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Thrombosis in spinal cord injury
THROMBOSIS RESEARCH 68; 357368,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
THROMBOSIS
IN SPINAL CORD INJURY
Yukihiko Fujii', Eberhard F. Mammen*, Abdelmonem Farag', Jaroslaw Muz3, Gino G. Salciccioli4, and Saul T. Weingarden'. Department of Neurosurgery, Niigata Brain Research Institute', Niigata, Japan and Departments of Patho+ogy*, Nuclear Medicine3, Orthopedics and Rehabilitation Medicine, Wayne State University, Detroit, MI, U.S.A. (Received 19.6.1992; accepted in original form 21.9.1992 by Editor F. MarkwarUt)
ABSTRACT Some traditional coagulation assays and several new molecular markers of hemostatic activation were meabured in 37 patients with spinal cord injury (SCI). TWenty one of the patients (57%) developed deep vein thrombosis (DVT). The radiofibrinogen uptake test (RFUT) was used to diagnose DVT. Thirty eight percent of quadriplegic and 88% of paraplegic patients developed DVT (p ~0.005). No significant differences were found in platelet counts, mean platelet volumes, fibrinogen levels, von Willebrand factor (Ag) levels, platelet factor 4 and beta thromboglobulin concentrations between the groups with and withIII Fibrinopeptide A, thrombin/antithrombin out DVT. (TAT) complexes and plasma D-dimer levels were significantly higher in the patients with thrombosis. Most with DVT had elevated TAT complex levels up patients D-dimer to three days before the RFUT became positive. levels were highest after the diagnosis had been made. INTRODUCTION Deep venous thrombosis (DVT) and pulmonary embolism (PE) are potentially fatal complications for hospitalized patient$, both Especially patients with acute spinal cord medical and surgical. injury (SCI) and paralysis are at considerable risk. The overall Key words: Spinal cord injury, venous thrombosis, A, thrombin/antithrombin III complexes, D-dimer.
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incidence of DVT was found to be from 70 to 100% radiofibrinogen uptake test (RFUT) was used (l-3).
when
the
Immobility due to paralysis is one of the major contributing The factors for the development of DVT in patients with SCI. resulting reduction in blood flow is due to a lack of pumping Blood seems to pool in action of the contracting muscle packages. the intramuscular sinuses of the calf and contribute to the development of DVT (4). Blood flow may further be influenced by viscosity (5) brought about by increased blood increased fibrinogen and von Willebrand factor elevated hematocrits, levels Increased platelet complex macromolecular (2,6). aggregation has also been measured (7). Decreased blood flow could of activation lead to endothelial damage, local accumulation products of coagulation, and local decreases in inhibitor levels. All would favor increased coagulability of the blood locally. Impaired fibrinolysis may be another predisposing factor (7). Of the non-invasive diagnostic tools to identify early DVT, the RFUT was a very sensitive procedure (4,8). Unfortunately this test was technically cumbersome, required elaborate personnel support and is no longer available. It would obviously be of advantage to have some reliable and relatively easy laboratory parameter(s) which would predict a developing thrombosis so that those patients could be treated with anticoagulants. Numerous attempts have been made to identify states of "hypercoagulability" by laboratory tests, but up to this point only elevations in the factor VIII macromolecular complex have been claimed to be of predictive value in SC1 patients (2,6). During the last few years a number of molecular markers of hemostasis activation have been introduced, using radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) technology. The markers are fibrinopeptide A (FpA), platelet factor 4 (Pf4), betathromboglobulin (BTG), thrombin-antithrombin III complexes (TAT), D-dimer (D-Di), and more recently prothrombin fragment 1+2. FpA is a small peptide released from the fibrinogen molecule predominantly by thrombin but possibly also by plasmin and tissue plasminogen activator (t-PA). Its elevation in plasma could signal that thrombin was present when the sample was drawn (9). Pf4 and BTG are two platelet specific proteins. Elevated plasma levels could indicate that platelets were activated in vivo and that they underwent a release reaction (lo), provided the blood sample was properly collected. Antithrombin III (AT) is one of the major inhibitors of clotting enzymes. It inactivates thrombin by forming equimolar thrombin-AT (TAT) complexes. Elevated levels of TAT in plasma mean that thrombin was present (11). D-Di is a fragment of fibrin (not fibrinogen). It is released when plasmin proteolytically digests fibrin. Its elevation in plasma indicates that both thrombin and plasmin were present (12).
