Characterization of Elevated Fibrin Split Products Following Thermal Injury P. WILLIAM CURRERI, M.D.,* MARY E. WILTERDINK, B.S., M.T., (ASCP), CHARLES R. BAXTER, M.D.
These studies establish that the staphylococcal clumping test is superior to the tanned red cell hemagglutination inhibition immunoassav for monitoring fibrin split product concentration in burn sera. It is strongly suggested that the principal circulating degradation product is a complex of soluble fibrin monomer with fragment D. Finally, there does not appear to be any effect on measured fibrin split product concentration in sera of burn patients receiving prophylactic heparin or aspirin.
E LEVATED) LEVELS of fibrin split produicts (FSP) have been reported to occur frequently in both thermally injuired patients and experimental burns. Both the staphylococcal clumping test (SCT) and the tanned red cell hemagglutination inhibition immunoassay (TRCHII) or Merskey methlod have been advocated for screening burn patients with suspected intravascular coagulation. The Merskey method has been utilized to detect the presence of X, Y, and D fragments in defibrinated plasma. It is only slightly reactive with fragment E. The staphylococcal cluimping test is positive in the presence of X and Y fragments, as well as soluble fibrin monomer or polymer complexes with fragments Y and D. In this study these two assays were uised to analyze the plasma fibrin split product concentration in burned patients during the first two postbuirn weeks.
From the Department of Surgery, The University of Texas Southwestern Medical School, Dallas, Texas
containing 3.8% sodiuim citrate (1:10 volume) and Trasylol (25 Christensen uinits of antiplasmin/ml). Samples were centriftuged, the plasma treated with thrombin, and the seruim immediately frozen for futuire testing. The fibrin split produicts level of each serum sample were determined according to the method of Merskey et al.,4 which depends on the neuitralization of antihuman fibrinogen antiserum by fibrinogen or fibrin degradation products. Neutralized antiseruim blocks the agglutination of tanned sheep red blood cells coated with huiman fibrinogen. Thus, the titer of fibrin split produicts is the highest dilution of test serum which clearly inhibits red cell aggluitination by the antiserum. The uise of known standards allows quantitation of fibrin split produict concentration and normal values range from 2 to 5
Ag/ml.
The blood samples for the staphylococcal clumping test were drawn into tubes containing soybean trypsin inhibitor (0.5 mg antiplasmin/ml blood) and thrombin (100 units/ml blood). The tuibes were mixed well and incubated at 37 C uintil clot retraction was evident, approximately two houirs. Seruim was then obtained by centrifugation and samples Methods frozen for fututre testing. The test was performed -on Blood samples for the tanned red cell hemagglutination microtiter plates as described by Leavelle and associates.2 inhibition immuinoassay were collected in vacutainer tubes The presence of a cluimping factor (bouind coagulase) on the cell walls of certain strains of Staphylococcus aureuis produices visible cluimping of the bacteria in the presence of Suibmitted for ptiblication Jtune 11, 1974. Stupported by the United States Ptublic Health Service Grants NIH 1- fibrinogen, some early high molecular weight breakdown RO1-GM19571 and NIH 5-PO1-GM14892 from the National Institute of produicts, and soluble fibrin monomer complexes. SiGeneral Medical Sciences. multaneouis determinations of known standards prepared * Recipient of Research Career Development Award from the Nafrom normal plasma allow the quantitation of the fibrin split tional Instittutes of Health. concentration. The mean valuie obtained from an produict Reprint reqtuests: P. William Ctirreri, M.D., Department of Surgery, control population was 2.7 ± 2.lug/ml. uninjtured The University of Washington School of Medicine, Seattle, Washington 98195. A prospective patient study was subsequiently instituted
157
158
CURRERI, WILTERDINK AND BAXTER
to test the effectiveness of prophylactic systemic anticoagtilation in preventing abnormal elevations of fibrin split produict concentration in the immediate post burn period. Thirty-one patients with greater than 30% total body suirface buirns, who had no gross contraindications for systemic anticoaguilation, were randomly assigned to one of three patient grouips. One grouip was administered Heparin, E 5,000 inits (suibcuitaneously) every six hours during the first two post-burn weeks. A second group received aspirin, 10 grains per rectuim, during the same time period. A control 0grouip of patients received neither heparin nor aspirin. acn Blood samples for the staphylococcal clumping test were drawn at least once every three days and the results grouped cn arbitrarily into three day periods.
