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Hematologic changes following burns
JOURNAL
OF SURGICAL
22,626-635
RESEARCH
Hematologic
(1977)
Changes
following
Burns1
JOSEPH A. CAPRINI, M.D., FACS,2 VIRGINIA LIPP, B.S., LEON ZUCKERMAN, PH.C., JOSEPH P. VAGHER, B.S., AND DAVID P. WINCHESTER, M.D., FACS Department of Surgery, Evanston Hospitul. und Northwestern Unilrersity Medical School, Evanston, Illinois 60201
Submittedfor publication November 9, 1976
Despite numerous investigations, the significance of hematologic changes following burns has not definitely been established. Debate continues regarding numerous hematologic issues including the intravascular or extracellular origin of fibrin degradation products (FDP), and the value of heparin therapy in these patients. The purpose of this study was to perform a wide variety of hemostatic and immunologic tests and relate the laboratory results to the clinical probability of survival. The stimulus for these investigations evolves from the recent evidence suggesting that coagulation and immunologic systems are activated through a common link, the Hageman factor (FXII), which is normally activated following intravascular contamination. If this contamination occurs following thermal injury, the extent of changes should be reflected in all of these blood systems. The results obtained indeed show significant correlation between the tests performed and the initial prognostic index and strongly support the concept of intravascular contamination. MATERIALS
AND METHODS
Patients entered into this study following their admission to Evanston Hospital’s Thermal Trauma Unit. Forty-five patients, with varying degrees of burn, were divided into three groups based on their prognostic ’ Supported in part by Dee and Moody Funds, Evanston Hospital, and the Upjohn Company. 2 Schweppe Foundation Investigator.
index (age vs percentage of total burn). The percentage range of each group was determined in an attempt to equalize the group numbers. Group I (12 patients) had a prognostic index survival rate of under 20%, Group II (11 patients) had a survival rate of 20 to 80%, and Group III (22 patients) had a survival rate of greater than 80% (Table 1). A blood sample was drawn from each patient within 48 hr postburn. Additional samples were drawn within the following time periods: 3-8, 9-16, and 17-25 days postburn. All blood samples were collected using a modified “two syringe technique.” Using a 19-gauge butterfly apparatus, the vein was entered cleanly with a minimum amount of trauma. The butterfly system was “cleaned’ ’ of tissue thromboplastin contamination by discarding the hrst 3 to 5 ml of blood. The plastic syringe was then connected and 20 ml of blood was gently withdrawn. Timing is started when the blood enters the sample syringe. The blood is gently expelled down the inside wall of the specific polypropylene test tubes. The test procedures performed required whole blood, plasma, and serum. Therefore, it was necessary to use specific test tubes for the various tests. The Clay-Adams microhematocrit [37] and the direct phase platelet count technique of Brecher and Cronkite [6] required an EDTA anticoagulant sample. One drop of EDTA was placed in a 10 x 75mm polypropylene test tube, and 1 ml of the blood
626 Copyright All rights
0 1977 by Academic Press, Inc. of reproductmn in any form reserved
ISSN 0022.4804
CAPRINI ET AL. : HEMATOLOGIC TABLE 1 PROGNOSTIC
INDEX
AND SURVIVAL
OF GROUPS
Group
Number of patients
Admission prognosis index survival rate (age vs percentage of bum)
I II III
12 11 22
<20% 20-80% >80%
Eventually lived/died 418 714 22/o
sample was added as described. The test tube was capped with parafilm and inverted gently five or six times to mix the blood and anticoagulant. The hematocrit and platelet counts were completed within 30 min of sample time. Within 4 min of sampling, two aliquots of whole blood (without anticoagulant and collected in a 10 x 75mm polypropylene test tube) were placed into the thrombelastograph (TEG) (Haemoscope Corporation, Albertson, New York). A solution of 1% (w/v) celite (Johns-Manville Co., New York) was prepared in physiologic saline at pH 7.0 and, by selective decantation of fines, a specific size distribution is selected. The thrombelastographic procedure involved placing 0.33 ml of whole blood in one cup with 0.03 ml of 1% celite solution, mixing by raising and lowering the TEG pin three times, and subsequently opening the TEG camera shutter. A second cup receives 0.36 ml of native whole blood, mixed in a similar fashion, and run at the same time. Additional details regarding the TEG procedure have previously been described [7,8, 13, 19, 381. The sample for fibrin split products [20, 341 by staph clumping (Sigma Chemical Co., St. Louis, Missouri), was placed in the thrombin vial provided with the kit. Whole blood is collected and added to excess thrombin to assure complete conversion of fibrinogen to fibrin and its subsequent removal in the clot. E-Aminocaproic acid (EACA) is added to the
CHANGES FOLLOWING
BURNS
627
thrombin prior to the whole blood addition to inhibit conversion of plasminogen to plasmin (fibrinolysis). The blood is then incubated at 37°C for 2 hr to assure complete coagulation, clot retraction, and inactivation of excess thrombin. Serum recovered from the clot is diluted with Trizma-saline buffer and mixed with staphylococcal cell suspensions. An estimate of fibrinogen/fibrin degradation products (FDP) present is obtained by comparing the degree of cell clumping produced by the diluted test serum with clumping observed using known amounts of fibrinogen standard. The anticoagulant which best preserves the labile coagulation factors V and VIII is accomplished with 0.3 ml of 0.1 M sodium citrate and 0.2 ml of 0.1 M citric acid solution pH 4.8-5.0. A 9:l ratio of blood to anticoagulant is kept constant. One milliliter of anticoagulant is added to a 16 x lOO-ml polypropylene test tube and 9 ml of whole blood is gently expelled down the inside wall of the test tube. The test tube was capped with Parafilm and inverted gently five or six times to mix the blood and anticoagulant. The anticoagulated whole blood sample was centrifuged at 4000 rpm at 4°C for 10 min, within 10 min of blood withdrawal. With a plastic pipet, the plasma was carefully removed from the sample, without disturbing the red cells. The plasma was placed in two 10 x 75mm polypropylene test tubes. One tube was stored at 4°C until the one stage prothrombin time [27, 281and activated partial thromboplastin time were completed [22] (coagulation profiler, model CP-8, Bio/Data Corp., Willow Creek, Pennsylvania). The remaining plasma was frozen at -70°C to be used for the following tests. C3, C4, fibrinogen, and plasminogen [4, 5, 17, 23, 361 were determined through immunodiffusion plates supplied by Behring Diagnostics (Sommerville, New Jersey). The patient’s plasma was thawed and placed in a well in each of the four specified plates. The protein antigens present in the plasma diffuse into the thin gel
628
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
matrix causing a precipitin ring to form. After 48 hr, the diffusion is complete and the diameter of the ring is measured. A standard reference curve, constructed from the three standards supplied by Behring, is drawn by plotting the squared diameters of the precipitin rings against their respective concentrations. The values of the patient’s samples are determined by referring their squared diameter to the cahbration curve. The pH stat assay as used by Snyder et al. [35] was used by our laboratory for the determination of spontaneous esterase activity (KO), prekallikrein (kaolin activatable) esterase (Kl’), and kallikrein (Cl-esterase) inhibitor activity (KS). The spontaneous esterase activity is determined by placing 0.2 ml of thawed plasma with 2 ml of TAMe in a vessel on the pH stat. The reaction is run for 10 min using 0.04 M NaOH as the titrant at 37°C with constant stirring. The slope for the last 2.5 min is calculated and multiplied by a factor to convert the answer to micromoles per hour per milliliter. The prekallikrein determination differs only by activating the plasma for 1 min with 3% kaolin prior to TAMe addition, The kallikrein inhibitor activity differs by activating with kaolin for 5 min prior to TAMe addition. The results in this paper are not converted into units of inhibitor activity because the levels of prekallikrein activity were low and, more often than not, the 5-min values were higher than the 1-min values. Therefore, K5’ is merely esterase activity resulting after 5 min of contact activation. Total hemolytic complement (CH,,) measures the ability of the patient’s plasma to lyse 50% of a standard suspension of sheep erythrocytes coated with rabbit antibody. The sheep cells are washed three times in saline and once in gelatin-veronal buffer (GVB). A working solution of GVB is made fresh every 24 hr by dissolving 150 mg of gelatin in a small amount of double-distilled water (DDW). Forty milliliters of stock GVB is added to this and the volume is increased