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The objective of this study was to apply some of these newly developed tests to spinal cord injury patients and correlate these data with RFUT. MATERIALS
AND METHODS
Thirty-seven patients, admitted to the Spinal Cord Unit of Detroit Receiving Hospital with acute spinal cord injury, completed the protocol. Of these, 8 were females; the age ranged from 18 to 73 years. Fifteen patients had paraplegia, 18 quadriplegia and 4 had spinal cord fracture with only temporary paralysis. All patients gave their consent which was approved by the Human Investigation Committee of Wayne State University and entered the study within 48 hours after spinal cord injuries. All had normal clotting screening tests and had similar routine physiotherapy during the course of the study. The following patients were excluded: 1) patients with trauma to the legs and left thorax (this could make the RFUT unreliable), 2) patients with recent myocardial infarctions or coronary artery insufficiency, 3) patients requiring plasma expanders of the dextran type, 4) patients who were on anticoagulants during the last ten days preceding the acute SCI, 5) patients with postphlebitic syhdrome, 6) patients in various forms of shock, 7) patients with known hypersensitivity to iodine or other contrast media used for venography, 8) patients with severe hepatic or renal diseases, 9) patients with known bleeding problems, and 10) pregnant patients. RFUT was initiated within the first three days after injury. At least 12 hours before and daily thereafter, the patients received 100 m potassium iodide to block the thyroid glands. About 100 uCi P fibrinogen (Ibrin, Amersham, Arlington Heights, IL) was of '*I injected intravenously. was The uptake of radiofibrinogen monitored over six distinct points of the thigh and six po'ints on the posterior popliteal fossa and calf of each leg and expressed as ratio of leg to precordial activity. An increase of 15 to 50% between any two points which persisted for 48 hours was considered as positive. All patients were scanned daily at the bedside for 30 days or when the test became positive. In case of a positive scan, we attempted to obtain a venography to confirm the diaignosis. Unfortunately this could not be accomplished in all patients so that we based our correlations on RFUT data. Before the initiation of the RFUT and every second day thereafter, blood samples were drawn for hemostasis studies, After clean venipuncture the first two ml of blood were discarded. Of the following 9 ml drawn, 4.5 ml were immediately transferred into a prechilled plastic tube containing 0.5 ml of a special anticoagulant (Diatube, American Bioproducts Company, Parsipanny, NJ) which is designed to prevent in vitro platelet activation and This sample was used for the assays of FpA, RTG and Pf4. release. After centrifugation at 2,500 x g for 30 minutes at 4'C, the top one-third of the plasma of this tube was recovered, placed in plastic tubes, immediately frozen and kept at -70°C for later batch The second 4.5 ml blood were transferred into a plastic analysis.
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IN SPINAL CORD INJURY
This blood tube containing 0.5 ml 3.8% sodium citrate solution. was used for platelet counts and mean platelet volumes; the plasma, for the above, described centrifugation, as obtained by determination of the other parameters. The following assays were performed by commercial RIA kits: ATG (Amersham, Arlington Heights, IL), Pf4 (Abbott Laboratories, The RIA assays were performed on an Abbott North Chicago, IL). ANSR Automated Gamma Counter. The following were determined and D-di (American Bioproducts), Marburg, Germany).
by commercial ELISA kits: FPA TAT complexes (Behringwerke,
Platelet counts (PLTC) and mean platelet measured on a Baker 810 Platelet Counter.
volumes
(MPV) were
Prothrombin Times (PT) and Activated Partial Fibrinogen, Thromboplastin Times (aPTT) were determined on an Automated Laboratory, (Instrumentation Coagulation Laboratory (ACL) Lexington, MA). von Willebrand factor antigen (vWFAg) was technique using Laurel1 immunoelectrophoresis Calbiochem-Behring (La Jolla, CA). The Student's
T-test was used for statistical
assayed by the antisera from analysis.
RESULTS Incidence
of DVT
Twenty-one of the 37 patients (57%) had a positive RFUT. One of these had a pulmonary embolism. Positive RFUTs were identified between the third and the 20th day post injury. Nine patients had a positive RFUT in the first week, 5 in the second week, 7 in the third week, but none after the 21st day. When the patients were divided into five groups according to their level of spinal cord injuries, 39% of the complete quadriplegic group, 25% of the incomplete quadriplegic group, 88% of the complete paraplegic group, 89% of the incomplete paraplegic group and none of the temporary paresis group had DVT (Table 1). The difference between the incidence of DVT in the paraplegia groups and the quadriplegia groups was highly significant (p < 0.005). There was no significant difference between the complete and the incomplete paresis groups. There also was no significant age difference between the 21 RFUT positive and the 16 RFUT negative patient (Table 1).
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TABLE 1 Clinical Summary of 37 Patients with SC1 DVT (+)
DVT (-1
Quadriplegia (complete)
(3i%)
(671%)
Quadriplegia (incomplete)
(251%)
(7i%)
Paraplegia (complete)
(8:%)
(IS%)
Paraplegia (incomplete)
(8:%)
(111%)
(E%)
(1040%)
Total 12 4 8 9
Temporary paresis
4
Total
37 (G%)
Age
41.2 (k18.8)
(4136%) 36.2 (219.3)
39.0 (218.9)
SC1 = spinal cord injury; DVT = deep venous thrombosis Changes
in Platelet
Counts and Mean Platelet Volumes
The average platelet counts of the two groups gradually increased after injury and reached maximum levels between 15 and 21 days after injury. There were no significant differences at any point between the two groups (Table 2). The mean platelet volumes gradually decreased following injury, and again no significant differences were found at any time. There was also no correlation between these two platelet parameters and the cases with a positive RFUT. Chanaes
in Fibrinogen
and vWFAq
The initial fibrinogen levels of the positive and negative DVT groups were 499 + 170 mg/dl and 453 f. 182 mg/dl, respectively. The levels gradually increased in both groups and reached maximum values on the 8th day post injury (Table 2). They plateaued during the second week. There were no significant differences between the two groups, however, and no correlation could be identified to onset of positive RFUT.
