 1.33 Results 11 4.50 3.25 Results are reported as the time in 12 6.50 3.50 hours or fraction of hours required for the 13 2.08 2.58 clot to dissolve. Of 25 ambulatory normal 14 2.75 2.75 15* 12.00 12.00* specimens, the individual results ranged Total 14 Females from 1.25 to 12.0 hours. The highest and Mean 3.44 hr 2.76 hr lowest values were dropped from the cal Range 1.33-6.50 0.92 - 4.92 culations of the means and standard 1 SD 1.05 2.76 deviations. Mean values for the 14 females are: pre-stasis 3.44 ± 1.05 (S.D.) * Result deleted from calculation. hours; post-stasis 2.76 ± 2.76 (S.D.) hours; and for the nine males, pre-stasis nant plasminogen activator, streptoki 3.68 ± 2.17 (S.D.) hours; post-stasis 3.64 nase, or urokinase. It is noted that of 25 ± 1.75 (S.D.) hours (tables I and II). ambulatory normal volunteers, five or 20 There was no statistically significant dif percent had ELTs equal to or less than ference between pre- and post-venostasis two hours. Functional assays, such as the ELT, and no significant difference ELT, are usually adequate for the clinical was noted between the male and situation, but if alpha 2 anti-plasmin defi female ELT. ciency is suspected, a specific immuno logic assay is recommended. Discussion In this study, venostasis, as produced The “euglobulin” fraction of plasma by a sphygmomanometer inflated to a contains plasminogen, fibrinogen, plas- point midway between the systolic and min, and fibrinolytic activators which are diastolic pressures of the normal volun precipitated when the sample is acidi teer, did not result in a statistically sig fied. Inhibitors of the fibrinolytic system nificant alteration of the ELT. This is which occur as a natural phenomenon are considered to be indirect evidence that discarded in the supernatant fraction. A significant fibrinolysis, as measured by short ELT of less than two hours is said to the ELT, is not apparent after up to 10 reflect acute episodes of fibrinolysis. minutes of venostasis. The ELT values These may accompany acquired disor for males and females are not statistically ders or result from the administration of different. The range of ELT extends from plasminogen activator, such as recombi- 1.25 to 12 hours. There was only a single 332 GLASSMAN, ABRAM, BAXTER, AND SWETT

TABLE II information about the action of plasmino gen activators and plasmin. Because of a Euglobulin Lysis Time: Males broad reference range, interpretation of Pre-stasis Lysis Post-stasis Lysis shortened ELT’s indicative of fibrino # Time in Hours Time in Hours lytic activity should be made with cau tion unless a baseline determination has 1 2.58 2.58 been made on the same individual under 2 2.33 1.75 non-stressed conditions. 3 4.10 4.10 4 7.50 7.50 Acknowledgment 5 7.00 7.00 The assistance of Ms. Debbie Warren in the 6 2.55 2.55 preparation of this manuscript is gratefully 7 1.58 1.33 acknowledged. 8 3.17 3.67 9 2.33 2.33 References 10* 1.25 0.83* 1. Jo h n s o n A. J ., S e m a r , M., and N e w m a n , J.: Estimation of fibrinolytic activity by whole Total 9 Males blood euglobulin clot lysis. In: Tocantins, Mean 3.68 hr 3.64 hr L. M., Kazal, L. A., eds. Blood Coagulation, Range 1.58-7.50 1.33-7.50 Hemorrhage and Thrombosis. New York, 1 SD 2.17 1.75 Grune and Stratton, Inc., 1964, pp. 46.5-467. 2. K o w a l s k i , E., K o p e c , M., and N i e w a r o w s k i , S.: Evaluation of euglobulin method for deter * Result deleted from calculation. mination of fibrinolysis. J. Clin. Path. 12:215, 1959. 3. M cG A N N , M. A. and TRIPLETT, D. A.: Labora value at 12 hours, from one individual. tory evaluation of the fibrinolytic system. Lab. The interpretation of the ELT should be Med. 14:18, 1983. made cautiously with this wide range 4. N g u y e n , G., H o r e l l o , M. H ., K r u i t h o f , K. O., C o n r a d , J., and S a m a m a , M. M.: Resid in m ind.6 ual plasminogen activator inhibitor activity after venostasis as a criterion for hypofibrinol- ysis: A study in 83 patients with confirmed Summary and Conclusions deep vein thrombosis. Blood 72:601-605,1988. 5. S h e r r y , S ., L i n d e m e y e r , R. I., F l e t c h e r , The mean ELT is approximately 3.5 A . P., and A l k j a e r s i g , N.: Studies on enhanced fibrinolytic activity in man. J. Clin. hours. The range, when dropping two Invest. 38:810, 1959. outliers, is from 1.33 to 7.5 hours. The 6 . V o n K a u l l a , K . N. and S c h u l t z , R. L: Meth ods for evaluation of human fibrinolysis: Stud ELT is a test that can be readily per ies with two combined technics. Am. J. Clin. formed in the laboratory. It provides Path. 29:104, 1958.