to 200 ml with DDW. This buffer is the only solution used for the remainder of the assay. A 1:2000 dilution of hemolysin is added to an equal amount of a 5% suspension of the cells. This is incubated in a 37°C water bath for 10 min. The suspension is centrifuged and the supernatant is removed. The cells are washed again and resuspended in a small amount of buffer. These are counted and their concentration is adjusted to 5 x 108cells/ml. They are kept on ice until used. For each thawed test plasma, a 1:51 dilution is made. This is rediluted to make four C’ dilutions of 1: 10, 1:20, 1:34, and 1:51. One milliliter of the prepared sheep cells is added to each of the four test tubes. The suspension is gently inverted three times. A GVB control to check for any spontaneous lysis and a control to give a complete lysis value are also set up. The tubes are incubated for 1 hr in a 37°C waterbath. Following incubation, they are centrifuged and the supernatant is carefully removed so as not to disturb the cell button. The optical density (OD) of the supernatant is read at a wavelength of 541 nm on the spectrophotometer using the GVB control as the cell blank. The percentage lysis is calculated by dividing the OD of the sample by the OD of the complete lysis value. The percentage lysis was plotted against C’ dilution using only those values between 20 and 80%. The CH,, is the reciprocal of the dilution at which the line crosses the 50% lysis point. For each group, the mean and standard error for the described tests in each of the four time periods were calculated. Group comparisons were made on the tests for statistical significance by a t test of the values in corresponding time periods. The statistically significant tests were plotted as bar graphs. RESULTS
Patients were grouped according to their prognostic index for the purpose of analyz-
CAPRINI ET AL. : HEMATOLOGIC
ing the data and, as seen in Table 1, the actual survival statistics are better than those predicted by the index. The calculated average prognostic indexes for the patients in these groups were: GI = 5%, GII = 51%, and GIII = 98%. The patients with the most severe burns (GI) had an average age of 62 years with an average of 69% of their body burned. The second group with moderate burns had a mean age of 50 and an average burn of 43%, while the third group (GIII) was much younger, with a mean age of 28, and an average burn of 21%. As can be seen in Table 2, patients with severe burns (GI) did not show appreciable drops in fibrinogen during the first 48 hr, despite the substantial volume replacement. After the first 48 hr, there was some elevation of their levels above the normal ranges. Although total complement activity by the hemolytic assay appeared to be within the normal range, immunoassay of C3 and C4 (Table 2, Fig. 1) appeared depressed throughout the course of their stay. The native kallikrein or esterase activity (0 min) of their plasma was always low or zero and reflected no circulating trypsin-like activity (Table 2). Activation of the plasma
CHANGES FOLLOWING
BURNS
629
with kaolin produced no detectable activity at 1 min while the 5-min (Fig. 2) incubation period with the contact activator showed some significant results. This is normally reported as the inhibitor levels, but since there is no detectable activity at the peak (Kl’) time, we offer this activity as a point of reference for further investigation. Plasminogen levels are also depressed initially and remain that way and are inversely related to the fibrinogen degradation products (Fig. 3) in this group. Thus, increased FSP is associated with decreased plasminogen (Fig. 4). Platelet counts remained within the normal range (Fig. 5), and the prothrombin time (PT) was prolonged. The partial thromboplastin time (PTT) seemed to correct after the first 48 hr, and then remain normal. The TEG index, which measures the interactions of platelets, RBC, and plasma factors in clot formation [S], showed accelerated coagulability during the study with the most severe acceleration occurring between the 9th and the 16th days. Thus, characteristic features of this population are significant depressions of plasminogen, kallikrein activity, C3, and C4, with elevation of FSP with normal or elevated fibrinogen levels.
TABLE 2 GROUP I: SEVERE BURN DATA~
Time periods (number of patients in group) <48 hr (10) Fibrinogen
470
2 62
c3 c4 C&o
68 26 24
-c8 +4 k4
KO Kl K5
1 21 22
2 1 *9 27
PT (PTJPT,) PTT W’TdP%) TEG (index)
1.29 2 0.07 1.29 + 0.13 5.0 k 1.6
3-8 days (5)
9- 16 days (5)
658
+ 41
535
k 109
61 29 35
k5 24 + 11
70 30 34
zk 6 k 3 -c 3
1 0 5
? 1 k 0 t 3
0 3 23
k f 2
1.25 k 0.06 1.00 2 0.03 8.6 k 2.3
1.21 f 1.09 2 12.3 2
17-25 days (0)b
0 2 8 0.68 0.13 2.5
a Values are means -C standard error of the mean. b Only one patient remained from the original group who was not being treated with heparin at this time.
630
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
The more dynamic nature of GII patients is attributable to marked changes in their blood components (Table 3). In Fig. 1 the rebound of the third component of complement is shown to occur within the tist week. It is also impressive in that this group started with the lowest FSP in the early period, achieved the highest peak between 3 and 8 days, and then slowly dropped (Fig. 3). Plasminogen levels recovered by the third time period and began to rise above normal by the 25th day (Fig. 4). However, kallikrein activity (Kl’) did not significantly recover from the initial fall of the 3- to g-day period. Platelets, PT and PTT, did not change significantly in this group. The overall effect, reflected by the TEG index, suggests a hypercoagulable condition as the result of high fibrinogen and platelets existing between the 9th and the 25th days. Fibrinogen levels among Group III burn victims tend to peak early and fall similar to the GII patients (Table 4). The fibrin split products (Fig. 3) were elevated above the normals and peaked at about the 3rd to the 8th days. Plasminogen (Fig. 4) levels have returned to normal by this period and remain stable. Complement levels show only modest depression during the hrst 48 hr and are well within normal limits for the next 14 days. It should be noted that group size changes from time period to time period. This attrition of subjects is caused by death, treatment with heparin, or release from the unit. The effect of these losses would tend to normalize the data toward the center group, i.e., the values of less severely burned Group III individuals and survivors of more severe burns both migrate toward one another with time. The initial kallikrein esterase activity (Kl’) of Group III is still lower than normal and seems to reflect the severity of the burn, in that Group I Q Group II I Group III. There was no significant free esterase activity (KO) measurable in any of these three groups. Therefore, there must
LWS
POST BURN
FIG. 1. Complement 3 levels for each group in each of the four time periods (mean values with SE).
be sufficient inhibitors to block any activity generated by intravascular contaminants or activation by plasmin. As previously mentioned with Group II, the TEG index reflects a possible hypercoagulable state by the ninth day which may be the result of elevated platelets (Fig. 5) and fibrinogen in the presence of intravascular burn contaminants. Some slight shortening of the
50
0
<48 t!Rs.