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TABLE 2 Highest Values of Hemostasis Normal PLTC (x109/L) MPV (pm3) Fibrinogen (mg/dl) vWFAg (%) Pf4 (ng/ml) BTG (ng/ml) FPA
(w/W
D-dimer (ng/ml) TAT (ng/ml)
200 6.8 200 60 0 0 0 0 0
- 350 - 7.8 - 350 - 200 - 10 - 50 -3 - 500 -5
DVT = deep venous thrombosis;
Parameters
in 37 Patients
DVT (+) fN=21)
DVT (-) fN=16)
3982147 7.720.8 9332248 605+478 13.726.9 57.4221.5 23.3k13.1 6361+3090 16.428.7
3602164 8.020.8 807+240 6272443 12.1k6.0 48.4k16.7 15.8k12.8 327823321 7.356.2
t-test NS NS NS NS NS NS
p
NS = not significant
increased in both groups. vWFAg levels also continuously Values peaked on the 14th day after injury in the RFUT negative group and increased until the 8th day in the DVT positive group. No significant differences After that they gradually decreased. were observed between the two groups (Table 2) and no correlation was found to the positive RFUT. Chanaes
in BTG. Pf4 and FPA
Pf4 and BTG levels were slightly above normal limits for most patients, with no significant differences between the groups (Table 2). In the negative RFUT group, mean FpA levels stayed under or around 10 ng/ml for the entire length of the study. In contrast, the mean FpA levels of the positive group increased gradually until the 8th day after injury, and gradually decreased thereafter (Figure 1). Changes
in TAT Comolexes
and D-dimer
Mean TAT complex levels for the positive RFUT group were always higher than those of the negative RFUT group. Already during the first week there were significant differences between the two groups (Figure 2). D-Dimer levels of both groups were elevated over the normal limit, but mean levels of the positive RFUT group were always significantly greater than those of the negative RFUT group (Figure 3).
THROMBOSIS
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FPA
q/ml
. ..*
ngiml
26 -
TAT
20 24
-
22
-
O-0 B-4
20
-
16
-
16
-
14
-
12
-
DvT(+)
16
~
DVT(-)
16
-
a
-
6
-
4
-
2
-
1
O-2
3-5
6-8 DAYS
9-11
12-14
AFTER
22-30
3-5
6-8 DAYS
9.11 AFTER
I
12-14
15-21
22~30
INJURY
Figure 2: Mean thromkpin/ antithrombin III (TAT) complex levels in SC1 patients with (n=21) and without (n=16) positive RFUT. = p < 0.02,
***
=
p < 0.01, **** = p < 0.001.
D-DIMER T-
--
L
o-2
INJURY
..
DVT(ti
_W-~DVT(-I
‘-I T
**
8000 t
i
1
15-21
Figure 1: Mean fibrinopeptide A (FpA) levels in SC1 patients with (n=21) and withaut (n=16) positive RFUT. ** = p < 0.02, *** = p < 0.01.
ng/ml
.-d
.. .
..
I
10
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.-.DVT(+)
A
H
DVT(-)
7000 -
6000 -
5000
-
4000 -
3000 -
2000
-
1000 ,___‘_--_,____.I.--__________------. I
I o-2
3-5
6-8 DAYS
9-l AFTER
1
12-14 INJURY
15-21
22-30
Figure 3: Mean D-dimer levels in SC1 patients with N(n=21) and without (n=16) positive *p < 0.05, ** = p RFUT. 0.02, *** p < 0.01.