Results Seruim from 55 bturned patients was simultaneouisly assayed serially during the first two post-burn weeks for fibrin split produicts by both the tanned red cell hemaggluitination inhibition immunoassay and the staphylococcal clumping test. Values obtained by the Merskey method were normal on all occasions in 23% of patients (mean = 2.90,ug/ml), minimally elevated in 49% (mean = 7.20,ug/ml) and significantly increased in 27% (mean = 16.33,ug/ml) (Table 1). Fibrin split product concentration measured by the staphylococcal clumping test were increased above normal in all patients at some period during the first two post-burn weeks. Only 7% had minimal elevation (mean = 6.77 ± 0.07,ug/ml), and 93% had at least fouir times maximuim normal concentration (mean = 41.33 ± 4.7,tg/ml). Fibrin split products observed by the staphylococcal cluimping test were consistently elevated between the fourth and eighth post-burn day, and although the levels of fibrin split produicts were somewhat more variable after this time, the concentration remained greater than accepted normal concentration throughouit the study period (Fig. 1). No significant differences in fibrin split product concentration were noted in the patients who received prophylactic heparin or aspirin when compared to those patients receiving no anticoaguilant therapy (Fig. 2). Serum from all three grouips contained markedly elevated levels of fibrin TABL. 1
Fibrin Split Product Concentration in Sera from Burn Patients as Measured by the TRCHII and SCT Methods Nuimber Per Cent Mean of of Range ± S.E. Total Patients Alg/ml
TRCHII (Normal = 2-5 mg/ml) SCT (Normal = (2.7 ± 2.1,Lg/
ml)
7.2 0-3.4 3.4-6.7
13 27 15 0 4
23 49 27 0 7
2.9 ± 0.38 7.2 ± 0 16.3 + 1.31
>6.7
51
93
41.3 ± 4.74
6.7 ± 0.07
Ann. Stirg. *
Febriiary 1975
36k 32k 28 -
24k 20-
16k 12 -
8k 4
O0
I
0
I
2
F i(;. 1. Fibrin split
I
4
10 8 6 DAYS POST BURN
12
14
produict concentration in sera from buirn patients test during the first two post-
measuired by the staphylococcal cluimping buirn weeks.
split produicts as measuired by the staphylococcal cluimping test. Discussion Interpretation of these results required knowledge of the normal degradation pathway of fibrinogen and fibrin by plasmin. In addition, the specificity of both the tanned red cell hemaggluitination inhibition immunoassay and the staphylococcal cluimping test in detecting each degradation produict muist be appreciated. The scheme of the degradation of fibrinogen and fibrin by plasmin is shown in Fig. 3. The initial digestion results in the conversion of fibrinogen (MW 340,000) to Fragment X (MW 270,000) pltus small polypeptide fragments. X tindergoes fuirther proteolysis and is degraded to Fragment Y (MW 165,000) and Fragment D (MW 85,000). Y is split fuirther, yielding Fragments D and E (MW 55,000) which are immtune to fuirther digestion by plasmin. The net resuilt of the degradation of one mole of fibrinogen by plasmin is 2 moles of D and an E fragment. During this proteolysis, fragments X, Y, and D also may complex with fibrin monomers, resuilting in non-clottable soluible fibrin monomer complexes (SFMC). Trasylol and soybean trypsin inhibitor are added to the
159
ELEVATED FIBRIN SPLIT PRODUCTS
Vol 181 * No 2
24 E Fic;. 2 Comparison of fibrin split produict concentration in sera from buirn patients receiving variouis prophylactic anticoagtilant regimens.
0
Tp
NO ANTI-COAGULANTS HEPARI N - 5000 units q 6hrs. CM ASA -X6 gr. q 6 hs.