3-0 ON9
9-l0[yIr
DAY9 POST BURN
FIG. 2. Kinin 5’ activity for each group in each of the four time periods (mean values with SE).
CAPRINI ET AL. : HEMATOLOGIC
CHANGES FOLLOWING
BURNS
631
group during the next 6 days. This also corresponds to the period of maximum plasminogen disappearance and the lowest esterase (KS’) and C3 levels of that same group of severe burns. Clinically, this period relates to a time when most of the edema of these patients has decreased. DISCUSSION
FIG.
A variety of hemostatic alterations have been observed following major thermal injury including thrombocytopenia, secondary thrombocytosis, increased platelet adhesiveness, decreased platelet survival time, elevated fibrinogen, factor V and VIII levels, and elevated fibrin split products [3, 10,16,21,25]. Depressed serum immunoglobulins and various complement components have been observed [l, 21. It has been suggested that the burn patient is more susceptible to infection [18], pulmonary embolism [9], and disseminated intravascular coagulation [24]. Heparin has 3. Fibrin split product levels for each group in been tried in experimental and clinical burns
each of the four time periods (mean values with SE).
PTT is evident by the second through the fourth periods, but there was too much variance during the 9th through the 16th day to correlate the results with the TEG index. Inhibition of coagulation as the result of elevation of FSP is not reflected by any of the coagulation tests (TEG, PT, PIT). The significance of this may be in the type of fragments which are peculiar to this type of trauma or may be that the degree of intravascular contamination and subsequent activation overrides the competitive inhibition of these FSP. This result is in marked contrast to that seen during the late phase of disseminated intravascular coagulation (DIC) where FSP are elevated, platelets are down, and all clotting tests are prolonged. It is also noteworthy that platelet levels (Fig. 5) are not excessively low within FIG. 4. Plasminogen levels for each group in each the first 48 hr, but fall in the high-risk of the four time periods (mean values with SE).
632
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
to reverse some of the clotting changes, lessen tissue loss by inhibiting vascular thrombosis, shorten healing time, prevent burn extension, decrease thromboembolic complications, and reduce pain. Unfortunately, uncontrolled studies and conflicting data make the role of heparin in burns unclear [12, 14, 15, 26, 30, 31, 32, 331. Failure to alter fibrin split product concentrations with heparin in animals prior to and following burn injury, and in randomly assigned burn patients suggests that these degradation products may be the result of extravascular plasmin digestion of fibrinogen rather than due to intravascular coagulation [ll]. These facts cast further doubt on the anticoagulant value of systemic heparin therapy in burns. The extravascular theory may be questioned, however, if fibrinolytic activity persists beyond the edema phase of the burn wound. Recently, evidence has appeared linking the blood clotting process with immunologic defenses [29]. Following intravascular contamination, it has been shown that coagulation, fibrinolytic, complement, and kinin pathways may be activated through the Hageman factor (FXII) by contact activation. If one assumes that intravascular contamination occurs in burn victims
j8 300
200
loo
0
<49
HRS
3-9 MY9 MY9Posr9lRN
9-16 OAYS
v-25
DAY!3
FIG. 5. Platelet counts for each group in each of the four time periods (mean values with SE).
early due to tissue damage, and later, possibly from sepsis, then changes in all of these systems should occur and be proportional to the extent of injury. The failure of heparin to modify FDP levels, or significantly modify the clinical burn course would not then be surprising since this anticoagulant in the usual pharmacologic dosages does not significantly inhibit Hage-
TABLE 3 GROUPII: MODERATE BURN DATA” Time periods (number of patients in group) <48 hr (10) Fibrinogen
3-8 days (4)
9- 16 days (5)
17-2.5 days (5)
387
f 56
676
f 124
573
f 79
520
2 35
c3 c4 CHsO
73 32 30
f 10 57 26
113 38 43
2 11 -c 4 2 4
103 32 45
29 +8 25
106 41 46
f 12 +7 27
KO Kl K.5
2 32 50
2 1 e 14 k5
2 2 35
0 0 34
k 0 + 0 +8
PT W’PL) PI-l- m-TIP=,) TEG (index)
1.10 2 0.05 1.03 k 0.07 0.6 k 2.0
2 f f
1.12 2 0.89 f 8.6 +
(2Values are means k standard error of the mean.
2 2 19 0.06 0.07 3.0
0
k 0
10
27
32
? 14
1.20 k 0.10 0.83 -t 0.12 5.7 2 4.5
1.13 % 0.11 1.11 k 10.8 f
0.12 2.1
CAPRINI
ET AL. : HEMATOLOGIC
CHANGES
TABLE
FOLLOWING
633
BURNS
4
GROUP III: MILD BURN DATA~ Time periods (number of patients in group) ~48 hr (14) Fibrinogen
416
3-8 days (10)
9- 16 days (10)
17-25 days (5)
2 27
614
2 48
592
f 33
583
f 35
106 39 48
k8 -+3 k4
117 39 45
27 *5 *5
121 40 50
2 10 k6 54
4 12 49
2 3 k4 *7
c3 c4 CHm
81 28 36
k7 24 k6
KO Kl KS
3 36 39
+ 2 -t7 k6
PT (PT,F”L) PI-I- (PTTJPTT,) TEG (index)
1.13 * 1.03 2 0.3 2
0.04 0.08 2.0
3 21 37
‘- 2 2 11 24
1.04 _’ 0.05 0.83 2 0.05 4.5 T 2.2
2 33 50
2 2 f 10 55
1.06 f 0.95 f 9.7 f
0.36 0.42 1.6
0.96 f 0.88 f 6.5 f
0.02 0.05 2.2
n Values are means f standard error of the mean
man factor, complement, or kinin activity. Hageman factor activated by intravascular contamination can trigger plasmin release directly with subsequent fibrinogen and fibrin degradation. The combination of Hageman factor and plasmin activation then results in activation of the coagulation complement and kinin sequences with aggregation of platelets, thrombin generation, and fibrin formation, liberation of vasoactive peptides that produce increased vascular permeability and edema, elaboration of anaphylatoxin triggering mast cell histamine release, and local pain. Endotoxin can be used in the absence of antigenantibody complexes classically known to activate this whole sequence. Thus, it is reasonable to assume that during massive burn wound sepsis, these changes should be present and positive test values obtained. Our results demonstrate statistically significant changes in at least one measurement from each of these systems that correlate with the expected survival. Early changes occur between these groups and with subsequent measurements up to 16 days postburn, the number of statistically significant parameters increase. This would suggest that intravascular contamination from tissue damage during the first 48
hr is not as important as the changes which occur later. The variety and multitude of changes observed at a time when sepsis, not edema, is the major clinical problem would support the evidence for intravascular origin of these changes. It is interesting to note that elevated FDP activity correlates with plasminogen suppression and these latter changes are most significant during the second postbum week. Since these investigations were completed, an additional 25 patients have been studied and the results will be reported later. This larger series has permitted evaluation of ultimate survival compared to all of these tests. Not only do the trends persist as previously seen using the prognostic index, but the number of statistically significant trends has also increased (31 statistically significant paired comparisons). SUMMARY
These studies demonstrate statistically significant changes between the platelet counts, FDP and plasminogen levels, and C3 and 5min kinin activity of three groups separated according to their prognostic indices. Subsequent analysis of survivors confirms these trends and reveals addi-
634
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
tional supporting test data. We postulate that intravascular contamination occurs following major burns due to tissue trauma, and continues as burn wound sepsis develops. Standard heparin therapy does not significantly inhibit Hageman factor, plasmin, complement, or kinin activation products. It is not surprising that this anticoagulant does not change FDP levels or modify the course of most burns; however, further studies are needed to clarify this situation. ACKNOWLEDGMENTS The authors acknowledge the efforts of Willard A. Fry, M.D., Thomas G. Soper, M.D., and the entire staff of the Evanston Hospital Thermal Trauma Unit.