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When highest TAT complex levels were correlated to the days where the RFUT became first positive, 11 patients had highest levels before the diagnosis was made (8 patients three days before, 3 patients one day before), 5 patients had highest levels on the day of diagnosis and 5 patients had highest levels over the next three days after the positive RFUT test. In contrast, 15 patients had highest D-dimer levels after the RFUT test became positive, 3 patients had highest levels on the day of diagnosis and 3 patients had highest levels before the RFUT test became positive (2 patients one day before, 1 patient three days before). It appears that the majority of SIC patients with thrombosis had TAT complex elevations up to three days before the clinical diagnosis of DVT was made, while the majority had highest D-dimer levels after having established the diagnosis. DISCUSSION Spinal cord injury patients with paralysis suffer a high incidence of DVT and PE post trauma. Of our 37 patients, 57% developed DVT, diagnosed by RFUT. This incidence is slightly lower in the literature using the same test than figures reported nodality (l-3). DVT was more frequently encountered in paraplegic than in quadriplegic patients (p < 0.005). None of the four patients with temporary paresis had DVT. These findings support the concept that immobility is a major factor in the development of 3VT (4). We used RFUT as the diagnostic modality and attempted to confirm DVT by venography. Unfortunately this was not possible in sll patients. In those patients in whom both tests were performed 3VT was confirmed. RFUT is sensitive and specific for thrombosis in the calf and lower thigh, but does not always reveal a process in the upper thigh and the iliac veins (13). We carefully excluded patients in whom false positive RFUTs could have been detected. It LS also recognized that in some patients the test may take 48 hours >r longer to become positive after the acute thrombosis may already 38 present. We attempted to further mimimize errors by having only two experienced health care workers perform the test. Many attempts have been made to employ laboratory procedures for the detection of a thrombotic process. Most of these have failed because they lack sensitivity (14). Many tests become positive as a result of an underlying disease, such as trauma. Our patients with acute SC1 did not have massive trauma thus minimizing potential erroneous test results. On the other hand, they had a Jery high incidence of DVT. Elevations in the factor VIII macronolecular complex components (factor VIII:C, von Willebrand factor) lave been described in SC1 patients (2,6) and were found to be of >redictive value. Increases in fibrinogen have also been reported in conjunction with developing thrombosis. We did not find a
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difference in von Willebrand factor levels in our RFUT positive and negative patients; fibrinogen levels were higher in the positive group (Table 2) but the difference was not significant. It will be noted that both factor levels were markedly elevated in both groups. Both proteins are acute phase reactants. Less information is in the literature concerning the use of newer molecular markers of hemostatic activation. These tests measure either released substances from platelet act~ivation [platelet factor 4 (Pf4) and beta thromboglobulin (DTG)] or released peptides or fragments from enzymatic actions [f:ibrinopeptide A (FpA) and D-dimer (D-di)] or activation complexes between enzymes and inhibitors [thrombin/antithrombin III (TAT) compllexes]. These tests are RIA or ELISA based, are sensitive and allow quantitation of very small amounts in plasma. They are not only sensitive for thrombosis, but rather reflect any state of in vivo activation of the hemostasis system. All of these markers are elevated in surgical or trauma patients and in the immediatie postpartum period. After major surgeries they remain elevated for about 48 to 72 hours (15). Even placement of a Swan-Ganz uatheter can elevate some of these markers (unpublished observation). Our patients did not have major trauma and none were subjected to a surgical procedure during the period of observation. These markers can also become elevated in vitro by poor blood drawing technique or improper sample handling. We minimizled this a special tube which cdntained blood collection by using and by anticoagulants plus inhibitors of platelet activation, having all samples with considerable drawn by one person experience. Our data demonstrated significant differences in the levels of FpA, TAT complexes and D-di between the patients with or without a positive RFUT. The levels of Pf4 and BTG were also higher in the positive patients, but this did not reach statistical significance (Table 2). FpA levels were significantly elevated during the first 8 days after injury (Figure l), TAT complex levels and Dddi were higher during the entire study period in the RFUT positive patients (Figures 2 and 3). FpA and TAT complexes are markers of dhrombin generation and elevated plasma levels would suggest increased thrombin generation. D-di are fibrin degradation products and Since both'patient suggest increased in vivo plasmin generation. groups were otherwise similar, our findings suggest that the RFUT positive patients had demonstrable activation of their clotting and fibrinolytic systems. Platelet release proteins (Pf4 and DTG) and also FpA were found to lack sensitivity and specificity in the diagnosi$ of DVT In contrast, both TAT complexes and D-di demonstrated (16,17). D-di had a sensitivity between 90-lOO%, while great sensitivity. TAT complexes had a shorter half-life, due to a markedly sensitivity < 90% (17-20). D-di lacks specificity and is therefore Its test for DVT a good screening not necessarily (20). sensitivity was found to be 98% but its specificity only 38% in
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patients with documented PE (21). Yet, both D-di and TAT complex determinations seem to be of some predictive value for ruling out DVT in the postoperative period (22,23), and normal levels of both seem to make DVT unlikely (24). Our observations that highest TAT complex levels were found in 11 of 21 RFUT positive patients before the RFUT became positive and highest D-di levels in 15 of these patients after the positive RFUT suggests a possible predictive value for TAT complexes. It can not be ruled out, however, that some of these TAT complex positive patients might have had a delayed RFUT positivity, as indicated above. ACKNOWLEDGEMENT This work was supported by a grant from the National Institute and on Disability Rehabilitation Research 35196), (GO085 Washington, D.C. REFERENCES 1.
TODD J.W., FRIESBIE J.H., ROSSIER A.B., ADAMS D.F., ALS A.V., ARMENIA R.J., SASAHARA A.A., TOW D.E. Deep venous thrombosis in acute spinal cord injury: A comparison of "'1 fibrinogen leg scanning, impedance plethysmography and venography. Paraoleaia 14:50, 1976.
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PETAJA J., MYLLYNEN P., ROKKANEN P., NOKELAINEN M. Fibrinolysis and spinal injury. Relationship to posttraumatic deep vein thrombosis. Br. J. Haematol. 45:143, 1980.