281
I
-
20 -
16
_
cn 0~
la
cn
12 _
,.,
.1
8_
41_ e
These studies establish that the staphylococcal clumping test is superior to the tanned red cell hemagglutination inhibition immunoassav for monitoring fibrin split product concentration in burn sera. It is strongly suggested that the principal circulating degradation product is a complex of soluble fibrin monomer with fragment D. Finally, there does not appear to be any effect on measured fibrin split product concentration in sera of burn patients receiving prophylactic heparin or aspirin.
E LEVATED) LEVELS of fibrin split produicts (FSP) have been reported to occur frequently in both thermally injuired patients and experimental burns. Both the staphylococcal clumping test (SCT) and the tanned red cell hemagglutination inhibition immunoassay (TRCHII) or Merskey methlod have been advocated for screening burn patients with suspected intravascular coagulation. The Merskey method has been utilized to detect the presence of X, Y, and D fragments in defibrinated plasma. It is only slightly reactive with fragment E. The staphylococcal cluimping test is positive in the presence of X and Y fragments, as well as soluble fibrin monomer or polymer complexes with fragments Y and D. In this study these two assays were uised to analyze the plasma fibrin split product concentration in burned patients during the first two postbuirn weeks.
From the Department of Surgery, The University of Texas Southwestern Medical School, Dallas, Texas
containing 3.8% sodiuim citrate (1:10 volume) and Trasylol (25 Christensen uinits of antiplasmin/ml). Samples were centriftuged, the plasma treated with thrombin, and the seruim immediately frozen for futuire testing. The fibrin split produicts level of each serum sample were determined according to the method of Merskey et al.,4 which depends on the neuitralization of antihuman fibrinogen antiserum by fibrinogen or fibrin degradation products. Neutralized antiseruim blocks the agglutination of tanned sheep red blood cells coated with huiman fibrinogen. Thus, the titer of fibrin split produicts is the highest dilution of test serum which clearly inhibits red cell aggluitination by the antiserum. The uise of known standards allows quantitation of fibrin split produict concentration and normal values range from 2 to 5
Ag/ml.
The blood samples for the staphylococcal clumping test were drawn into tubes containing soybean trypsin inhibitor (0.5 mg antiplasmin/ml blood) and thrombin (100 units/ml blood). The tuibes were mixed well and incubated at 37 C uintil clot retraction was evident, approximately two houirs. Seruim was then obtained by centrifugation and samples Methods frozen for fututre testing. The test was performed -on Blood samples for the tanned red cell hemagglutination microtiter plates as described by Leavelle and associates.2 inhibition immuinoassay were collected in vacutainer tubes The presence of a cluimping factor (bouind coagulase) on the cell walls of certain strains of Staphylococcus aureuis produices visible cluimping of the bacteria in the presence of Suibmitted for ptiblication Jtune 11, 1974. Stupported by the United States Ptublic Health Service Grants NIH 1- fibrinogen, some early high molecular weight breakdown RO1-GM19571 and NIH 5-PO1-GM14892 from the National Institute of produicts, and soluble fibrin monomer complexes. SiGeneral Medical Sciences. multaneouis determinations of known standards prepared * Recipient of Research Career Development Award from the Nafrom normal plasma allow the quantitation of the fibrin split tional Instittutes of Health. concentration. The mean valuie obtained from an produict Reprint reqtuests: P. William Ctirreri, M.D., Department of Surgery, control population was 2.7 ± 2.lug/ml. uninjtured The University of Washington School of Medicine, Seattle, Washington 98195. A prospective patient study was subsequiently instituted
157
158
CURRERI, WILTERDINK AND BAXTER
to test the effectiveness of prophylactic systemic anticoagtilation in preventing abnormal elevations of fibrin split produict concentration in the immediate post burn period. Thirty-one patients with greater than 30% total body suirface buirns, who had no gross contraindications for systemic anticoaguilation, were randomly assigned to one of three patient grouips. One grouip was administered Heparin, E 5,000 inits (suibcuitaneously) every six hours during the first two post-burn weeks. A second group received aspirin, 10 grains per rectuim, during the same time period. A control 0grouip of patients received neither heparin nor aspirin. acn Blood samples for the staphylococcal clumping test were drawn at least once every three days and the results grouped cn arbitrarily into three day periods.