REFERENCES 1. Arturson, G., and Fjellstrom, K. E. Complement active serum protein in clinical bums. In A. B. Wallace and A. W. Wilkinson (Eds.), Research in Burns, p. 401. E. & S. Livingstone, Edinburgh, 1966. 2. Arturson, G., Hogman, C. F., Johnsson, S. G. O., and Killander, J. Changes in immunoglobulin levels in severely burned patients. Lancet 1: 546, 1%9. 3. Baxter, C. R. The response to initial fluid and electrolyte therapy of bum shock. In J. B. Lynch and S. R. Lewis (Eds.), Symposium on the Treatment of Burns, p. 42. C. V. Mosbey, St. Louis, 1973. 4. Becker, W. Determination of antisera titres using the single radial immunodiffusion method. Zmmunochemistry 6: 539, 1%9. 5. Becker, W., et al. Z. Klin. Chem. Biochem. 6: 113, 1968. 6. Brecher, G., and Cronkite, E. P. Morphology and enumeration of human blood platelets. J. Appl. Physiol. 3: 365, 1950. 7. Caprini, J. A., Eckenhoff, J. B., Ramstack, M. J., Zuckerman, L., and MO&OS, L. F. Contact activation of heparinized plasma. Thromb. Res. 5: 379, 1974. 8. Caprini, J. A., Zuckerman, L., Cohen, E., Vagher, J. P., and Lipp, V. The identification of accelerated coagulability. Thromb. Res. 9: 167, 1976. 9. Coleman, J. B., and Chang, F. C. Pulmonary embolism: An unrecognized event in severely burned patients. Amer. J. Surg. 130: 697, 1975. 10. Curreri, P. W., et al. Coagulation abnormalities in the thermally injured patient. Current Topics Surg. Res. 2: 401, 1970.
11. Curreri, P. W., Rayfield, D. L., Vaught, M., and Baxter, C. R. Extravascular fibrinogen degradation in experimental bum wounds. Surgery 77: 86, 1975. 12. Curreri, P. W., Wilterdink, M. E., and Baxter, C. R. Coagulation dynamics following thermal injury: Effect of heparin and protamine sulfate. Ann. Surg. 181: 161, 1975. 13. DeNicola, P. Thrombelastography. Charles C Thomas, Springfield, Ill., 1957. 14. Dougherty, T. F., and Dolowitz, D. A. Physiologic actions of heparin not related to blood clotting. Amer. J. Cardiol. 18: 24, 1964. 15. Elrod, P. D., McCleery, R. S., and Ball, C. 0. T. An experimental study of the effect of heparin on survival time following lethal bums. Surg. Gynecol. Obstet. 92: 35, 1951. 16. Eurenius, K., Mortensen, R. F., Meserol, P. M., and Curreri, P. W. Platelet and megakaryocyte kinetics following thermal injury. J. Lab. Clin. Med. 79: 247, 1972. 17. Fahey, J. L., and McKolvey, E. M. Quantitative determination of seum immunoglobulins in antibody-agar plates. J. Zmmunol. 94: 84, 1%5. 18. Foley, F. D., Greenawald, K. A., Nash, G., and Pruitt, B. A., Jr. Herpes virus infection in burned patients. New Engl. J. Med. 282: 652, 1972. 19. Haid, M., Zuckerman, L., Caprini, J. A., Kurtides, E. S., and Vagher, J. P. Thrombelastographic changes is carcinoma: An animal model. J. Med., in press. 20. Hawiger, J., Niewiarowski, S., Gurewich, U., and Thomas, D. P. Measurement of fibrinogen and fibrin degradation products in serum by staphylococcal clumping test. J. Lab. C/in. Med. 75: 93, 1970. 21. Hergt, K. Blood levels of thrombocytes in burned patients. J. Trauma 12: 599, 1972. 22. Hirsh, J., and Gallus, A. S. The activated partial thromboplastin time. N. Engl. J. Med. 288: 1410, 1963. 23. Mancini, G., Carbonara, A. O., and Heremons, J. F. Immunochemical quantitation of antigens by single radial immunodiffusion. Zmmunochemistry 2: 235, 1965. 24. McManus, W. F., Eurenius, K., and Pruitt, B. A., Jr. Disseminated intravascular coagulation in burned patients. J. Trauma 13: 416, 1973. 25. Meyers, A. Fibrin split products in the severely burned patient. Arch. Surg. 105: 404, 1972. 26. Parsons, R., Jr., Alrich, E. M., and Lehman, R. P. Studies on bums: Experimental study of the effect of heparinization and gravity on tissue loss resulting from third degree bums. Surg. Gynecol. Obstet. 90: 722, 1950. 27. Quick, A. J. Hemorrhagic Diseases. Lea and Febiger, Philadelphia, 1957. 28. Quick, A. J. Hemorrhagic Diseases and Throm-
CAPRINI ET AL. : HEMATOLOGIC basis, 2nd ed. Lea and Febiger, Philadelphia, 1966. 29. Ratnoff, 0. D. The interrelationship of clotting and immunologic mechanisms. Hosp. Prac. 6: 119, 1971. 30. Saliba, M. J. Heparin efficacy in burns, II. Aerospce Med. 41: 1302, 1970. 31. Saliba, M. J., Dempsey, W. C., and Kruggel, J. L. Large bums in humans: Treatment with heparin. .I. Amer. Med. Assoc. 225: 261, 1973.
32. Saliba, M. J., and Griner, L. A. Heparin efficacy in burns, I. Aerospace Med. 41: 179, 1970. 33. Saliba, M. J., and Saliba, R. J. Heparin in bums. Thromb. Diath. Haemorrh.