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BOUNAMEAUX H., SCHNEIDER P.A., REBER G., De MOERLOOSE P., KRAHENBUHL B. Measurement of plasma D-dimer for diagnosis of Am. J. Clin. Pathol. 91:82, 1989. deep venous thrombosis.
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19.
ROWBOTHAM B.J., CARROLL P., WHITAKER A.N., BRUNCE I.H., COBCROFT R.G., ELMS M.J., MASCI P.P.P., BUNDESEN P.G., RYLATT D.B., WEBER A.J. Measurement of crosslinked fibrin derivatives. Use in the diagnosis of venous thrombosis. Thromb. Haemostas. 57:59, 1987.
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THROMBOSIS
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Yukihiko Fujii', Eberhard F. Mammen*, Abdelmonem Farag', Jaroslaw Muz3, Gino G. Salciccioli4, and Saul T. Weingarden'. Department of Neurosurgery, Niigata Brain Research Institute', Niigata, Japan and Departments of Patho+ogy*, Nuclear Medicine3, Orthopedics and Rehabilitation Medicine, Wayne State University, Detroit, MI, U.S.A. (Received 19.6.1992; accepted in original form 21.9.1992 by Editor F. MarkwarUt)
ABSTRACT Some traditional coagulation assays and several new molecular markers of hemostatic activation were meabured in 37 patients with spinal cord injury (SCI). TWenty one of the patients (57%) developed deep vein thrombosis (DVT). The radiofibrinogen uptake test (RFUT) was used to diagnose DVT. Thirty eight percent of quadriplegic and 88% of paraplegic patients developed DVT (p ~0.005). No significant differences were found in platelet counts, mean platelet volumes, fibrinogen levels, von Willebrand factor (Ag) levels, platelet factor 4 and beta thromboglobulin concentrations between the groups with and withIII Fibrinopeptide A, thrombin/antithrombin out DVT. (TAT) complexes and plasma D-dimer levels were significantly higher in the patients with thrombosis. Most with DVT had elevated TAT complex levels up patients D-dimer to three days before the RFUT became positive. levels were highest after the diagnosis had been made. INTRODUCTION Deep venous thrombosis (DVT) and pulmonary embolism (PE) are potentially fatal complications for hospitalized patient$, both Especially patients with acute spinal cord medical and surgical. injury (SCI) and paralysis are at considerable risk. The overall Key words: Spinal cord injury, venous thrombosis, A, thrombin/antithrombin III complexes, D-dimer.
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incidence of DVT was found to be from 70 to 100% radiofibrinogen uptake test (RFUT) was used (l-3).
when
the
Immobility due to paralysis is one of the major contributing The factors for the development of DVT in patients with SCI. resulting reduction in blood flow is due to a lack of pumping Blood seems to pool in action of the contracting muscle packages. the intramuscular sinuses of the calf and contribute to the development of DVT (4). Blood flow may further be influenced by viscosity (5) brought about by increased blood increased fibrinogen and von Willebrand factor elevated hematocrits, levels Increased platelet complex macromolecular (2,6). aggregation has also been measured (7). Decreased blood flow could of activation lead to endothelial damage, local accumulation products of coagulation, and local decreases in inhibitor levels. All would favor increased coagulability of the blood locally. Impaired fibrinolysis may be another predisposing factor (7). Of the non-invasive diagnostic tools to identify early DVT, the RFUT was a very sensitive procedure (4,8). Unfortunately this test was technically cumbersome, required elaborate personnel support and is no longer available. It would obviously be of advantage to have some reliable and relatively easy laboratory parameter(s) which would predict a developing thrombosis so that those patients could be treated with anticoagulants. Numerous attempts have been made to identify states of "hypercoagulability" by laboratory tests, but up to this point only elevations in the factor VIII macromolecular complex have been claimed to be of predictive value in SC1 patients (2,6). During the last few years a number of molecular markers of hemostasis activation have been introduced, using radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) technology. The markers are fibrinopeptide A (FpA), platelet factor 4 (Pf4), betathromboglobulin (BTG), thrombin-antithrombin III complexes (TAT), D-dimer (D-Di), and more recently prothrombin fragment 1+2. FpA is a small peptide released from the fibrinogen molecule predominantly by thrombin but possibly also by plasmin and tissue plasminogen activator (t-PA). Its elevation in plasma could signal that thrombin was present when the sample was drawn (9). Pf4 and BTG are two platelet specific proteins. Elevated plasma levels could indicate that platelets were activated in vivo and that they underwent a release reaction (lo), provided the blood sample was properly collected. Antithrombin III (AT) is one of the major inhibitors of clotting enzymes. It inactivates thrombin by forming equimolar thrombin-AT (TAT) complexes. Elevated levels of TAT in plasma mean that thrombin was present (11). D-Di is a fragment of fibrin (not fibrinogen). It is released when plasmin proteolytically digests fibrin. Its elevation in plasma indicates that both thrombin and plasmin were present (12).