Results Seruim from 55 bturned patients was simultaneouisly assayed serially during the first two post-burn weeks for fibrin split produicts by both the tanned red cell hemaggluitination inhibition immunoassay and the staphylococcal clumping test. Values obtained by the Merskey method were normal on all occasions in 23% of patients (mean = 2.90,ug/ml), minimally elevated in 49% (mean = 7.20,ug/ml) and significantly increased in 27% (mean = 16.33,ug/ml) (Table 1). Fibrin split product concentration measured by the staphylococcal clumping test were increased above normal in all patients at some period during the first two post-burn weeks. Only 7% had minimal elevation (mean = 6.77 ± 0.07,ug/ml), and 93% had at least fouir times maximuim normal concentration (mean = 41.33 ± 4.7,tg/ml). Fibrin split products observed by the staphylococcal cluimping test were consistently elevated between the fourth and eighth post-burn day, and although the levels of fibrin split produicts were somewhat more variable after this time, the concentration remained greater than accepted normal concentration throughouit the study period (Fig. 1). No significant differences in fibrin split product concentration were noted in the patients who received prophylactic heparin or aspirin when compared to those patients receiving no anticoaguilant therapy (Fig. 2). Serum from all three grouips contained markedly elevated levels of fibrin TABL. 1
Fibrin Split Product Concentration in Sera from Burn Patients as Measured by the TRCHII and SCT Methods Nuimber Per Cent Mean of of Range ± S.E. Total Patients Alg/ml
TRCHII (Normal = 2-5 mg/ml) SCT (Normal = (2.7 ± 2.1,Lg/
ml)
7.2 0-3.4 3.4-6.7
13 27 15 0 4
23 49 27 0 7
2.9 ± 0.38 7.2 ± 0 16.3 + 1.31
>6.7
51
93
41.3 ± 4.74
6.7 ± 0.07
Ann. Stirg. *
Febriiary 1975
36k 32k 28 -
24k 20-
16k 12 -
8k 4
O0
I
0
I
2
F i(;. 1. Fibrin split
I
4
10 8 6 DAYS POST BURN
12
14
produict concentration in sera from buirn patients test during the first two post-
measuired by the staphylococcal cluimping buirn weeks.
split produicts as measuired by the staphylococcal cluimping test. Discussion Interpretation of these results required knowledge of the normal degradation pathway of fibrinogen and fibrin by plasmin. In addition, the specificity of both the tanned red cell hemaggluitination inhibition immunoassay and the staphylococcal cluimping test in detecting each degradation produict muist be appreciated. The scheme of the degradation of fibrinogen and fibrin by plasmin is shown in Fig. 3. The initial digestion results in the conversion of fibrinogen (MW 340,000) to Fragment X (MW 270,000) pltus small polypeptide fragments. X tindergoes fuirther proteolysis and is degraded to Fragment Y (MW 165,000) and Fragment D (MW 85,000). Y is split fuirther, yielding Fragments D and E (MW 55,000) which are immtune to fuirther digestion by plasmin. The net resuilt of the degradation of one mole of fibrinogen by plasmin is 2 moles of D and an E fragment. During this proteolysis, fragments X, Y, and D also may complex with fibrin monomers, resuilting in non-clottable soluible fibrin monomer complexes (SFMC). Trasylol and soybean trypsin inhibitor are added to the
159
ELEVATED FIBRIN SPLIT PRODUCTS
Vol 181 * No 2
24 E Fic;. 2 Comparison of fibrin split produict concentration in sera from buirn patients receiving variouis prophylactic anticoagtilant regimens.
0
Tp
NO ANTI-COAGULANTS HEPARI N - 5000 units q 6hrs. CM ASA -X6 gr. q 6 hs.
281
I
-
20 -
16
_
cn 0~
la
cn
12 _
,.,
.1
8_
41_ e