33: 113, 1974.
34. Staphylococcal clumping test: A visual test for the
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35.
36. 37. 38.
BURNS
635
semiquantitative estimation of fibrinogen/fibrin degradation products in serum. Sigma Technical Bulkfin No. 8.50. Sigma Chemical Company, St. Louis, MO. Revised May 1975. Snyder, A., Hand, M. R., and Gewurz, H. A rapid pH stat assay for plasma prekallikrein and fluctuations in disease. Allergy 36: 1, 1974. Storiko, K. Blur 16: 200, 1968. Wintrobe, M. M. Clinical Hematology. Lea & Febiger, Philadelphia, 1974. Zuckerman, L., Ramstack, M. J., Vagher, J. P., Carpini, J. A., and Mockros, L. F. Neutralization of heparin by cellular blood elements. Thromb. Res. 7: 149, 1975.
OF SURGICAL
22,626-635
RESEARCH
Hematologic
(1977)
Changes
following
Burns1
JOSEPH A. CAPRINI, M.D., FACS,2 VIRGINIA LIPP, B.S., LEON ZUCKERMAN, PH.C., JOSEPH P. VAGHER, B.S., AND DAVID P. WINCHESTER, M.D., FACS Department of Surgery, Evanston Hospitul. und Northwestern Unilrersity Medical School, Evanston, Illinois 60201
Submittedfor publication November 9, 1976
Despite numerous investigations, the significance of hematologic changes following burns has not definitely been established. Debate continues regarding numerous hematologic issues including the intravascular or extracellular origin of fibrin degradation products (FDP), and the value of heparin therapy in these patients. The purpose of this study was to perform a wide variety of hemostatic and immunologic tests and relate the laboratory results to the clinical probability of survival. The stimulus for these investigations evolves from the recent evidence suggesting that coagulation and immunologic systems are activated through a common link, the Hageman factor (FXII), which is normally activated following intravascular contamination. If this contamination occurs following thermal injury, the extent of changes should be reflected in all of these blood systems. The results obtained indeed show significant correlation between the tests performed and the initial prognostic index and strongly support the concept of intravascular contamination. MATERIALS
AND METHODS
Patients entered into this study following their admission to Evanston Hospital’s Thermal Trauma Unit. Forty-five patients, with varying degrees of burn, were divided into three groups based on their prognostic ’ Supported in part by Dee and Moody Funds, Evanston Hospital, and the Upjohn Company. 2 Schweppe Foundation Investigator.
index (age vs percentage of total burn). The percentage range of each group was determined in an attempt to equalize the group numbers. Group I (12 patients) had a prognostic index survival rate of under 20%, Group II (11 patients) had a survival rate of 20 to 80%, and Group III (22 patients) had a survival rate of greater than 80% (Table 1). A blood sample was drawn from each patient within 48 hr postburn. Additional samples were drawn within the following time periods: 3-8, 9-16, and 17-25 days postburn. All blood samples were collected using a modified “two syringe technique.” Using a 19-gauge butterfly apparatus, the vein was entered cleanly with a minimum amount of trauma. The butterfly system was “cleaned’ ’ of tissue thromboplastin contamination by discarding the hrst 3 to 5 ml of blood. The plastic syringe was then connected and 20 ml of blood was gently withdrawn. Timing is started when the blood enters the sample syringe. The blood is gently expelled down the inside wall of the specific polypropylene test tubes. The test procedures performed required whole blood, plasma, and serum. Therefore, it was necessary to use specific test tubes for the various tests. The Clay-Adams microhematocrit [37] and the direct phase platelet count technique of Brecher and Cronkite [6] required an EDTA anticoagulant sample. One drop of EDTA was placed in a 10 x 75mm polypropylene test tube, and 1 ml of the blood
626 Copyright All rights
0 1977 by Academic Press, Inc. of reproductmn in any form reserved
ISSN 0022.4804
CAPRINI ET AL. : HEMATOLOGIC TABLE 1 PROGNOSTIC
INDEX
AND SURVIVAL
OF GROUPS
Group
Number of patients
Admission prognosis index survival rate (age vs percentage of bum)
I II III
12 11 22
<20% 20-80% >80%
Eventually lived/died 418 714 22/o
sample was added as described. The test tube was capped with parafilm and inverted gently five or six times to mix the blood and anticoagulant. The hematocrit and platelet counts were completed within 30 min of sample time. Within 4 min of sampling, two aliquots of whole blood (without anticoagulant and collected in a 10 x 75mm polypropylene test tube) were placed into the thrombelastograph (TEG) (Haemoscope Corporation, Albertson, New York). A solution of 1% (w/v) celite (Johns-Manville Co., New York) was prepared in physiologic saline at pH 7.0 and, by selective decantation of fines, a specific size distribution is selected. The thrombelastographic procedure involved placing 0.33 ml of whole blood in one cup with 0.03 ml of 1% celite solution, mixing by raising and lowering the TEG pin three times, and subsequently opening the TEG camera shutter. A second cup receives 0.36 ml of native whole blood, mixed in a similar fashion, and run at the same time. Additional details regarding the TEG procedure have previously been described [7,8, 13, 19, 381. The sample for fibrin split products [20, 341 by staph clumping (Sigma Chemical Co., St. Louis, Missouri), was placed in the thrombin vial provided with the kit. Whole blood is collected and added to excess thrombin to assure complete conversion of fibrinogen to fibrin and its subsequent removal in the clot. E-Aminocaproic acid (EACA) is added to the
CHANGES FOLLOWING
BURNS
627
thrombin prior to the whole blood addition to inhibit conversion of plasminogen to plasmin (fibrinolysis). The blood is then incubated at 37°C for 2 hr to assure complete coagulation, clot retraction, and inactivation of excess thrombin. Serum recovered from the clot is diluted with Trizma-saline buffer and mixed with staphylococcal cell suspensions. An estimate of fibrinogen/fibrin degradation products (FDP) present is obtained by comparing the degree of cell clumping produced by the diluted test serum with clumping observed using known amounts of fibrinogen standard. The anticoagulant which best preserves the labile coagulation factors V and VIII is accomplished with 0.3 ml of 0.1 M sodium citrate and 0.2 ml of 0.1 M citric acid solution pH 4.8-5.0. A 9:l ratio of blood to anticoagulant is kept constant. One milliliter of anticoagulant is added to a 16 x lOO-ml polypropylene test tube and 9 ml of whole blood is gently expelled down the inside wall of the test tube. The test tube was capped with Parafilm and inverted gently five or six times to mix the blood and anticoagulant. The anticoagulated whole blood sample was centrifuged at 4000 rpm at 4°C for 10 min, within 10 min of blood withdrawal. With a plastic pipet, the plasma was carefully removed from the sample, without disturbing the red cells. The plasma was placed in two 10 x 75mm polypropylene test tubes. One tube was stored at 4°C until the one stage prothrombin time [27, 281and activated partial thromboplastin time were completed [22] (coagulation profiler, model CP-8, Bio/Data Corp., Willow Creek, Pennsylvania). The remaining plasma was frozen at -70°C to be used for the following tests. C3, C4, fibrinogen, and plasminogen [4, 5, 17, 23, 361 were determined through immunodiffusion plates supplied by Behring Diagnostics (Sommerville, New Jersey). The patient’s plasma was thawed and placed in a well in each of the four specified plates. The protein antigens present in the plasma diffuse into the thin gel