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The objective of this study was to apply some of these newly developed tests to spinal cord injury patients and correlate these data with RFUT. MATERIALS
AND METHODS
Thirty-seven patients, admitted to the Spinal Cord Unit of Detroit Receiving Hospital with acute spinal cord injury, completed the protocol. Of these, 8 were females; the age ranged from 18 to 73 years. Fifteen patients had paraplegia, 18 quadriplegia and 4 had spinal cord fracture with only temporary paralysis. All patients gave their consent which was approved by the Human Investigation Committee of Wayne State University and entered the study within 48 hours after spinal cord injuries. All had normal clotting screening tests and had similar routine physiotherapy during the course of the study. The following patients were excluded: 1) patients with trauma to the legs and left thorax (this could make the RFUT unreliable), 2) patients with recent myocardial infarctions or coronary artery insufficiency, 3) patients requiring plasma expanders of the dextran type, 4) patients who were on anticoagulants during the last ten days preceding the acute SCI, 5) patients with postphlebitic syhdrome, 6) patients in various forms of shock, 7) patients with known hypersensitivity to iodine or other contrast media used for venography, 8) patients with severe hepatic or renal diseases, 9) patients with known bleeding problems, and 10) pregnant patients. RFUT was initiated within the first three days after injury. At least 12 hours before and daily thereafter, the patients received 100 m potassium iodide to block the thyroid glands. About 100 uCi P fibrinogen (Ibrin, Amersham, Arlington Heights, IL) was of '*I injected intravenously. was The uptake of radiofibrinogen monitored over six distinct points of the thigh and six po'ints on the posterior popliteal fossa and calf of each leg and expressed as ratio of leg to precordial activity. An increase of 15 to 50% between any two points which persisted for 48 hours was considered as positive. All patients were scanned daily at the bedside for 30 days or when the test became positive. In case of a positive scan, we attempted to obtain a venography to confirm the diaignosis. Unfortunately this could not be accomplished in all patients so that we based our correlations on RFUT data. Before the initiation of the RFUT and every second day thereafter, blood samples were drawn for hemostasis studies, After clean venipuncture the first two ml of blood were discarded. Of the following 9 ml drawn, 4.5 ml were immediately transferred into a prechilled plastic tube containing 0.5 ml of a special anticoagulant (Diatube, American Bioproducts Company, Parsipanny, NJ) which is designed to prevent in vitro platelet activation and This sample was used for the assays of FpA, RTG and Pf4. release. After centrifugation at 2,500 x g for 30 minutes at 4'C, the top one-third of the plasma of this tube was recovered, placed in plastic tubes, immediately frozen and kept at -70°C for later batch The second 4.5 ml blood were transferred into a plastic analysis.
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IN SPINAL CORD INJURY
This blood tube containing 0.5 ml 3.8% sodium citrate solution. was used for platelet counts and mean platelet volumes; the plasma, for the above, described centrifugation, as obtained by determination of the other parameters. The following assays were performed by commercial RIA kits: ATG (Amersham, Arlington Heights, IL), Pf4 (Abbott Laboratories, The RIA assays were performed on an Abbott North Chicago, IL). ANSR Automated Gamma Counter. The following were determined and D-di (American Bioproducts), Marburg, Germany).
by commercial ELISA kits: FPA TAT complexes (Behringwerke,
Platelet counts (PLTC) and mean platelet measured on a Baker 810 Platelet Counter.
volumes
(MPV) were
Prothrombin Times (PT) and Activated Partial Fibrinogen, Thromboplastin Times (aPTT) were determined on an Automated Laboratory, (Instrumentation Coagulation Laboratory (ACL) Lexington, MA). von Willebrand factor antigen (vWFAg) was technique using Laurel1 immunoelectrophoresis Calbiochem-Behring (La Jolla, CA). The Student's
T-test was used for statistical
assayed by the antisera from analysis.
RESULTS Incidence
of DVT
Twenty-one of the 37 patients (57%) had a positive RFUT. One of these had a pulmonary embolism. Positive RFUTs were identified between the third and the 20th day post injury. Nine patients had a positive RFUT in the first week, 5 in the second week, 7 in the third week, but none after the 21st day. When the patients were divided into five groups according to their level of spinal cord injuries, 39% of the complete quadriplegic group, 25% of the incomplete quadriplegic group, 88% of the complete paraplegic group, 89% of the incomplete paraplegic group and none of the temporary paresis group had DVT (Table 1). The difference between the incidence of DVT in the paraplegia groups and the quadriplegia groups was highly significant (p < 0.005). There was no significant difference between the complete and the incomplete paresis groups. There also was no significant age difference between the 21 RFUT positive and the 16 RFUT negative patient (Table 1).