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JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
matrix causing a precipitin ring to form. After 48 hr, the diffusion is complete and the diameter of the ring is measured. A standard reference curve, constructed from the three standards supplied by Behring, is drawn by plotting the squared diameters of the precipitin rings against their respective concentrations. The values of the patient’s samples are determined by referring their squared diameter to the cahbration curve. The pH stat assay as used by Snyder et al. [35] was used by our laboratory for the determination of spontaneous esterase activity (KO), prekallikrein (kaolin activatable) esterase (Kl’), and kallikrein (Cl-esterase) inhibitor activity (KS). The spontaneous esterase activity is determined by placing 0.2 ml of thawed plasma with 2 ml of TAMe in a vessel on the pH stat. The reaction is run for 10 min using 0.04 M NaOH as the titrant at 37°C with constant stirring. The slope for the last 2.5 min is calculated and multiplied by a factor to convert the answer to micromoles per hour per milliliter. The prekallikrein determination differs only by activating the plasma for 1 min with 3% kaolin prior to TAMe addition, The kallikrein inhibitor activity differs by activating with kaolin for 5 min prior to TAMe addition. The results in this paper are not converted into units of inhibitor activity because the levels of prekallikrein activity were low and, more often than not, the 5-min values were higher than the 1-min values. Therefore, K5’ is merely esterase activity resulting after 5 min of contact activation. Total hemolytic complement (CH,,) measures the ability of the patient’s plasma to lyse 50% of a standard suspension of sheep erythrocytes coated with rabbit antibody. The sheep cells are washed three times in saline and once in gelatin-veronal buffer (GVB). A working solution of GVB is made fresh every 24 hr by dissolving 150 mg of gelatin in a small amount of double-distilled water (DDW). Forty milliliters of stock GVB is added to this and the volume is increased
to 200 ml with DDW. This buffer is the only solution used for the remainder of the assay. A 1:2000 dilution of hemolysin is added to an equal amount of a 5% suspension of the cells. This is incubated in a 37°C water bath for 10 min. The suspension is centrifuged and the supernatant is removed. The cells are washed again and resuspended in a small amount of buffer. These are counted and their concentration is adjusted to 5 x 108cells/ml. They are kept on ice until used. For each thawed test plasma, a 1:51 dilution is made. This is rediluted to make four C’ dilutions of 1: 10, 1:20, 1:34, and 1:51. One milliliter of the prepared sheep cells is added to each of the four test tubes. The suspension is gently inverted three times. A GVB control to check for any spontaneous lysis and a control to give a complete lysis value are also set up. The tubes are incubated for 1 hr in a 37°C waterbath. Following incubation, they are centrifuged and the supernatant is carefully removed so as not to disturb the cell button. The optical density (OD) of the supernatant is read at a wavelength of 541 nm on the spectrophotometer using the GVB control as the cell blank. The percentage lysis is calculated by dividing the OD of the sample by the OD of the complete lysis value. The percentage lysis was plotted against C’ dilution using only those values between 20 and 80%. The CH,, is the reciprocal of the dilution at which the line crosses the 50% lysis point. For each group, the mean and standard error for the described tests in each of the four time periods were calculated. Group comparisons were made on the tests for statistical significance by a t test of the values in corresponding time periods. The statistically significant tests were plotted as bar graphs. RESULTS
Patients were grouped according to their prognostic index for the purpose of analyz-
CAPRINI ET AL. : HEMATOLOGIC
ing the data and, as seen in Table 1, the actual survival statistics are better than those predicted by the index. The calculated average prognostic indexes for the patients in these groups were: GI = 5%, GII = 51%, and GIII = 98%. The patients with the most severe burns (GI) had an average age of 62 years with an average of 69% of their body burned. The second group with moderate burns had a mean age of 50 and an average burn of 43%, while the third group (GIII) was much younger, with a mean age of 28, and an average burn of 21%. As can be seen in Table 2, patients with severe burns (GI) did not show appreciable drops in fibrinogen during the first 48 hr, despite the substantial volume replacement. After the first 48 hr, there was some elevation of their levels above the normal ranges. Although total complement activity by the hemolytic assay appeared to be within the normal range, immunoassay of C3 and C4 (Table 2, Fig. 1) appeared depressed throughout the course of their stay. The native kallikrein or esterase activity (0 min) of their plasma was always low or zero and reflected no circulating trypsin-like activity (Table 2). Activation of the plasma
CHANGES FOLLOWING
BURNS
629
with kaolin produced no detectable activity at 1 min while the 5-min (Fig. 2) incubation period with the contact activator showed some significant results. This is normally reported as the inhibitor levels, but since there is no detectable activity at the peak (Kl’) time, we offer this activity as a point of reference for further investigation. Plasminogen levels are also depressed initially and remain that way and are inversely related to the fibrinogen degradation products (Fig. 3) in this group. Thus, increased FSP is associated with decreased plasminogen (Fig. 4). Platelet counts remained within the normal range (Fig. 5), and the prothrombin time (PT) was prolonged. The partial thromboplastin time (PTT) seemed to correct after the first 48 hr, and then remain normal. The TEG index, which measures the interactions of platelets, RBC, and plasma factors in clot formation [S], showed accelerated coagulability during the study with the most severe acceleration occurring between the 9th and the 16th days. Thus, characteristic features of this population are significant depressions of plasminogen, kallikrein activity, C3, and C4, with elevation of FSP with normal or elevated fibrinogen levels.
TABLE 2 GROUP I: SEVERE BURN DATA~
Time periods (number of patients in group) <48 hr (10) Fibrinogen
470
2 62
c3 c4 C&o
68 26 24
-c8 +4 k4
KO Kl K5
1 21 22
2 1 *9 27
PT (PTJPT,) PTT W’TdP%) TEG (index)
1.29 2 0.07 1.29 + 0.13 5.0 k 1.6
3-8 days (5)
9- 16 days (5)
658
+ 41
535
k 109
61 29 35
k5 24 + 11
70 30 34
zk 6 k 3 -c 3
1 0 5
? 1 k 0 t 3
0 3 23
k f 2
1.25 k 0.06 1.00 2 0.03 8.6 k 2.3
1.21 f 1.09 2 12.3 2
17-25 days (0)b
0 2 8 0.68 0.13 2.5
a Values are means -C standard error of the mean. b Only one patient remained from the original group who was not being treated with heparin at this time.
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JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
The more dynamic nature of GII patients is attributable to marked changes in their blood components (Table 3). In Fig. 1 the rebound of the third component of complement is shown to occur within the tist week. It is also impressive in that this group started with the lowest FSP in the early period, achieved the highest peak between 3 and 8 days, and then slowly dropped (Fig. 3). Plasminogen levels recovered by the third time period and began to rise above normal by the 25th day (Fig. 4). However, kallikrein activity (Kl’) did not significantly recover from the initial fall of the 3- to g-day period. Platelets, PT and PTT, did not change significantly in this group. The overall effect, reflected by the TEG index, suggests a hypercoagulable condition as the result of high fibrinogen and platelets existing between the 9th and the 25th days. Fibrinogen levels among Group III burn victims tend to peak early and fall similar to the GII patients (Table 4). The fibrin split products (Fig. 3) were elevated above the normals and peaked at about the 3rd to the 8th days. Plasminogen (Fig. 4) levels have returned to normal by this period and remain stable. Complement levels show only modest depression during the hrst 48 hr and are well within normal limits for the next 14 days. It should be noted that group size changes from time period to time period. This attrition of subjects is caused by death, treatment with heparin, or release from the unit. The effect of these losses would tend to normalize the data toward the center group, i.e., the values of less severely burned Group III individuals and survivors of more severe burns both migrate toward one another with time. The initial kallikrein esterase activity (Kl’) of Group III is still lower than normal and seems to reflect the severity of the burn, in that Group I Q Group II I Group III. There was no significant free esterase activity (KO) measurable in any of these three groups. Therefore, there must
LWS
POST BURN
FIG. 1. Complement 3 levels for each group in each of the four time periods (mean values with SE).
be sufficient inhibitors to block any activity generated by intravascular contaminants or activation by plasmin. As previously mentioned with Group II, the TEG index reflects a possible hypercoagulable state by the ninth day which may be the result of elevated platelets (Fig. 5) and fibrinogen in the presence of intravascular burn contaminants. Some slight shortening of the
50
0
<48 t!Rs.
3-0 ON9
9-l0[yIr
DAY9 POST BURN
FIG. 2. Kinin 5’ activity for each group in each of the four time periods (mean values with SE).
CAPRINI ET AL. : HEMATOLOGIC
CHANGES FOLLOWING
BURNS
631
group during the next 6 days. This also corresponds to the period of maximum plasminogen disappearance and the lowest esterase (KS’) and C3 levels of that same group of severe burns. Clinically, this period relates to a time when most of the edema of these patients has decreased. DISCUSSION
FIG.
A variety of hemostatic alterations have been observed following major thermal injury including thrombocytopenia, secondary thrombocytosis, increased platelet adhesiveness, decreased platelet survival time, elevated fibrinogen, factor V and VIII levels, and elevated fibrin split products [3, 10,16,21,25]. Depressed serum immunoglobulins and various complement components have been observed [l, 21. It has been suggested that the burn patient is more susceptible to infection [18], pulmonary embolism [9], and disseminated intravascular coagulation [24]. Heparin has 3. Fibrin split product levels for each group in been tried in experimental and clinical burns
each of the four time periods (mean values with SE).
PTT is evident by the second through the fourth periods, but there was too much variance during the 9th through the 16th day to correlate the results with the TEG index. Inhibition of coagulation as the result of elevation of FSP is not reflected by any of the coagulation tests (TEG, PT, PIT). The significance of this may be in the type of fragments which are peculiar to this type of trauma or may be that the degree of intravascular contamination and subsequent activation overrides the competitive inhibition of these FSP. This result is in marked contrast to that seen during the late phase of disseminated intravascular coagulation (DIC) where FSP are elevated, platelets are down, and all clotting tests are prolonged. It is also noteworthy that platelet levels (Fig. 5) are not excessively low within FIG. 4. Plasminogen levels for each group in each the first 48 hr, but fall in the high-risk of the four time periods (mean values with SE).
632
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
to reverse some of the clotting changes, lessen tissue loss by inhibiting vascular thrombosis, shorten healing time, prevent burn extension, decrease thromboembolic complications, and reduce pain. Unfortunately, uncontrolled studies and conflicting data make the role of heparin in burns unclear [12, 14, 15, 26, 30, 31, 32, 331. Failure to alter fibrin split product concentrations with heparin in animals prior to and following burn injury, and in randomly assigned burn patients suggests that these degradation products may be the result of extravascular plasmin digestion of fibrinogen rather than due to intravascular coagulation [ll]. These facts cast further doubt on the anticoagulant value of systemic heparin therapy in burns. The extravascular theory may be questioned, however, if fibrinolytic activity persists beyond the edema phase of the burn wound. Recently, evidence has appeared linking the blood clotting process with immunologic defenses [29]. Following intravascular contamination, it has been shown that coagulation, fibrinolytic, complement, and kinin pathways may be activated through the Hageman factor (FXII) by contact activation. If one assumes that intravascular contamination occurs in burn victims
j8 300
200
loo
0
<49
HRS
3-9 MY9 MY9Posr9lRN
9-16 OAYS
v-25
DAY!3
FIG. 5. Platelet counts for each group in each of the four time periods (mean values with SE).
early due to tissue damage, and later, possibly from sepsis, then changes in all of these systems should occur and be proportional to the extent of injury. The failure of heparin to modify FDP levels, or significantly modify the clinical burn course would not then be surprising since this anticoagulant in the usual pharmacologic dosages does not significantly inhibit Hage-
TABLE 3 GROUPII: MODERATE BURN DATA” Time periods (number of patients in group) <48 hr (10) Fibrinogen
3-8 days (4)
9- 16 days (5)
17-2.5 days (5)
387
f 56
676
f 124
573
f 79
520
2 35
c3 c4 CHsO
73 32 30
f 10 57 26
113 38 43
2 11 -c 4 2 4
103 32 45
29 +8 25
106 41 46
f 12 +7 27
KO Kl K.5
2 32 50
2 1 e 14 k5
2 2 35
0 0 34
k 0 + 0 +8
PT W’PL) PI-l- m-TIP=,) TEG (index)
1.10 2 0.05 1.03 k 0.07 0.6 k 2.0
2 f f
1.12 2 0.89 f 8.6 +
(2Values are means k standard error of the mean.