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TABLE 1 Clinical Summary of 37 Patients with SC1 DVT (+)
DVT (-1
Quadriplegia (complete)
(3i%)
(671%)
Quadriplegia (incomplete)
(251%)
(7i%)
Paraplegia (complete)
(8:%)
(IS%)
Paraplegia (incomplete)
(8:%)
(111%)
(E%)
(1040%)
Total 12 4 8 9
Temporary paresis
4
Total
37 (G%)
Age
41.2 (k18.8)
(4136%) 36.2 (219.3)
39.0 (218.9)
SC1 = spinal cord injury; DVT = deep venous thrombosis Changes
in Platelet
Counts and Mean Platelet Volumes
The average platelet counts of the two groups gradually increased after injury and reached maximum levels between 15 and 21 days after injury. There were no significant differences at any point between the two groups (Table 2). The mean platelet volumes gradually decreased following injury, and again no significant differences were found at any time. There was also no correlation between these two platelet parameters and the cases with a positive RFUT. Chanaes
in Fibrinogen
and vWFAq
The initial fibrinogen levels of the positive and negative DVT groups were 499 + 170 mg/dl and 453 f. 182 mg/dl, respectively. The levels gradually increased in both groups and reached maximum values on the 8th day post injury (Table 2). They plateaued during the second week. There were no significant differences between the two groups, however, and no correlation could be identified to onset of positive RFUT.
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TABLE 2 Highest Values of Hemostasis Normal PLTC (x109/L) MPV (pm3) Fibrinogen (mg/dl) vWFAg (%) Pf4 (ng/ml) BTG (ng/ml) FPA
(w/W
D-dimer (ng/ml) TAT (ng/ml)
200 6.8 200 60 0 0 0 0 0
- 350 - 7.8 - 350 - 200 - 10 - 50 -3 - 500 -5
DVT = deep venous thrombosis;
Parameters
in 37 Patients
DVT (+) fN=21)
DVT (-) fN=16)
3982147 7.720.8 9332248 605+478 13.726.9 57.4221.5 23.3k13.1 6361+3090 16.428.7
3602164 8.020.8 807+240 6272443 12.1k6.0 48.4k16.7 15.8k12.8 327823321 7.356.2
t-test NS NS NS NS NS NS
p
NS = not significant
increased in both groups. vWFAg levels also continuously Values peaked on the 14th day after injury in the RFUT negative group and increased until the 8th day in the DVT positive group. No significant differences After that they gradually decreased. were observed between the two groups (Table 2) and no correlation was found to the positive RFUT. Chanaes
in BTG. Pf4 and FPA
Pf4 and BTG levels were slightly above normal limits for most patients, with no significant differences between the groups (Table 2). In the negative RFUT group, mean FpA levels stayed under or around 10 ng/ml for the entire length of the study. In contrast, the mean FpA levels of the positive group increased gradually until the 8th day after injury, and gradually decreased thereafter (Figure 1). Changes
in TAT Comolexes
and D-dimer
Mean TAT complex levels for the positive RFUT group were always higher than those of the negative RFUT group. Already during the first week there were significant differences between the two groups (Figure 2). D-Dimer levels of both groups were elevated over the normal limit, but mean levels of the positive RFUT group were always significantly greater than those of the negative RFUT group (Figure 3).
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FPA
q/ml
. ..*
ngiml
26 -
TAT
20 24
-
22
-
O-0 B-4
20
-
16
-
16
-
14
-
12
-
DvT(+)
16
~
DVT(-)
16
-
a
-
6
-
4
-
2
-
1
O-2
3-5
6-8 DAYS
9-11
12-14
AFTER
22-30
3-5
6-8 DAYS
9.11 AFTER
I
12-14
15-21
22~30
INJURY
Figure 2: Mean thromkpin/ antithrombin III (TAT) complex levels in SC1 patients with (n=21) and without (n=16) positive RFUT. = p < 0.02,
***
=
p < 0.01, **** = p < 0.001.
D-DIMER T-
--
L
o-2
INJURY
..
DVT(ti
_W-~DVT(-I
‘-I T
**
8000 t
i
1
15-21
Figure 1: Mean fibrinopeptide A (FpA) levels in SC1 patients with (n=21) and withaut (n=16) positive RFUT. ** = p < 0.02, *** = p < 0.01.
ng/ml
.-d
.. .
..
I
10
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.-.DVT(+)
A
H
DVT(-)
7000 -
6000 -
5000
-
4000 -
3000 -
2000
-
1000 ,___‘_--_,____.I.--__________------. I
I o-2
3-5
6-8 DAYS
9-l AFTER
1
12-14 INJURY
15-21
22-30
Figure 3: Mean D-dimer levels in SC1 patients with N(n=21) and without (n=16) positive *p < 0.05, ** = p RFUT. 0.02, *** p < 0.01.
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When highest TAT complex levels were correlated to the days where the RFUT became first positive, 11 patients had highest levels before the diagnosis was made (8 patients three days before, 3 patients one day before), 5 patients had highest levels on the day of diagnosis and 5 patients had highest levels over the next three days after the positive RFUT test. In contrast, 15 patients had highest D-dimer levels after the RFUT test became positive, 3 patients had highest levels on the day of diagnosis and 3 patients had highest levels before the RFUT test became positive (2 patients one day before, 1 patient three days before). It appears that the majority of SIC patients with thrombosis had TAT complex elevations up to three days before the clinical diagnosis of DVT was made, while the majority had highest D-dimer levels after having established the diagnosis. DISCUSSION Spinal cord injury patients with paralysis suffer a high incidence of DVT and PE post trauma. Of our 37 patients, 57% developed DVT, diagnosed by RFUT. This incidence is slightly lower in the literature using the same test than figures reported nodality (l-3). DVT was more frequently encountered in paraplegic than in quadriplegic patients (p < 0.005). None of the four patients with temporary paresis had DVT. These findings support the concept that immobility is a major factor in the development of 3VT (4). We used RFUT as the diagnostic modality and attempted to confirm DVT by venography. Unfortunately this was not possible in sll patients. In those patients in whom both tests were performed 3VT was confirmed. RFUT is sensitive and specific for thrombosis in the calf and lower thigh, but does not always reveal a process in the upper thigh and the iliac veins (13). We carefully excluded patients in whom false positive RFUTs could have been detected. It LS also recognized that in some patients the test may take 48 hours >r longer to become positive after the acute thrombosis may already 38 present. We attempted to further mimimize errors by having only two experienced health care workers perform the test. Many attempts have been made to employ laboratory procedures for the detection of a thrombotic process. Most of these have failed because they lack sensitivity (14). Many tests become positive as a result of an underlying disease, such as trauma. Our patients with acute SC1 did not have massive trauma thus minimizing potential erroneous test results. On the other hand, they had a Jery high incidence of DVT. Elevations in the factor VIII macronolecular complex components (factor VIII:C, von Willebrand factor) lave been described in SC1 patients (2,6) and were found to be of >redictive value. Increases in fibrinogen have also been reported in conjunction with developing thrombosis. We did not find a
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difference in von Willebrand factor levels in our RFUT positive and negative patients; fibrinogen levels were higher in the positive group (Table 2) but the difference was not significant. It will be noted that both factor levels were markedly elevated in both groups. Both proteins are acute phase reactants. Less information is in the literature concerning the use of newer molecular markers of hemostatic activation. These tests measure either released substances from platelet act~ivation [platelet factor 4 (Pf4) and beta thromboglobulin (DTG)] or released peptides or fragments from enzymatic actions [f:ibrinopeptide A (FpA) and D-dimer (D-di)] or activation complexes between enzymes and inhibitors [thrombin/antithrombin III (TAT) compllexes]. These tests are RIA or ELISA based, are sensitive and allow quantitation of very small amounts in plasma. They are not only sensitive for thrombosis, but rather reflect any state of in vivo activation of the hemostasis system. All of these markers are elevated in surgical or trauma patients and in the immediatie postpartum period. After major surgeries they remain elevated for about 48 to 72 hours (15). Even placement of a Swan-Ganz uatheter can elevate some of these markers (unpublished observation). Our patients did not have major trauma and none were subjected to a surgical procedure during the period of observation. These markers can also become elevated in vitro by poor blood drawing technique or improper sample handling. We minimizled this a special tube which cdntained blood collection by using and by anticoagulants plus inhibitors of platelet activation, having all samples with considerable drawn by one person experience. Our data demonstrated significant differences in the levels of FpA, TAT complexes and D-di between the patients with or without a positive RFUT. The levels of Pf4 and BTG were also higher in the positive patients, but this did not reach statistical significance (Table 2). FpA levels were significantly elevated during the first 8 days after injury (Figure l), TAT complex levels and Dddi were higher during the entire study period in the RFUT positive patients (Figures 2 and 3). FpA and TAT complexes are markers of dhrombin generation and elevated plasma levels would suggest increased thrombin generation. D-di are fibrin degradation products and Since both'patient suggest increased in vivo plasmin generation. groups were otherwise similar, our findings suggest that the RFUT positive patients had demonstrable activation of their clotting and fibrinolytic systems. Platelet release proteins (Pf4 and DTG) and also FpA were found to lack sensitivity and specificity in the diagnosi$ of DVT In contrast, both TAT complexes and D-di demonstrated (16,17). D-di had a sensitivity between 90-lOO%, while great sensitivity. TAT complexes had a shorter half-life, due to a markedly sensitivity < 90% (17-20). D-di lacks specificity and is therefore Its test for DVT a good screening not necessarily (20). sensitivity was found to be 98% but its specificity only 38% in
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patients with documented PE (21). Yet, both D-di and TAT complex determinations seem to be of some predictive value for ruling out DVT in the postoperative period (22,23), and normal levels of both seem to make DVT unlikely (24). Our observations that highest TAT complex levels were found in 11 of 21 RFUT positive patients before the RFUT became positive and highest D-di levels in 15 of these patients after the positive RFUT suggests a possible predictive value for TAT complexes. It can not be ruled out, however, that some of these TAT complex positive patients might have had a delayed RFUT positivity, as indicated above. ACKNOWLEDGEMENT This work was supported by a grant from the National Institute and on Disability Rehabilitation Research 35196), (GO085 Washington, D.C. REFERENCES 1.
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