2 2 19 0.06 0.07 3.0
0
k 0
10
27
32
? 14
1.20 k 0.10 0.83 -t 0.12 5.7 2 4.5
1.13 % 0.11 1.11 k 10.8 f
0.12 2.1
CAPRINI
ET AL. : HEMATOLOGIC
CHANGES
TABLE
FOLLOWING
633
BURNS
4
GROUP III: MILD BURN DATA~ Time periods (number of patients in group) ~48 hr (14) Fibrinogen
416
3-8 days (10)
9- 16 days (10)
17-25 days (5)
2 27
614
2 48
592
f 33
583
f 35
106 39 48
k8 -+3 k4
117 39 45
27 *5 *5
121 40 50
2 10 k6 54
4 12 49
2 3 k4 *7
c3 c4 CHm
81 28 36
k7 24 k6
KO Kl KS
3 36 39
+ 2 -t7 k6
PT (PT,F”L) PI-I- (PTTJPTT,) TEG (index)
1.13 * 1.03 2 0.3 2
0.04 0.08 2.0
3 21 37
‘- 2 2 11 24
1.04 _’ 0.05 0.83 2 0.05 4.5 T 2.2
2 33 50
2 2 f 10 55
1.06 f 0.95 f 9.7 f
0.36 0.42 1.6
0.96 f 0.88 f 6.5 f
0.02 0.05 2.2
n Values are means f standard error of the mean
man factor, complement, or kinin activity. Hageman factor activated by intravascular contamination can trigger plasmin release directly with subsequent fibrinogen and fibrin degradation. The combination of Hageman factor and plasmin activation then results in activation of the coagulation complement and kinin sequences with aggregation of platelets, thrombin generation, and fibrin formation, liberation of vasoactive peptides that produce increased vascular permeability and edema, elaboration of anaphylatoxin triggering mast cell histamine release, and local pain. Endotoxin can be used in the absence of antigenantibody complexes classically known to activate this whole sequence. Thus, it is reasonable to assume that during massive burn wound sepsis, these changes should be present and positive test values obtained. Our results demonstrate statistically significant changes in at least one measurement from each of these systems that correlate with the expected survival. Early changes occur between these groups and with subsequent measurements up to 16 days postburn, the number of statistically significant parameters increase. This would suggest that intravascular contamination from tissue damage during the first 48
hr is not as important as the changes which occur later. The variety and multitude of changes observed at a time when sepsis, not edema, is the major clinical problem would support the evidence for intravascular origin of these changes. It is interesting to note that elevated FDP activity correlates with plasminogen suppression and these latter changes are most significant during the second postbum week. Since these investigations were completed, an additional 25 patients have been studied and the results will be reported later. This larger series has permitted evaluation of ultimate survival compared to all of these tests. Not only do the trends persist as previously seen using the prognostic index, but the number of statistically significant trends has also increased (31 statistically significant paired comparisons). SUMMARY
These studies demonstrate statistically significant changes between the platelet counts, FDP and plasminogen levels, and C3 and 5min kinin activity of three groups separated according to their prognostic indices. Subsequent analysis of survivors confirms these trends and reveals addi-
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JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
tional supporting test data. We postulate that intravascular contamination occurs following major burns due to tissue trauma, and continues as burn wound sepsis develops. Standard heparin therapy does not significantly inhibit Hageman factor, plasmin, complement, or kinin activation products. It is not surprising that this anticoagulant does not change FDP levels or modify the course of most burns; however, further studies are needed to clarify this situation. ACKNOWLEDGMENTS The authors acknowledge the efforts of Willard A. Fry, M.D., Thomas G. Soper, M.D., and the entire staff of the Evanston Hospital Thermal Trauma Unit.
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11. Curreri, P. W., Rayfield, D. L., Vaught, M., and Baxter, C. R. Extravascular fibrinogen degradation in experimental bum wounds. Surgery 77: 86, 1975. 12. Curreri, P. W., Wilterdink, M. E., and Baxter, C. R. Coagulation dynamics following thermal injury: Effect of heparin and protamine sulfate. Ann. Surg. 181: 161, 1975. 13. DeNicola, P. Thrombelastography. Charles C Thomas, Springfield, Ill., 1957. 14. Dougherty, T. F., and Dolowitz, D. A. Physiologic actions of heparin not related to blood clotting. Amer. J. Cardiol. 18: 24, 1964. 15. Elrod, P. D., McCleery, R. S., and Ball, C. 0. T. An experimental study of the effect of heparin on survival time following lethal bums. Surg. Gynecol. Obstet. 92: 35, 1951. 16. Eurenius, K., Mortensen, R. F., Meserol, P. M., and Curreri, P. W. Platelet and megakaryocyte kinetics following thermal injury. J. Lab. Clin. Med. 79: 247, 1972. 17. Fahey, J. L., and McKolvey, E. M. Quantitative determination of seum immunoglobulins in antibody-agar plates. J. Zmmunol. 94: 84, 1%5. 18. Foley, F. D., Greenawald, K. A., Nash, G., and Pruitt, B. A., Jr. Herpes virus infection in burned patients. New Engl. J. Med. 282: 652, 1972. 19. Haid, M., Zuckerman, L., Caprini, J. A., Kurtides, E. S., and Vagher, J. P. Thrombelastographic changes is carcinoma: An animal model. J. Med., in press. 20. Hawiger, J., Niewiarowski, S., Gurewich, U., and Thomas, D. P. Measurement of fibrinogen and fibrin degradation products in serum by staphylococcal clumping test. J. Lab. C/in. Med. 75: 93, 1970. 21. Hergt, K. Blood levels of thrombocytes in burned patients. J. Trauma 12: 599, 1972. 22. Hirsh, J., and Gallus, A. S. The activated partial thromboplastin time. N. Engl. J. Med. 288: 1410, 1963. 23. Mancini, G., Carbonara, A. O., and Heremons, J. F. Immunochemical quantitation of antigens by single radial immunodiffusion. Zmmunochemistry 2: 235, 1965. 24. McManus, W. F., Eurenius, K., and Pruitt, B. A., Jr. Disseminated intravascular coagulation in burned patients. J. Trauma 13: 416, 1973. 25. Meyers, A. Fibrin split products in the severely burned patient. Arch. Surg. 105: 404, 1972. 26. Parsons, R., Jr., Alrich, E. M., and Lehman, R. P. Studies on bums: Experimental study of the effect of heparinization and gravity on tissue loss resulting from third degree bums. Surg. Gynecol. Obstet. 90: 722, 1950. 27. Quick, A. J. Hemorrhagic Diseases. Lea and Febiger, Philadelphia, 1957. 28. Quick, A. J. Hemorrhagic Diseases and Throm-
CAPRINI ET AL. : HEMATOLOGIC basis, 2nd ed. Lea and Febiger, Philadelphia, 1966. 29. Ratnoff, 0. D. The interrelationship of clotting and immunologic mechanisms. Hosp. Prac. 6: 119, 1971. 30. Saliba, M. J. Heparin efficacy in burns, II. Aerospce Med. 41: 1302, 1970. 31. Saliba, M. J., Dempsey, W. C., and Kruggel, J. L. Large bums in humans: Treatment with heparin. .I. Amer. Med. Assoc. 225: 261, 1973.
32. Saliba, M. J., and Griner, L. A. Heparin efficacy in burns, I. Aerospace Med. 41: 179, 1970. 33. Saliba, M. J., and Saliba, R. J. Heparin in bums. Thromb. Diath. Haemorrh.
33: 113, 1974.
34. Staphylococcal clumping test: A visual test for the
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35.
36. 37. 38.
BURNS
635
semiquantitative estimation of fibrinogen/fibrin degradation products in serum. Sigma Technical Bulkfin No. 8.50. Sigma Chemical Company, St. Louis, MO. Revised May 1975. Snyder, A., Hand, M. R., and Gewurz, H. A rapid pH stat assay for plasma prekallikrein and fluctuations in disease. Allergy 36: 1, 1974. Storiko, K. Blur 16: 200, 1968. Wintrobe, M. M. Clinical Hematology. Lea & Febiger, Philadelphia, 1974. Zuckerman, L., Ramstack, M. J., Vagher, J. P., Carpini, J. A., and Mockros, L. F. Neutralization of heparin by cellular blood elements. Thromb. Res. 7: 149, 1975.